{'fraction_non_alphanumeric': 0.04511515406457041, 'fraction_numerical': 0.023451467031233568, 'mean_word_length': 5.111503856041131, 'pattern_counts': {'":': 0, '<': 4, '<?xml version=': 0, '>': 1, 'https://': 1, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Characterization of wild-type and STAT3 signaling-suppressed mesenchymal stem cells obtained from hemovac blood concentrates

Background Venous blood drained from the knee joint after total knee arthroplasty (TKA) using a hemovac line is a potential source of bone marrow components, including stem cells, from the cutting surface of cancellous bones of the knee joint. However, the function of mesenchymal stem cells (MSCs) in patients with osteoarthritis (OA-MSCs) can be disrupted by inflammation of the joint. Further, to override the invasive nature of the currently used methods to obtain stem cells, their functional modification is necessary for therapeutic applications. Methods The effects of signal transducer and activator of transcription 3 (STAT3) signaling suppression on MSCs (iSTAT3-MSCs) were evaluated by comparative analyses of the characteristics of OA-MSCs and iSTAT3-MSCs from 20 patients who underwent TKA. Results OA-MSCs and iSTAT3-MSCs were adherent, with fibroblast-like appearance and high rates of expression of MSC-specific markers, including CD73, CD90, and CD105 (>90%). Both OA-MSCs and iSTAT3-MSCs were able to differentiate into osteogenic, adipogenic, and chondrogenic cells; however, iSTAT3-MSCs showed higher levels of osteogenic and chondrogenic differentiation markers than OA-MSCs. Additionally, the anti-inflammatory and chondroprotective cytokine levels were higher in iSTAT3-MSCs than in OA-MSCs. Conclusions These findings indicate that iSTAT3-MSCs after TKA are potentially effective for stem cell therapy in the context of bone and cartilage disorders.


Introduction
Total knee arthroplasty (TKA) is the gold standard treatment for end-stage osteoarthritis (OA); a large number of patients undergo this procedure every year (1)(2)(3)(4)(5)(6). A large components from the cutting surface of cancellous bones of the distal femur and proximal tibia can be found in hemovac blood (HVB) (e.g., stromal cells, including stem cells) (15,16). As HVB is mostly venous blood, stromal components from the bone marrow must be isolated from HVB concentrates (HVBCs) by centrifugation (17). Importantly, stem cells isolated from HVB are multipotent, with the ability to differentiate into osteogenic, adipogenic, and chondrogenic cells (17).
However, patients who undergo TKA are typically elderly and/or have inflammatory joint conditions (18,19). Consequently, their stem cells express inflammatory signals (20,21). Therefore, to use HVB from such patients for stem cell therapy, it is necessary to modify MSCs aiming to reduce the expression of inflammatory factors.
We hypothesized that the inhibition of signal transducer and activator of transcription 3 (STAT3) in MSCs derived from HVB collected from patients subjected to TKA would improve their functions in the context of OA. Therefore, in this study, to improve the therapeutic potential of MSCs, we treated OA-MSCs with STA21, a STAT3 inhibitor. The anti-inflammatory effects, marker expression, and the differentiation ability of MSCs (wild type vs. STA21-treated) were then evaluated in vitro. We present the following article in accordance with the MDAR reporting checklist (available at https://dx.doi. org/10.21037/atm-21-791).

Patients and ethical statement
HVB was collected from 20 patients who underwent TKA.

Isolation and culture of OA-MSCs from HVBCs
Just before wound closure, a hemovac line was inserted through the superolateral aspect of the patella and placed in the lateral gutter of the knee joint. The line was connected to the hemovac and an average of 300 mL blood was collected within 3 h after surgery.
HVB (40 mL), including 4 mL of ACD solution, was aseptically aspirated from the hemovac after TKA. The BMC Kit (Revmed, Seongnam, Korea) was used to concentrate the blood aspirate via two cycles of centrifugation ( Figure 1). HVBCs and PBS (Gibco Invitrogen, Grand Island, NY, USA) were then mixed at a 1:2 ratio. The mixture was carefully loaded on the top of Histopaque solution (1.077 g/m; Sigma Chemical co., St. Louis, MO, USA). Mononuclear cells were separated by density gradient centrifugation (400 ×g, 25 min, room temperature), washed three times with PBS, and filtered using a 70-µm cell strainer (Becton Dickinson, Franklin Lakes, NJ, USA). Resuspended cells were incubated at 37 ℃ with 5% CO 2 in basal medium (αMEM containing 10% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin). The medium was changed at 3-day intervals and non-adherent cells were removed by gently pipetting.

Flow cytometry
The surface markers of OA-MSCs and iSTAT3-MSCs were analyzed using a FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA, USA). Briefly, the harvested cells were fixed in 4% paraformaldehyde at 4 ℃ for 30 min and washed in cold flow cytometry buffer (BD Biosciences). Aliquots of 1×10 6 cells were incubated with specific antibodies for 20 min in the dark. After incubation, the cells were washed in PBS, and the expression of the different surface markers was assessed by flow cytometry.

Quantitative real-time polymerase chain reaction
Total RNA was extracted from OA-MSCs and iSTAT3-MSCs using the RNeasy Mini Kit (74104; QIAGEN, Hilden, Germany). The RNA samples (≥20 ng/µL) were reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit (205311, Qiagen) following the manufacturer's instructions. All samples were then subjected to qPCR using SYBR Green (A6001; Promega, Madison, WI, USA) and the Promega qPCR system. The relative expression levels were determined using the 2 −∆∆Ct method.

Evaluation of the differentiation potential of OA-MSCs and iSTAT3-MSCs
The osteogenic, adipogenic, and chondrogenic differentiation potential of OA-MSCs and iSTAT3-MSCs were evaluated. OA-MSCs and iSTAT3-MSCs were plated at a density of 2×10 4 cells/well in 6-well plates.
For osteogenic differentiation, cells were incubated in osteogenic medium (A1007201; Gibco, Life Technologies, Karlsruhe, Germany); the medium was changed every 2-3 days. After 14 days of culture, cells were washed thrice with PBS (Gibco) and fixed in 4% paraformaldehyde at 4 ℃ for 20 min. The mineralization of the extracellular matrix as an indicator of osteogenic differentiation was determined using Alizarin red S and alkaline phosphatase (ALP) staining.
For adipogenic differentiation, cells were incubated in adipogenic differentiation medium (A1007001; Gibco); the medium was changed every 2-3 days. After 14 days, the cells were washed 3 times with PBS (Gibco) and fixed in 4% paraformaldehyde at 4 ℃ for 20 min. The cells were then treated with the Oil red O dye to visualize the lipids. For quantitative measurement, Oil red O was dissolved in isopropanol, and the absorbance was measured at 520 nm.
For chondrogenic differentiation, a pellet culture method was used. Briefly, 2.5×10 5 cells (passage 3) were resuspended in chondrogenic differentiation medium (A1007101; Gibco) in 15 mL polypropylene tubes. After centrifugation at 500 ×g for 5 min for aggregation, the cells were incubated at 37 ℃ in a humidified atmosphere containing 5% CO 2 . The aggregates were cultivated for 21 days, and the medium was changed every 2-3 days. On day 21, the cell pellets were fixed in 10% formalin and embedded in paraffin. Then, 4-µm-thick tissue sections were obtained and stained with type II collagen (1:200, ab185430; Abcam, Cambridge, MA, USA), Alcian blue (pH 2.5), and toluidine blue to reveal the collagen and sulfated proteoglycan content in the extracellular matrix. Hematoxylin-eosin staining was also performed for morphological observations.

Statistical analysis
All statistical analyses were performed in triplicate. The results are shown as the mean ± standard deviation (SD). For comparison, the t-test and ANOVA were used (IBM SPSS, version 20.0). Statistical significance was defined as P<0.05.

Morphology of OA-MSCs and iSTAT3-MSCs
OA-MSCs were successfully isolated from HVBCs in all cases. The cells readily expanded in vitro, and fibroblastlike cells could be observed attached to the culture flasks, 4 to 8 days after initial plating ( Figure 2A). The cells were maintained in αMEM supplemented with 10% FBS and penicillin. No morphological changes were observed after 11 days of culture ( Figure 2B). Additionally, iSTAT3-MSCs showed no morphological differences ( Figure 2C).

Multi-lineage differentiation (osteogenic, adipogenic, and chondrogenic) of OA-MSCs and iSTAT3-MSCs
Osteogenic differentiation was confirmed by determining the ALP and Alizarin red S staining after 14 days of culture ( Figure 4A,4B). The ALP staining was not observed in undifferentiated OA-MSCs and iSTAT3-MSCs [ Figure 4A  Chondrogenic differentiation was observed using hematoxylin-eosin (to evaluate the cell morphology), Alcian blue (pH 2.5) and toluidine blue (to evaluate pericellular matrix proteoglycans), and type II collagen staining. Many round cells with a lacuna-like structure were observed in the context of OA-MSCs ( Figure 5A; black arrow) and iSTAT3-MSCs ( Figure 5B; red arrow) cultured in chondrogenic differentiation medium, as per hematoxylin-eosin staining. A rich extracellular matrix surrounding the cells was observed in both differentiated OA-MSCs and iSTAT3-MSCs. But, undifferentiated OA-MSCs and iSTAT3-MSCs not observed lacuna-like structure and round shape like chondrocytes ( Figure 5C,5D). Differentiated OA-MSCs and iSTAT3 OA-MSCs showed strong Alcian blue ( Figure  5E; black *, Figure 5F; red *), while undifferentiated OA-MSCs and iSTAT3-MSCs did not show any staining ( Figure  5G,5H). In toluidine blue staining, differentiated OA-MSCs and iSTAT3 OA-MSCs showed strong purple ( Figure  5I; yellow *, Figure 5J; yellow **), while undifferentiated OA-MSCs and iSTAT3-MSCs did not show staining ( Figure 5K,5L). Notably, the Alcian blue (pH 2.5) and toluidine blue staining intensities were stronger in differentiated iSTAT3-MSCs ( Figure 5F,5J) than OA-MSCs ( Figure 5E,5I). Type II collagen is the main collagen component of the extracellular matrix in cartilage. Here, the expression of type II collagen in OA-MSCs ( Figure 5M; black *) and iSTAT3-MSCs ( Figure 5N; black **) was detected as brown color. In line with the above results, while type II collagen was not expressed in undifferentiated cells ( Figure 5O,5P), the levels of type II collagen were detected in both differentiated iSTAT3-MSCs and OA-MSCs (to a greater extent in the former).

Gene expression analyses
The gene expression levels in OA-MSCs and iSTAT3-MSCs were evaluated on day 14 of culture after osteogenic and adipogenic differentiation and on day 21 after chondrogenic differentiation.

Inhibition of STAT3 signaling induces the antiinflammatory response of OA-MSCs
The levels of different cytokines in the culture supernatants of OA-MSCs and iSTAT3-MSCs were evaluated using ELISA (Figure 7). The levels of pro-inflammatory cytokines, including IL-6, IL-1β, IL-8, and VEGF, were significantly lower in the supernatants of iSTAT3-MSCs than in those of OA-MSCs (IL-6 and IL-1β; **, P<0.01; IL-8 and VEGF; ***, P<0.001). In contrast, the levels of the anti-inflammatory cytokines, IL-10 and TGF-β, were significantly higher in the culture supernatants of iSTAT3-MSCs than in those of OA-MSCs (***, P<0.001). Altogether, these data show that STAT3 signaling inhibition may increase the anti-inflammatory response and improve the therapeutic effects of MSCs.

Discussion
Stem cells collected from different sources, including the bone marrow, adipose tissue, cord blood, and synovium have clearly established clinical applications (22)(23)(24). To utilize autologous cells, surgical or invasive procedures are necessary to obtain the tissues. Traditionally, the bone marrow has been an important source of stem cells; however, bone marrow aspiration is a very painful procedure (25). Nevertheless, clinical studies with bone marrow aspirates are ongoing owing to their simple culture process and favorable stemness characteristics (26)(27)(28)(29).
Additionally, large numbers of cells can be isolated from the adipose tissues, with a higher chondrogenic potential than MSCs from other sources; however, an invasive procedure is still required to harvest these cells (30,31). To avoid such procedures, allogenic cells are a commercially available alternative. Nonetheless, it is difficult and time-consuming to obtain approval from the FDA and there are many ethical concerns associated (32).
HVB can be obtained easily after bone surgery, such as TKA, without special procedures, pain, or related ethical issues. The volume of HVB obtained varies among patients; nevertheless, a sufficient number of MSCs can be obtained by concentration and cultivation. Accordingly, HVB is a potential stem cell source. Importantly, HVB has bone marrow components, although stromal components are less abundant in these samples than in bone marrow aspirates. However, the issue of low numbers of stem cells or stromal cells in HVB can be resolved by repeated concentration after sample collection following TKA. As the stromal component of the bone marrow can be used for the treatment of musculoskeletal diseases, centrifugation and concentration of the stromal component are inevitable.
The hemovac allows the collection of up to 400 mL of blood; the kit used in this study handled a volume of 40 mL. Theoretically, 800 mL from the first and second hemovac drains can be concentrated (8). Contrastingly, the contents of the third hemovac drain are dominated by venous blood; therefore, concentration is not effective. Notably, through the method used in this study, it is possible to produce 4 mL of HVBC and obtain 20-fold increased cell counts, allowing the culture of HVBC cells for clinical applications.
OA is a degenerative joint disease that presents with inflammation and, consequently, with elevated levels of proinflammatory cytokines (33,34). MSCs can modulate the inflammatory response; nonetheless, their application for the treatment of OA may be associated with a few problems (35,36). For instance, it has been reported that MSCs can differentiate into several proinflammatory cells, failing to decrease the inflammatory immune response (37). Therefore, the development of optimization strategies to regulate the immunomodulatory function of MSCs, such as the inhibition of STAT3 signaling, may be necessary before the clinical application of MSCs in the treatment of OA can be implemented.
A concern regarding this newly developed method is that the quality of stem cells from old individuals and patients with severe inflammation may be low (38)(39)(40). STAT3 is a transcription factor belonging to the STAT protein family (41,42) that is activated by inflammatory cytokines, including in diseases associated with chronic inflammation and fibrosis (42)(43)(44)(45)(46)(47). The restoration of stem cell function by STAT3 signaling inhibition has been previously evaluated (48)(49)(50)(51). For instance, STAT3 signaling inhibition in MSCs from the adipose tissue resulted in improved antiinflammatory response and reduced inflammation (50). Reduced OA severity in a rat model and pain relief have also been reported (50). However, these studies were based on cells from the adipose tissue or other sources; studies using HVBs have not been conducted to date. Notably, the stem cell characteristics or function could differ according to the source or environmental conditions of the harvested tissue (e.g., with respect to age and degree of inflammation) (20,37,52,53).
Importantly, to consider the use of STAT3 signalingsuppressed HVB cells for stem cell research, three key points must be addressed. First, stem cell marker expression should be maintained to ensure the lack of changes in the characteristics of stem cells after STAT3 signaling suppression. In this study, we showed that iSTAT3-MSCs maintained the expression levels of stem cell markers (high expression of CD73, CD90, and CD105) as well as the fibroblast-like morphology observed in the context of the "mother cells" (OA-MSCs) (Figures 2,3). Therefore, these results suggest the above pre-requisite can be fulfilled.
Second, iSTAT3-MSCs should maintain a multi-lineage differentiation potential. In this study, the osteogenic, adipogenic, and chondrogenic differentiation potentials of OA-MSCs and iSTAT3-MSCs were evaluated (Figure 4,5). Higher expression levels of RUNX2, ALP, osteocalcin, and osteopontin (osteogenic markers) were observed in OA-MSCs and iSTAT3-MSCs in the differentiated state than in the undifferentiated state ( Figure 6A). Additionally, the ALP activity ( Figure 4A) and Alizarin red S staining intensity (calcium deposition and mineralization; Figure 4B) of OA-MSCs and iSTAT3-MSCs were higher in differentiated than undifferentiated cells. Moreover, the expression of PPARγ and C/EBPα, adipocyte differentiation marker levels ( Figure 6B), and lipid droplet formation (as per Oil red O staining) were increased in differentiated OA-MSCs and iSTAT3-MSCs ( Figure 4C). The same was true in the context of the expression of different chondrogenic markers, including SOX9, aggrecan, and type II collagen ( Figure 6C) as well as Alcian blue (pH 2.5), toluidine blue, and type II collagen staining ( Figure 5). Altogether, these results support the notion that iSTAT3-MSCs show a multi-lineage differentiation potential. Interestingly, both osteogenic and chondrogenic potentials were higher in iSTAT3-MSCs than in OA-MSCs ( Figure 6A,6C). Therefore, our data support the future clinical applications of iSTAT3-MSCs. If iSTAT3-MSCs, generated after TKA surgery are stored in a freezer, they can be used to resolve bone disorders, including fractures, non-union fractures, and avascular necrosis (51,(54)(55)(56). Furthermore, since after TKA, patients mainly use the non-operated leg, which can lead to injury in the articular cartilage of the knee joint, iSTAT3-MSCs, with enhanced chondrogenic potential, can be used to treat potential cartilage defects of non-operated knees.
Third, similar to iSTAT3-MSCs from other sources, such as the adipose tissue (50) anti-inflammatory properties must be demonstrated. Here, iSTAT3-MSCs obtained from HVBCs showed lower pro-inflammatory and higher anti-inflammatory activity than OA-MSCs, as per the determined cytokine levels ( Figure 7). Therefore, these results indicate that STAT3 suppression is effective for the optimization of MSCs isolated in the context of inflammatory and degenerative conditions.
To the best of our knowledge, no studies on MSCs with STAT3 signaling suppression derived from HVB have been conducted to date. Overall, the results of the current study show the advantages of iSTAT3-MSCs over OA-MSCs with respect to osteogenic and chondrogenic differentiation ability as well as anti-inflammatory potential. Therefore, in conclusion, our results suggest that iSTAT3-MSCs obtained from HBV after TKA are promising for stem cell research purposes and the treatment of musculoskeletal diseases.