Non‐invasive in vivo monitoring of transplanted stem cells in 3D‐bioprinted constructs using near‐infrared fluorescent imaging

Abstract Cell‐based tissue engineering strategies have been widely established. However, the contributions of the transplanted cells within the tissue‐engineered scaffolds to the process of tissue regeneration remain poorly understood. Near‐infrared (NIR) fluorescence imaging systems have great potential to non‐invasively monitor the transplanted cell‐based tissue constructs. In this study, labeling mesenchymal stem cells (MSCs) using a lipophilic pentamethine indocyanine (CTNF127, emission at 700 nm) as a NIR fluorophore was optimized, and the CTNF127‐labeled MSCs (NIR‐MSCs) were printed embedding in gelatin methacryloyl bioink. The NIR‐MSCs‐loaded bioink showed excellent printability. In addition, NIR‐MSCs in the 3D constructs showed high cell viability and signal stability for an extended period in vitro. Finally, we were able to non‐invasively monitor the NIR‐MSCs in constructs after implantation in a rat calvarial bone defect model, and the transplanted cells contributed to tissue formation without specific staining. This NIR‐based imaging system for non‐invasive cell monitoring in vivo could play an active role in validating the cell fate in cell‐based tissue engineering applications.


| INTRODUCTION
Tissue engineering involves the combination of cells with natural or synthetic biomaterials to create implantable tissue constructs, and it offers an attractive approach for the treatment of damaged tissues and organs. A large number of successful tissue engineering strategies require the use of mesenchymal stem cells (MSCs). MSCs hold great therapeutic promise as a cell source, and they can differentiate into multiple cell types, such as adipocytes, osteoblasts, and chondrocytes. 1 The ability of MSCs to evade immunosurveillance after cell transplantation and to suppress the immune response has made MSCs attractive candidates for clinical use. While the therapeutic effects of stem cells are widely accepted, 2 the specific mechanisms of tissue formation and the role of MSCs in the tissue-engineered complex are poorly understood. To better understand the therapeutic effects of tissue-engineered constructs after transplantation, monitoring of cell behaviors such as migration, survival and death, and differentiation in vivo should be performed. Recent advances in molecular imaging have allowed for the non-invasive monitoring of transplanted cells, [3][4][5] tissue formation, 6,7 and scaffold degradation 6,[8][9][10][11][12][13] in vivo without retrieving the implants. Non-destructive and longitudinal consecutive monitoring provides more reliable information and is both more ethical and economical than invasive methods.
Bioprinting is a relatively novel technology that creates 3D structures consisting of biomaterials, living cells, and biomolecules. It has brought about a revolution in the field of tissue engineering, allowing for the fabrication of precise and sophisticated structures in a patientspecific manner. Bioink is printable soft biomaterials loaded with living cells and is one of the main components of bioprinting technology.
Bioink materials should be printable, degradable, non-toxic, cytocompatible, and possess high mechanical properties. Hydrogels, which can be produced from proteins and extracellular matrix components (e.g., collagen and hyaluronic acid), provide environmental cues that directly aid stem cell growth. Hydrogels have been chosen as materials for 3D bioprinting due to their cell-friendly and cross-linkable properties. [14][15][16] Hydrogel precursor solution is typically the state of a bioink during 3D printing. Crosslinking step is necessary to make a hydrogel network, and covalent crosslinking, unlike relatively weak physical crosslinking, increases strength and durability after 3D printing, and many studies have been conducted to impart covalent functionalization to natural hydrogels. Functionalized gelatin methacryloyl (GelMA) is relatively stable at body temperature, more resistant to degradation than gelatin, and at the same time retains the bioactive properties of gelatin such as cell attachment and enzymatic degradation. [16][17][18][19] Near-infrared (NIR) fluorescence has advantages for biomedical imaging, including relatively low tissue absorption, reduced scattering, and minimal autofluorescence. 20 Unlike visible light, which penetrates living tissue to a depth of less than a millimeter, NIR light can penetrate living tissue to a depth ranging between millimeters and centimeters. Thus, the use of NIR fluorophores produces a high signal that is detected with low background. In addition to imaging, there is a recent report that NIR has been used as a light source enabling non-invasive 3D bioprinting due to its penetration into deep tissue and biocompatibility, 21 and its application range is gradually expanding.
Previously, we reported that NIR fluorescence could be used to non-invasively monitor scaffold degradation in vivo by employing zwitterionic NIR fluorophore (ZW800-1) or highly charged cyanine 6,22 and cell growth in vitro using functional polymethine indocyanines. 23 In this study, we tried to create a system that can be used to track cells embedded in a 3D bioprinted tissue-engineered construct in vivo. GelMA bioink that impregnates NIR fluorophore-labeled mesenchymal stem cells (NIR-MSCs-GelMA) was used as a bioink and analyzed signal stability and reliability after 3D bioprinting in vitro.
Finally, we proposed that NIR-MSCs-GelMA could be an effective tool for non-invasive tracking of transplanted cells in vivo and confirming the role of transplanted cells in tissue formation without specific staining ( Figure 1).

| Intracellular trafficking of NIR fluorophores
We utilized placenta-derived mesenchymal stem cells (PMSCs) because they are a promising cell source for regenerative medicine.
PMSCs are obtained from the placenta, a tissue that is involved in multiple essential roles, including fetal development, tolerance, and serving as a reservoir of progenitor/stem cells. PMSCs have a remarkable ability to both differentiate into multiple cell types (encompassing the three germ layers), and to sustain undifferentiated proliferation for prolonged periods. 24,25 A high yield of PMSCs can be obtained from placentas discarded after childbirth. In addition, PMSCs expand quickly in vitro, produce a subdued immune response in recipients, and present enhanced immunosuppressive properties compared with bone marrow-derived stem cells.
Several lipophilic organic fluorophores have been developed to label cytoplasm membrane, mitochondria, lysosomes, and so forth.
Tagging membranes can interfere with cell migration and tagging mitochondria can affect membrane potential changes. On the other hand, tagging lysosomes as an intracellular digestive system minimizes the effect on cellular activity. 26,27 CTNF127 is a functionalized fluorophore that is selectively targeted to lysosomes and has excellent physicochemical and optical properties in serum. Furthermore, this dye is available at an NIR wavelength, and this enables the longitudinal tracking of living cells in vivo with minimal absorption, scattering, and autofluorescence. 27,28 Before the main experiments, we optimized the CTNF127 label-

| Synthesis and characterization of GelMA
GelMA has natural cell-binding motifs, hydrophilicity, and matrix metalloproteinase degradation sites. 29 GelMA is water-soluble, enhances cellular adhesion, and can degrade gradually in the body.
The methacryloyl (MA) groups enable the GelMA to be light-cured with UV treatment. GelMA has different physical properties (i.e., elastic modulus, water swelling, degradation, etc.) depending on the synthesis process; therefore, it is important to establish appropriate conditions according to the purpose of tissue engineering. In particular, the degree of methacryloyl substitution that directly affects the degree of crosslinking and pore size of hydrogel can be controlled by the amount of MA during synthesis. 30,31 F I G U R E 1 Schematic of the non-invasive near-infrared (NIR) imaging system used to track stem cells (a) Bioink prepared by mixing GelMAbased materials (including other excipients) and NIR-mesenchymal stem cells (MSCs). (i) GelMA synthesis, (ii) NIR-MSCs preparation. NIR-MSCs: Fluorescence-emitting (700 nm) CTNF127-labeled placenta-derived mesenchymal stem cells (PMSCs) (b) 3D bioprinting of hybrid constructs; PCLG-copolymer for a framework and NIR-MSCs-GelMA for cells were printed alternately. (c) Transplantation into rat calvarial defect and noninvasive stem cell imaging We synthesized GelMA using various amounts of methacrylic anhydride. The GelMA solution before lyophilization was analyzed by 1 H NMR that is often used to evaluate the replacement of free amino groups on gelatin by methacrylate groups ( Figure S1). In the NMR peaks, the grafted methacryloyl group was verified using signals at δ = 5.4 ppm, δ = 5.7 ppm (acrylic protons, 2H), and by another peak at δ = 1.8 ppm (methyl group, 3H) ( Figure S1a). In addition, the MA modification of lysine residues, according to the addition of methacrylic anhydride, can be confirmed by the decrease of the lysine signal at δ = 2.9 ppm (lysine methylene, 2H). The degree of substitution of methacrylamide on gelatin could be calculated using the lysine signal among NMR peaks. 32 Consequently, it increased from 22% to 93% depending on the amount of methacrylic anhydride added (0.05-4 ml) ( Table 2). We chose 0.6 ml of methacrylic anhydride based on stiffness of hydrogel fabricated with 5% GelMA (data not shown).
GelMA (hydrogels at concentrations (2.5% and 5%) were prepared to assess the gel stability depending on UV treatment time ( Figure S1b). Nozzle clogging or pressure fluctuations were less in 5% GelMA than 2.5% GelMA (data not shown). As the GelMA  concentration and UV treatment time increased, the opacity of the gel that was formed also increased, which indicated highly crosslinked gel formation. Furthermore, the formed gel maintained stable status after 15 days of incubation in PBS. For the rest study, 5% GelMA was mixed with the other components of bioink and crosslinked by UV for 90 s per one side following 3D printing. In other studies, 5% GelMA hydrogel showed high cell viability in vitro 33 and excellent result for bone tissue regeneration. 34 High crosslinking and high concentrations of polymer are more suitable to print structures with good shape fidelity and will be a hindrance to cell growth. More fine-tuning is required in order to find the optimal biofabrication window. 35

| 3D bioprinting using bioink NIR-MSCs-GelMA
The  In quantitative analysis (Figure 4b-iii and iv), the entire NIR fluorescence signal intensity and its signal area in the ROI was significantly invariable during test. Unlike indirect labeling using such as cell transfection with fluorescent proteins (e.g., green fluorescent protein), many direct labeling technologies commonly undergo halving of dye following cell division. 38 Our result disproves that the fluorescence leakage from cells was minimized, which is expected to be These results demonstrate that CTNF127 can be a reliable fluorophore for use in 3D culture environments and would be suitable for long-term cell imaging in 3D construct.

| Stem cell tracking by histological analysis
The contribution of PMSCs to tissue formation is shown in Figure 6. The main two modes of stem cell therapy are a direct replacement of dead cells and indirect healing through the release of paracrine factors. 44 In order for stem cells to work in any way, understanding and monitoring the fate and regenerative potential of transplanted stem cells is essential to expediting the clinical application of stem cells by improving the safety and therapeutic efficacy of stem cell-based therapies. 45,46 To accurately determine the degree of utilization of stem cells placed into the scaffold, a deeper study is required to detect the bio-distribution, cell survival, and fate at the whole organism level. Nevertheless, the current study shows that the MSC encapsulated in the scaffold has achieved successful colonization and tissue formation, more importantly, our imaging system contributed to identifying this.
Preclinically, the effect of PMSCs have been proved in the various bone diseases such as osteogenesis imperfecta, 47 [53][54][55][56] We also expect that the newly formed tissue is bone extracellular matrix, however, the detail tissue characterization should be performed.
Injecting additional fluorophores targeting specific tissues such as bone and cartilage 57,58 or targeting specific molecules such as fibrin 59 and cathepsin B 60 will be helpful for characterizing tissue formation or responses.
Although it can vary depending on the type and site of the injury, it was reported that the average bone healing time is between 6 and 8 weeks. 61 In rodents, the calvarial defects can be filled by soft fibrous tissue suggesting the critical period of restoring bone is between 4 and 8 weeks. 62,63 As such, the 8-week period is clinically relevant but is also a remarkable period for in vivo cell tracking studies. This period is longer than previous in vivo studies with labeled stem cells, including IR-780 iodide-labeled multipotent stem cells derived from rat skin dermis in rats (1 week), 64 PKH26 fluorescent-labeled human bone marrow-derived MSCs transplanted in nude mice (4 weeks), 65 and ultrasmall superparamagnetic iron oxide-labeled human-derived stem cells in nude mice (4 weeks). 5 Even though studies that use labels that are bound with DNA (such as BrdU) 66 or membrane proteins (such as GFP) 67 have tracked cells for similar or longer timeframes (8-12 weeks), these compounds limit clinical translation.

| Cultivation of PMSCs
Consent for acquiring the human placenta was obtained from mothers before giving birth and the fresh placenta was transported to Wake Forest Institute for Regenerative Medicine for tissue isolation. 68 Briefly, fetal cells were isolated from chorionic placental tissue cells at full term. For placental cells, the whole placenta was collected, and a biopsy isolating the chorion was digested for fetal placental cells. 38 We received a donation of quality-controlled human placenta-derived

| Cellular labeling with NIR fluorophore
The 700 nm NIR fluorescence-emitting fluorophore (CTNF127) was synthesized based on the previous method. 27

| 3D bioprinting using NIR-MSCs-GelMA
The 3D bioprinter (Integrated Tissue-Organ Printer, ITOP) and CAD software, including slicing, tool path generation, and motion program generation, used in this study were developed in-house. 70 The 3D bioprinter is composed of six cartridges that enable the printing of  During the procedure, the animal was positioned on a warming pad.
The hair over the skull was shaved, and the underlying skin was aseptically prepared using povidone-iodine/betadine scrub. The skin was roundly incised, and the subcutaneous tissue was dissected along the same line as the skin. The underlying periosteum was sharply incised and subsequently elevated off the skull to obtain sufficient exposure for defect creation. A cooled stainless-steel trephine (7 mm diameter) and a surgical drill were used to remove a full-thickness section of bone. The 3D-printed complex was implanted after separating the bone flap from the dura mater underneath with a Malis dissector.
Meticulous hemostasis was maintained throughout the procedure via epinephrine gauze and manual compression. The implants were secured by snug placement and by the closure of the overlying fascia.
For non-invasive fluorescent imaging, the periosteum and subcutaneous tissue were closed with 5-0 absorbable suture, and the skin was closed with 4-0 non-absorbable suture in an interrupted pattern. The suture was removed 3 days after surgery. Animals were hosted in the WFIRM animal care center.

| NIR fluorescence imaging
Micro-fluorescence images were obtained using a fluorescence micro- Macro-fluorescence images were taken using a small animal in vivo imaging system (Pearl® Impulse, LI-COR Biosciences, Lincoln, NE, United States), which was equipped with NIR channels (700 and 800 nm) and a white channel. The intensity of the NIR fluorescence signal from rats was monitored over the skin weekly for 8 weeks, while the rats were anesthetized with 3% isoflurane. In this study, the imaging system was remodeled to be used with rats (to avoid size limitations). Rats were sacrificed at 8 weeks for imaging after skin removal and histological analysis. Imaging data were collected and quantified by Pearl Software Images (LI-COR Biosciences). The fluorescence region seen in the earliest sample was determined by the ROI. The mean of the fluorescence intensity (FL Mean; arbitrary unit) was presented in the results.

| Histological analysis
Transplanted sites including implants from rats were harvested and decalcified in Richard Allan Scientific Decalcifying Solution (Thermo Scientific) for 24 h following fixation with 10% neutral-buffered formalin for 48 h. Decalcified tissues were rinsed with DW, dipped in 30% sucrose for 1 day, and frozen in liquid nitrogen for cryo-embedding. Frozen samples were sequentially cryo-sectioned (10 μm per slice). One of the slides was used to observe the NIR fluorescence signal at 700 nm. Another slide was used to assess DAPI staining (nuclei).
To visualize tissue formation, the other sectioned slide was stained with H&E after fixation with 4% paraformaldehyde for 10 min. All the images were obtained using the NIR fluorescence microscope described above Section 4.8.

| Statistical analysis
The samples were assessed in triplicate for each group to analyze the statistical data. The data are presented as the mean value ± SD. Statistical analysis of the experimental results was performed using one-way analysis of variance followed by the Tukey multiple comparisons test. The reported p values were considered statistically significant at p < 0.05.

ACKNOWLEDGMENTS
This study was supported by the following the US National Institutes of Health (NIH) grants: #P41EB023833, #R01EB022230, and #R01HL143020. This work was also supported by the National