Stem Cell-Mediated Accelerated Bone Healing Observed with In Vivo Molecular and Small Animal Imaging Technologies in a Model of Skeletal Injury

Abstract

Adult stem cells are promising therapeutic reagents for skeletal regeneration. We hope to validate by molecular imaging technologies the in vivo life cycle of adipose-derived multipotent cells (ADMCs) in an animal model of skeletal injury. Primary ADMCs were lentivirally transfected with a fusion reporter gene and injected intravenously into mice with bone injury or sham operation. Bioluminescence imaging (BLI), [¹⁸F]FHBG (9-(fluoro-hydroxy-methyl-butyl-guanine)-micro-PET, [¹⁸F]Fluoride ion micro-PET and micro-CT were performed to monitor stem cells and their effect. Bioluminescence microscopy and immunohistochemistry were done for histological confirmation. BLI showed ADMC’s traffic from the lungs then to the injury site. BLI microscopy and immunohistochemistry confirmed the ADMCs in the bone defect. Micro-CT measurements showed increased bone healing in the cell-injected group compared to the noninjected group at postoperative day 7 (p <0.05). Systemically administered ADMC’s traffic to the site of skeletal injury and facilitate bone healing, as demonstrated by molecular and small animal imaging. Molecular imaging technologies can validate the usage of adult adipose tissue-derived multipotent cells to promote fracture healing. Imaging can in the future help establish therapeutic strategies including dosage and administration route.

Five to 10% of the fractures that occur annually in the United States demonstrate delayed healing or nonunion.¹ Numerous approaches, including autologous and allogeneic bone, osteoinductive scaffolds, and growth factors, have been used to facilitate the healing of fractures with various degree of success.^2–4 A promising alternative is to deliver mesenchymal stem cells (MSCs) harvested from the patient’s own tissues to the site of injury. Injury/trauma evoke the release of MSCs into peripheral blood, and the circulating stem cells are thought to participate in healing by homing to damaged and unhealthy tissues in a mechanism akin to leukocyte trafficking to sites of inflammation that are mediated via adhesion molecules such as selectin, chemokine receptors, and integrins; MSC trafficking to sites of injury are also characterized by cytokine production and differentiation after arrival at the target tissue.⁵ Thus, the therapeutic response can potentially be increased by enhancing the endogenous MSC pool with exogenously administered MSCs.

The majority of the studies on the stem cell therapy have been on myocardial or neural regeneration after injury or ischemia, where the method to follow the life, death, and the effect of the stem cells, namely the trafficking, has been by nuclear medicine or magnetic resonance imaging (MRI).^6–9 Bioluminescence imaging (BLI) has been used to monitor hematopoietic stem cells (HSCs) and embryonic stem cells (ESCs) in mice model of sublethal irradiation and myocardiac infarction, where the cells survived and reconstituted the bone marrow or improved the cardiac function, respectively.^10,11 There are proof of principle studies in using MSCs including fracture repair or osteogenesis imperfecta,^12,13 but long-term systemic trafficking of systemically administered MSCs in living subjects by BLI have not been reported thusfar. As the progress in the characterization of the harvested MSCs increases, increased clinical use of these cells will be facilitated by imaging methods that longitudinally monitor the biodistribution, viability and action of engrafted cells in living subjects.¹⁴

To study MSC trafficking to a site of injury, we have used adipose-derived multipotent cells (ADMCs), which show higher cell yield, less complication, and similar differentiation potential.¹⁵ Using an animal model of skeletal injury, the goals of this work are to determine (1) whether engrafted ADMCs aid in the healing of the injury, and (2) whether molecular and small animal imaging technologies can be successfully utilized to monitor these effects in vivo. In this study, we have labeled ADMCs using a fusion reporter gene and applied small animal and molecular imaging technologies to repeatedly study the function of exogenously administered cells in a reliable animal fracture model over time. Biological parameters such as and bone metabolism/mineralization, cell trafficking, and cell survival were longitudinally monitored and quantified with [¹⁸F]Fluoride ion small animal positron emission tomography (micro-PET), small animal computed tomography (micro-CT), and BLI. This is the first example where a population of systemically administered reporter ADMCs have been tracked in living subjects to home to a musculoskeletal injury and shown to promote healing of the damaged tissue.

METHODS AND METHODS

Cell Harvest and Culture Conditions

All experiments followed the protocols approved by the IACUC at Stanford University. All primary cell cultures were derived from adult (2- to 3-month-old) female FVB mice. Adipose-derived stem cells were harvested from the subcutaneous fat of anterior abdominal wall. The fat pads were excised, washed in PBS, and finely minced. Tissues were digested with 0.075% collagenase (Sigma-Aldrich, St. Louis, MO) at 37°C for 1 h. Neutralized cells were centrifuged to eliminate mature adipocytes and fibrovascular fraction. Floating adipocytes were removed, and pelleted stromal cells were passed through a 100-μm cell strainer before plating. All cell types were cultured in DMEM, containing 10% FBS and 100 IU/mL penicillin, 100 IU/mL streptomycin, and 0.25 μg/mL amphotericin at 37°C in an atmosphere of 5% CO₂. Cells were passaged by standard methods of trypsinization. Only cells of passage 2 and 3 were used for all experiments. Based upon the followed protocol for ADMC harvest, multiple washings, and centrifugation immediately after removes most of the fibrovascular fraction of the fat tissue, and after two or three passages of plastic adherence, cells show homogeneity reaching 80–90%.

Transducing Cells with the Reporter Gene

Lentiviral transfer vector was produced by transfection of 293T cells as described previously.¹⁶ ADMCs were trypsinized upon subconfluence, and at 24 h after second passage, the cells were labeled with CMV promoter-driven fusion reporter system encoding firefly luciferase (fluc)-monomeric red fluorescent protein (mrfp)-herpes simplex virus truncated thymidine kinase (hsvttk) using lentivirus-delivered stable transduction protocol. Using FACS analysis, about 35% of the cells were confirmed to be transduced with the lentiviral vector. Transfected cells underwent luciferase assay, fluorescence microscopy, and thymidine kinase assay to confirm the transfection.

Surgery

Fifteen adult female FBV mice were anesthetized with intra-peritoneal injection of a 20-mL Ketamine–Xylazine mixture; the right thigh was shaved. The animal was placed under inhalation anesthesia, and after surgical cleansing, an incision was made on the lateral aspect of the right thigh to expose the right femur. A 0.5 mm-sized defect was made through the proximal shaft of the right femur using a drill bit, under saline irrigation, and the skin was sutured closed. To create a soft tissue (sham) injury model, five mice underwent the same procedure including the incision through skin and muscle. Instead of the femoral defect, the surface of the bone was gently touched with the blunt side of the scalpel. Subsequently, the soft tissue wound was sutured close.

Cell Injection

The ADMSCs were stably transduced with the fusion reporter gene 72 h prior to delivery. On the day of injection, the cells were suspended in 250 mL Hanks balanced salt solution, and 250,000 cells were injected per mouse via lateral tail vein. They were injected twice at postoperative day 2 and day 5, into 15 animals, five each in the bone injury group, sham injury group, and those that had received no procedure.

Imaging

Bioluminescence

Ten minutes after intraperitoneal administration of D-luciferin (3 mg/mouse, Biosynth International, IL), 5-min images were taken with an in vivo imaging system employing a cooled charge-coupled device camera (IVIS; Xenogen, CA). Prone and right lateral images were obtained from each animal at each time point to better determine the origin of photon emission. The animals were imaged on days 0, 3, 6, 10, 15 postinjection. For clarification, the postinjection day 0 is the day of the injection, which is postoperative day 2. The postinjection day 0 animals were imaged immediately after systemic injection of ADMCs. Of note, the postinjection day 3 bioluminescence image was taken just prior to the second boost of injection. Bioluminescence was quantified by drawing ovoid regions of interest (ROIs), uniformly sized 3.19 × 2.20 cm throughout the whole experiment, over the wound on the lateral images of the mice, and the average radiance measured (photon/cm²/sec/steradian). Statistical analyses were done with a Student t-test.

Micro-PET

Animals underwent micro-PET imaging with [¹⁸F]FHBG (9-fluoro-hydroxy-methyl-butyl-guanine). Imaging was performed on postinjection days 2, 3, 6, and 7, after intravenous injection of 200 mCi FHBG per mouse 1 h before scanning. A subset of animals were imaged after intravenous injection of 140 mCi [¹⁸F]Fluoride on days 1, 4, and 10 with or without the stem cell injection (n = 2 each). Mice were anesthetized with isoflurane 1 h after the injection of a tracer, placed in a prone position on the gantry, and imaged using an R4 micro-PET scanner (Concorde Microsystems, TN). Acquisition time was 10 min, and images were reconstructed by using OSEM algorithm using Micro-PET Manager.

The uptake in the injury site was obtained by putting an ellipsoid ROIs, which were normalized by the uptake from an ROI in the upper thoracic spine.

Micro-CT

On postinjection days −2, 5, 12, and 25 (PODs 0, 7, 14, and 27), the animals underwent computed tomography under isoflurane anesthesia. The animals were placed into the scanner (eXplore RS Micro-CT System, GE Medical Systems, Raleigh, NC), and the lower body was imaged with 80-kVp, 450-μA cone beam CT with the 400-ms exposure time, 400 number of views, and two frame averages. The estimated skin dose was 0.20 Gy. The image acquisition and reconstruction were done using eXplore Evolver and eXplore Reconstruction Interface software, respectively. The reconstructed images were viewed using GEMS Microview. The resolution of the image voxel was 45 × 45 × 45 mm³. The change of bone density in the femoral defect was followed by measuring Hounsfield units in three 0.2285 mm³-sized cylindrical ROIs within the defect, one in the centermost part and two in the periphery of the defect, and the results were compared using a Student’s t-test.

Histologic Analysis

At postinjection days 8 and 16 a subset from each group of animals was sacrificed and their right thigh harvested immediately after intravenous injection of 100 mL of D-luciferin. The thigh was snap frozen in liquid nitrogen and stored in −80°C until ready for sectioning. Twelve micron-thick frozen sections were cut from OCT-embedded tissues using a tungsten carbide knife (Thermo Shandon, Waltham, MA) in an HM 500 series Microtome (Microm, Heidelberg, Germany). For the detection of bioluminescence, the sections were placed under a light tight chamber of inverted microscope (Axiovert 200, Carl Zeiss, Germany) and imaged using Living Image software. To eliminate the possibility of irregular substrate distribution, 4 mL of a mixture of D-luciferin (30 mg/mL) and ATP (20 μmol/200 μL) was applied onto the tissue section and imaged (a Hamamatsu C2300 CCD camera with an intensifier, Hamamatsu, Japan). For the detection of fluorescent cells within the tissue, the slides were put under the fluorescence microscope (Carl Zeiss) with the maximum excitation wavelength of 514 nm. For immunohistochemistry, fixed and decalcified tissues were frozen-sectioned at 6–8 μM thickness. The sections were blocked with a goat antimouse blocking serum (1:10 dilution). Serial sections were stained for immunofluorescence with rabbit polyconal anti-RFP antibody (1:100, Clontech, Mountainview, CA). Primary antibodies were detected by using goat antirabbit immunoglobulin conjugated with biotin (1:500). Endogenous RFP signals were amplified by staining with diaminobenzedine.

Statistical Methods

The data from the ROIs were plotted onto an Excel spreadsheet, and were analyzed for statistical significance using a Student t-test. A p-value below 0.05 was used for statistical significance.

RESULTS

Harvested ADMCs can be transformed into reporter ADMCs using a multimodality reporter gene. The stromal cells harvested from adipose tissue resembled fibroblasts in shape. After passage and expansion, ADMCs were transformed using a lentiviral vector or plasmid encoding luciferase and fluorescence protein. Luciferase assay confirmed the successful transformation of these cells. The transfection efficiency was 35% as confirmed by fluorescence activated cell sorting (FACS) analysis. Once prepared, reporter ADMCs were systemically injected into a variety of animal injury models and controls via tail vein. “Injured” animals are animals that had undergone surgery, whether it was a femoral 0.5-mm diameter bicortical trephine defect or a “sham operation” (i.e., wound in the thigh down to the femoral periosteum without trephination of the femur).

A pattern of trafficking of the reporter ADMCs was noted in injured animals. Systemically administered reporter ADMCs initially traffic to the lung. In days immediately following, a BLI signal could be seen arising from the injury site then to injury site in the thigh. Repeated BLI was performed on injured and noninjured mice starting on day 0 after the systemic administration of reporter cells (Fig. 1a). For “day 0,” BLI was performed immediately after systemic intravenous injection of reporter ADMCs. On this day, injured mice showed bright bioluminescence over the thorax, suggesting the stem cells predominantly localized to the lungs after injection. In contrast, animals that did not receive reporter ADMCs did not show any signal in the chest. Also on day 0, no significant BLI signal difference was seen in the operated thigh in injured animals between those that received and those that did not receive reporter ADMCs.

Figure 1.

BLI. BLI of mice after femoral injury (first row) and sham-operated animal (second row), both after systemic injection of ADMCs. Mice with femoral injury without injection of ADMCs (third row). Uninjured mice with injection of ADMCs (fourth row). When the cells are injected into the mice on day 0 (first column), the luminescent cells were initially found in the lungs. In mice with bone injury and sham-operation, BLI signal is observed in the injured leg (white arrows, days 3 and 6), suggesting trafficking of reporter cells to this region. Eventually, by day 15, decreased BLI signal is observed in the area of injury. In mice with bone injury without cell injection, there is low background signal in the wound area that decreases over time. Quantification of the luminescence signal (photon/second/cm²/steradian) shows that the mice with systemically injected ADMCs show higher signal in the wound compared to controls, but the increased signal observed then decreases over time, suggesting transgene loss or reporter cell death (b).

On postinjection day 3, the wounded right thigh showed localized, positive BLI signal, and the thoracic BLI signal disappeared. By quantifying the luminescence signal (photon/second/cm²/steridian) from an ROI drawn over the thigh, values from the cell-injected injured group were higher than that of the noninjected injured group (p <0.05). Injured animals (bone or soft tissue injury) consistently had higher BLI signal in the right thigh than uninjured animals (p <0.05), until postinjection day 15, when the signal from all the groups disappeared. Subsequent histologic evaluation with bioluminescence microscopy confirmed the presence of reporter ADMCs in the fracture callus (Fig. 2).

Figure 2.

Histological analysis. Bioluminescence microscopy from the mice with ADMCs expressing luciferase show increased luminescent signal in the bone defect compared to the control (a, b, white arrows). Immumohistochemistry using antibody against RFP shows positive cells in the injured bone of the cell-injected group (c) and not in the control (d).

Healing of the trephine defect as measured by micro-CT is faster in stem cell-injected animals compared to noninjected controls. Bicortical trephination of the proximal right femur was easily appreciated on micro-CT scans through the defect. The bone density within the defect, as measured in average Hounsfield Units (HU), could be measured using a volumetric, cylindrical ROI drawn to encompass the defect. Immediate postoperative images show that the defect corresponded to a cylindrically shaped low-density area in the proximal femoral diaphysis. Small amounts of dense material could be found within the trephine defect, but are most likely due to osseous debris created by the drilling procedure. By postoperative day 7, the average HU within the defect increased, suggesting healing of the bone injury. The defect was completed healed by postoperative day 27 (postinjection day 25), and trabecular bone could be visualized (Fig. 3a and b). Average HU measurements of the defect demonstrated that the ADMC-injected animals showed higher density in the area on postoperative day 7 (postinjection day 5) compared to noninjected animals (p ≤ 0.015). On postoperative day 14, however, there was no significant difference in the HU of the defect area between the two groups, suggesting that both cell-injected and noninjected animals had achieved an equivalent level of healing by this time point (Fig. 3c).

Figure 3.

Micro-CT and micro-PET imaging. HU unit measurement in the bone defect (white arrow) shows that the defect is healing faster in the cell-injected group compared to control, as indicated by the increased bony bridge in the fracture gap. Fluoride ion micro-PET imaging shows increasing fluoride ion uptake in the injured femur (white arrows) compared to the intact contralateral femur after the surgery on POD 4 and 10 (p <0.001) (d). Quantification of the uptake in the femoral wound showed higher uptake compared to the contralateral femur (e).

Bone mineral metabolism can be quantitatively monitored using [¹⁸F]fluoride ion micro-PET. On fluoride ion micro-PET images, which shows fluoride uptake wherever bone mineralization takes place, increased radiotracer uptake was seen in the injured proximal femoral area. The injured bone showed significantly higher [¹⁸F]fluoride ion uptake compared to the contralateral intact femur after the operation (Fig. 3d and e). The injured bone showed significantly higher [¹⁸F]fluoride ion uptake compared to the contralateral intact femur after the operation (p <0.00029 on POD 4, p <0.000175 on POD 10). The bone-injured group with and without cell injection did not show statistically significant difference in [¹⁸F]fluoride ion uptake (10.16 vs. 15.4% injected dose/g body weight, p = 0.89). [¹⁸F]FHBG-PET data did not show definite uptake from the site of injury relative to background measurements, and we hypothesize the reason to be low number of reporter ADMCs and reporter expression in the ROI.

Fluorescence component of the fusion reporter product was not visible by fluorescence microscopy of frozen tissue sections. Reports have suggested transgene silencing, or free radical-associated cellular phototoxicity by the excitation of the fluorescent protein.^17,18 Additionally, bone autofluorescence contributed to the difficulty in identifying fluorescent proteins. Immunohistochemistry revealed the presence of RFP-expressing ADMCs in the fracture callus after being injected into the bone (Fig. 2).

DISCUSSION

MSCs are well-known candidates for an effective therapeutic approach in clinically challenging scenarios such as fracture nonunion, delayed union, segmental defect repair, and osteoporosis.¹⁹ ADMCs, which are abundant in the subcutaneous abdominal fat and require less traumatic procedure than bone marrow-derived counterparts, were used in our study. Following systemic infusion of the cells into living subjects possessing a bone injury, accelerated bone healing was noted within a week of the injury in the injected group compared to controls.

The presence of stem cells is critical for the bone repair. Several ongoing clinical trials are based upon this premise.²⁰ MSCs are attracted to and retained in the injured tissue and promote healing by a few proposed mechanisms. For example, MSCs are attracted to the site of injury via adhesion molecules, chemokines, and specific receptors or ligands within a certain window of time.^21,22 Additionally, the biology of fracture healing is essentially a recapitulation of bone formation seen during embryogenesis, where MSCs are important in callus formation.²³ In both fracture healing and endochondral ossification, there are a series of events that include immature mesenchymal cell condensation, cartilage development, chondrogenic hypertrophy, vascular invasion, osteoblast recruitment and, ultimately, bone formation. Studies focusing on long bone repair suggest that the source of immature mesenchymal cells is indeed pluripotent MSCs, which can be isolated from the surrounding soft tissues (e.g., adipose) and the hematopoietic system, including the bone marrow compartment and the systemic circulation.^24–26 MSCs with considerable plasticity are found in a variety of tissues,²⁷ and give rise to the chondrocytic and osteoblastic elements eventually required in the fracture generate. In the case of tissue injury or inflammation, MSCs are attracted to the site, and the migrated stem cells proliferate and differentiate, becoming normal constituents of the host cytoarchitecture and supporting stroma of the injury. Furthermore, other studies have observed that stem cells may not need to differentiate into the regenerating tissue type, but rather are responsible for the local production of growth factors or stromal elements, such as guiding strands in the injured area.²⁸ These characteristics of the MSCs form the premise for the development of novel, minimally invasive cell-based approaches toward nonhealing or poorly healing fractures. However, much less is known about there behavior in vivo with regard to survival, distribution, and fate.⁵

Although we were able to serially demonstrate that systemically delivered reporter ADMCs do indeed track to the injury in a living animal model, a number of important considerations and challenges, which have risen from this study, are given below. The majority of the ADMCs, for example, are first trapped in the pulmonary vasculature after systemic injection, which is most likely related to the large size of ADMCs and “activated” state.^5,29 The BLI signal from the lung soon disappears and signal in the injury appears in the days following. Explanations for this progressive increase in BLI signal from the injury can be explained either by the reporter ADMCs migrating from the lung or other sites to the injury or by the initial survival and proliferation of the reporter ADMCs at the site of injury. Although some ADMCs clearly home to the injury site, the overall BLI signal in the body drops precipitously. The result implies that there is significant death of the donor cells. The mechanism for this is not entirely clear, but is consistent with observations from other studies; for example, others have found that the survival rate of transplanted cells range from near 0 to 5% of baseline values after transfusion.^30,31 The protective functional niche provided by the injured tissue is known to be essential for engraftment of the stem cells into the host tissue, the window of which niche could be quite short lived.³² Other explanations for the BLI signal loss after day 0 include the fact that reporter gene silencing may occur after cells differentiate and proliferate.^31,33,34 Also, the heterogeneity of the cell colony and the presence of confounding, preexisting disease conditions, such as the degree of tissue injury, affect the behavior of cells.³⁵ Devine et al.³³ reported that MSCs systemically infused into irradiated baboons distributed randomly to a variety of tissues including the genitourinary and gastrointestinal tracts, liver, and lung. In additional experiments they performed, they detected GFP-expressing cells in bone marrow following GFP-transduced bMSC infusion, but they failed to identify GFP expression in the other tissues by immunohistochemistry.^31,33 They suggested that gene silencing, cell death, and immunologic rejection played a role in the loss reporter activity and/or the actual loss of reporter cells. The masking of the reporter expression of the stem cells poses is a challenge for the reporter gene technology and gene therapy, for which further studies are necessary.

A major reason to further develop reporter gene imaging is its ability to distinguish viable from nonviable cells. Current clinical imaging methods currently exist that can monitor the trafficking of cells to target sites, including radionuclide methods (¹¹¹In-oxine, ¹¹¹In-tropolonate, and ^99mTc-HMPAO)^6,7 or MRI.^8,9 However, all of these suffer from an inability to distinguish viable from nonviable cells. Furthermore, previously marked cells can lose their molecular “tag” to phagocytic cells such as macrophages, which compromises the specificity of such studies.

Our predominant cell tracking system is the optical luciferase reporter system that has been utilized by a number of other investigators in studies of stem cell reconstitution in vivo.¹⁰ A significant amount of the emitted light generated from the reporters, Firefly or Renilla luciferase, is absorbed by endogenous molecules such as hemoglobin and melanin. There is also considerable scattering of the light through the tissue, which necessarily limits the spatial resolution. As a result, optical BLI is limited to small subjects such as mice, and its translation into larger species is limited.³⁶ One related interesting finding is that the sham-operated with ADMCs group has a higher BLI signal at day 6 than bone injury group with ADMCs. It appears that the pool of viable, circulating ADMCs had to home to two injured environments in the bone injury model, namely, the bone and soft tissue injury. By comparison, only a single site of injury is present in the sham-operated model. Thus, assuming equal affinity for the osseous and soft tissue site, a higher number of ADMCs should home to the soft tissue in the sham-operated model than the soft tissues in the bone injury model. Given the fact that optical signal is attenuated with increasing tissue depth, an osseous injury may “hide” a fair number of ADMCs. Additionally, if the osseous injury is able to differentially “attract” more ADMCs than a soft tissue injury for whatever reason, the differences in the bioluminescence image will be further accentuated.

Another important consideration in this study is the radiation dose administered to the animal during micro-CT acquisition and [¹⁸F]-fluoride ion dose administration.³⁷ The dose administered may have effects on fracture healing and ADMC mortality and morbidity. The radiation dose caused by our study protocol is 20 cGy on the mouse surface for a single micro-CT study. Dose estimation for the PET radioisotope have been previously calculated by others using mouse phantom, according to which the whole-body dose ranged from 60 to 170 mGy depending upon the biologic half-time for the tracer.³⁸ The dose that can stimulate gene expression is 200 mGy, and the dose at which 50% of the mice perish without medical intervention (LD₅₀) is 5 Gy. Depending on the time schedule over which the radiation dose is delivered, the effect will be a complex mix of radiation-induced damage (proportional to the delivered dose) and the stimulation of repair mechanism.³⁹ Further studies need to be performed to address the effect on cell viability and bone healing and to optimize the imaging sequences.

Despite some challenges, it is certain that exogenously administered ADMCs reached the fracture site as shown through BLI microscopy and immunohistochemistry. In turn, the addition of ADMCs augmented healing, because the micro-CT could follow the physiologic outcome and confirmed the accelerated mineralization observed in the ADMC-treated group. In the future, we hope to minimize the challenges with reporter gene imaging and broaden the application of the reporter system so that biodistribution, safety, and efficacy of cell-based therapies can be more accurately assessed. Future iterations of reporter gene imaging and small animal imaging technologies are certain to aid in the assessment and validation of cell-based therapies in living animal models.

CONCLUSIONS

After MSCs were injected in living subjects with bone injury, the course of the cells and the efficacy of the cellular therapy were followed longitudinally by labeling the cells with a multimodality reporter gene. Micro-CT showed that the systemically administered MSCs promoted healing of the injury compared to controls. The reporter gene technology provided a useful tool to purify or locate the cells in vivo and ex vivo, but there were some limitations in trafficking the cells at later time points and from deep tissues. Overall, we can conclude that the adipose-derived MSCs are promising therapeutic reagent for osseous injuries.