The incision length, operative time , intraoperative radiation exposure dose, changes in hemoglobin before and after surgery, postoperative hospital stay, visual analog scale (VAS) score for low back pain and leg, and Oswestry disability index (ODI) were statistically analyzed.We collected the clinical data of patients with L5/S1 lumbar disc herniation who had undergone endoscopic surgery at our center from January 2020 to July 2021, and 92 patients were included. They were divided into UBE/BESS (P < 0.01). VAS and ODI scores , the overall operative time, and postoperative hospital stay were not significantly different between the two groups (P > 0.05). No statistical difference in intraoperative radiation exposure dose was noted between the two groups (P > 0.05). The surgical incision length was greater in the UBE/BESS group (P < 0.01), and pre- and postoperative hemoglobin changes were more pronounced in the UBE/BESS group (P < 0.01). The UBE/BESS group had a longer extracanal operative time and shorter intracanal decompression time (P < 0.01).One case incurred dural tear in the UBE/BESS group, and one case developed recurrence in the IELD group. Postoperatively, the VAS score and ODI index decreased significantly in both groups (The clinical efficacy of UBE/BESS for L5/S1 lumbar disc herniation is comparable to that of IELD. Intraoperative radiation exposure doses were similar in both techniques. UBE/BESS required more time to identify tissue structures and a larger working space when operating outside the spinal canal; however, the efficiency of nucleus pulposus removal and nerve root release inside the spinal canal superseded that in IELD. Furthermore, the surgical incision in the UBE/BESS technique was longer, with greater actual blood loss during surgery, thus rendering UBE/BESS inferior to the IELD technique in terms of surgical trauma. Nonetheless, no significant difference was noted between the two techniques in the postoperative recovery time of patients. The exclusion criteria were as follows: (1) history of lumbar spine surgery at the L5/S1 segment; (2) lumbar spine infection, tumor, or trauma; (3) presence of lumbar instability and/or lumbar isthmic fracture; (4) concomitant severe psychiatric disorders; and (5) inability to tolerate general anesthesia.via the interlaminar approach were collected. The patients were recruited based on the inclusion and exclusion criteria, and they were all followed up for ≥12 months, mean 13.26 months. The preoperative clinical manifestations were low back pain, lower extremity radiating pain, and lower extremity motor and/or sensory dysfunction, and all patients had preoperative MRIs confirming the diagnosis of lumbar disc herniation (L5/S1).This was a retrospective cohort study wherein the data of 92 patients with L5/S1 lumbar disc herniation treated at our center from January 2020 to July 2021 using spinal endoscopic surgery UBE/BESS group: 23men, 19women, mean age was 45.57 ± 11.15 years (25–66 years), mean body mass index (BMI) was 24.53 ± 2.96. IELD group: 31men, 19women, mean age was 46.68 ± 12.09 years (22–67 years), mean BMI was 24.57 ± 3.71.The study was conducted in accordance with the principles of the Declaration of Helsinki, and patients or their families provided written informed consent for the procedure.All surgical procedures were performed by the same experienced surgeon. Patients in both groups were placed in the prone position and underwent surgery under general anesthesia, and no postoperative drains were utilized in either group.The inferior border of the affected L5 pedicle and superior border of the S1 pedicle were located using a C-arm and marked on the body surface. A longitudinal skin incision of approximately 5 mm was made at the proximal marker point, and an endoscopic puncture sheath was placed. A transverse skin incision of approximately 10 mm was made at the distal marker point, and a progressive dilator was placed. The puncture sheath and the dilator met together at the L5 spinous process and plate migration, and C-arm fluoroscopy was used to confirm the position. The dilator was subsequently removed and a periosteum detacher placed. After placement of the endoscope, the inferior margin of the L5 lamina was identified using radiofrequency hemostasis, followed by exposure of the interlaminar window. If necessary, the lamina was partially shaped using a grinding drill. The ligamentum flavum was incised at the medial edge of the articular eminence under endoscopic surveillance, and the canal was entered. After entering the spinal canal, the lateral margin of the S1 nerve root was revealed and the S1 nerve root can be retracted in the midline using a nerve puller to reveal the herniated disc. The herniated disc was removed, and the procedure was completed with adequate neurological decompression and hemostasis .Using a 6.9-mm endoscopic system, the center of the L5/S1 interlaminar window on the affected side was positioned under the C-arm and marked on the body surface. A longitudinal skin incision of approximately 7 mm was made at the marking point, a stepwise dilator and working tube were placed directly on the surface of the L5/S1 interlaminar window, and the position of the working tube was confirmed using C-arm fluoroscopy. After placement of the endoscope, the interlaminar window and ligamentum flavum were exposed. The superficial ligamentum flavum was removed using nucleus pulposus forceps. Subsequently, the deep ligamentum flavum was bluntly separated using a nerve stripper, and the ligamentum flavum was split using a working tube to facilitate entry into the spinal canal. The ligamentum flavum was removed to the medial edge of the facet joint using punches, and the dural sac and nerve root were exposed. Based on the location of the herniated disc, the disk fragment was removed in the axilla of the nerve root or shoulder .Postoperative analgesic treatment was routinely administered. If no dural tear occurred, the patient could walk after 4 h post surgery; however, if a dural tear occurred, no special treatment was administered to asymptomatic patients, and bed rest for 5–7 days was prescribed for symptomatic patients. The patient's lumbar spine MRI was reviewed before discharge from the hospital. The patient was advised to wear a lumbar brace for 1 month and avoid strenuous activities for 3 months after surgery. Routine blood tests were performed 3 days after surgery, and the hemoglobin (Hb) level was recorded.The operative time was recorded for all patients and categorized as follows: (1) overall operative time, (2) extracanal operative time , (3) intracanal decompression time , (4) intraoperative radiation exposure dose, (5) operative incision length , (6) operative related complications, (7) postoperative hospital stay, and (8) preoperative and postoperative day 3 Hb levels. The visual analog scale (VAS) scores of back/leg pain before and 3 days, 3 months, 6 months, and 12 months after surgery as well as the Oswestry disability index (ODI) before and 3 months, 6 months, and 12 months after surgery were recorded.t-test or independent-samples nonparametric test. Hb levels, as well as VAS and ODI scores at different time points, were compared within groups using the paired-samples t-test or paired-samples nonparametric test. The χ2 test was used to compare results between sexes among the patients in the two groups. Statistical significance was set at P < 0.05.SPSS  software was used for statistical analyses. Normally distributed measures are expressed as the mean ± standard deviation ( ± s). Patient age, operative time, incision length, Hb, and postoperative hospital stay were compared between groups using the independent-samples P > 0.05), as shown in Baseline information, such as age, sex, preoperative low back/leg VAS score, and ODI were not statistically significantly different between the two groups (P < 0.01). A significant difference in Hb level before and after surgery was noted in the UBE/BESS group compared with that in the IELD group (P < 0.01), suggesting that the actual blood loss in the UBE/BESS group was greater than that in the IELD group. No statistical difference in the total operative time was observed between the two groups (P > 0.05); however, the extracanal operative time was significantly longer in the UBE/BESS group than in the IELD group (P < 0.01), and the operative time for intracanal decompression was significantly shorter in the UBE/BESS group than in the IELD group (P < 0.01). The Hb level on postoperative day 3 was significantly different from the preoperative Hb level (P < 0.01) in both groups, as shown in Patients in both groups underwent surgery successfully. On comparing the two groups, the surgical incision length in the UBE/BESS group was significantly longer than that in the IELD group (P < 0.01). No statistically significant differences in VAS and ODI scores at 3 days, 3 months, 6 months, and 12 months after surgery were noted upon comparing the two groups (P > 0.05) (Postoperative VAS and ODI scores decreased significantly in the two groups compared with their preoperative scores ( > 0.05) .The distribution and comparison of the VAS scores for low back/leg pain in the UBE/BESS group at each postoperative time point are shown in One case incurred a dural tear in the UBE/BESS group. The patient had no postoperative symptoms and did not complain of discomfort upon wearing a lumbar brace to enable mobility on the second day after surgery. Moreover, no special treatment was administered. In the IELD group, one case developed recurrence 7 months after surgery, with symptoms similar to those before surgery, and the diagnosis was confirmed by MRI. The patient recovered well after the revised endoscopic surgery.via the interlaminar approach, achieving favorable clinical results. Since then, IELD has rapidly developed and emerged as a reliable technique for minimally invasive spine surgery. Because the operating channel of uniportal endoscopy is integrated with the endoscope, the surgical instruments involved are more slender than traditional surgical instruments, and the surgical procedure differs significantly from traditional surgery. Studies have increasingly shown that uniportal endoscopy is more difficult to master  In UBE/BESS, conventional arthroscopes and surgical instruments can be utilized to complete the surgical procedure without purchasing a special uniportal endoscopic system or supporting surgical instruments. (2) UBE/BESS involves two channels. The endoscope and operating instruments are in different channels, which can move independently and freely. This significantly increases the observation range of the endoscope and working area of the surgical instruments. (3) The surgical path and decompression process of UBE/BESS are similar to those of conventional microscopic lumbar discectomy, and studies have demonstrated that the learning curve of the UBE/BESS technique for lumbar disc herniation is 14 cases . (4) TheDue to the lack of a rigid cannula to dilate the soft tissue in the UBE/BESS technique, blunt dissection of the muscle is required to create a working space before decompression of the spinal canal. Therefore, theoretically, UBE/BESS should result in greater blood loss and worse postoperative back pain than IELD. Certain studies have attempted to address these issues. Hao  retrospevia the lateral foramen, which differed from the surgical path of the UBE/BESS technique; the control group was operated on under local anesthesia, whereas the trial group was operated on under general anesthesia; the trial group was operated on in the prone position in one study, whereas the control group was operated on in the lateral position; and the surgical segments in one of the studies were not similar in both groups. In the present study, we limited the surgical segment to the L5/S1 segment, and patients in both groups were operated on in the prone position under general anesthesia; moreover, both groups used a posterior trans-interlaminar approach to increase homogeneity, reduce trial bias, and improve the accuracy of the study.To the best of our knowledge, these are the only two studies to have compared the clinical efficacy of UBE/BESS with that of uniportal endoscopy in the treatment of lumbar disc herniation. However, the above two studies had certain shortcomings in terms of trial design. For example, patients in the control group were operated According to our data, both the UBE/BESS and IELD groups achieved favorable clinical results, and postoperative low back pain, leg pain, and ODI scores significantly improved. No significant differences were noted between the two groups, exhibiting consistency with the two studies mentioned above , 35. We In terms of surgical trauma, the difference in postoperative hospitalization time between the two was not significant. The surgical incision length in the UBE/BESS group was significantly longer than that in the IELD group, and the pre- and postoperative Hb change was significantly greater in the UBE/BESS group than in the IELD group, indicating that the actual blood loss in the UBE/BESS group was greater than that in the IELD group. Because both endoscopic surgical techniques require intraoperative saline irrigation, the intraoperative blood loss could not be accurately estimated. Furthermore, the postoperative “hidden” blood loss could not be estimated because no drainage tube was used after surgery. Therefore, in this study, we selected the method of dynamic Hb monitoring to evaluate the actual postoperative blood loss. Certain studies have shown that dynamic monitoring of hematocrit and Hb can effectively and accurately reflect blood loss in surgical patients. Both are potentially useful in calculating the actual blood loss after surgery , 37. To This study has certain limitations. First, it is a retrospective study with a short follow-up time and a small sample size. Second, only L5 and S1 segments were compared in this study. In addition, IELD was the exclusive control procedure in this study, whereas microscopic discectomy is also an effective, minimally invasive method for the treatment of lumbar disc herniation. Therefore, these minimally invasive surgical techniques should be discussed together in future studies.In conclusion, the UBE/BESS and IELD techniques are both safe and effective in the treatment of lumbar disc herniation. The introduction of dynamic Hb in this study revealed that IELD involves less actual blood loss and trauma for the patient than UBE/BESS, suggesting that UBE/BESS requires optimization in the future to further reduce trauma. The rigid cannula used in IELD potentially reduces the extracanal operative time. The surgical equipment used in UBE/BESS is more efficient in removing the nucleus pulposus and releasing the nerve roots.


However, the in vitro induction of both the angiogenesis and adipogenesis differentiations from stem cells yields limited maturation states for these two pathways. To overcome these issues, we report a novel method for obtaining a fully vascularized adipose tissue reconstruction using supporting bath bioprinting. For the first time, directly isolated mature adipocytes encapsulated in a bioink containing physiological collagen microfibers (CMF) were bioprinted in a gellan gum supporting bath. These multilayered bioprinted tissues retained high viability even after 7 days of culture. Moreover, the functionality was also confirmed by the maintenance of fatty acid uptake from mature adipocytes. Therefore, this method of constructing fully functional adipose tissue regeneration holds promise for future clinical applications.The development of soft tissue regeneration has recently gained importance due to safety concerns about artificial breast implants. Current autologous fat graft implantations can result in up to 90% of volume loss in long-term outcomes due to their limited revascularization. Adipose tissue has a highly vascularized structure which enables its proper homeostasis as well as its endocrine function. Mature adipocytes surrounded by a dense vascular network are the specific features required for efficient regeneration of the adipose tissue to perform host anastomosis after its implantation. Recently, bioprinting has been introduced as a promising solution to recreate  Soft tissue can be damaged by trauma, disease, high-grade burns, deep wounds, congenital deformities, or tumor resection. In such cases, large fatty tissue reconstruction should be readily available to provide an aesthetic structural and functional restoration, as well as for ameliorating patients' psychological distress. These large defective adipose tissues, in particular following mastectomy, were traditionally replaced by artificial prostheses because of their easy and fast implantation, with a relatively short recovery time. Unfortunately, they have a limited lifespan of 10–20 years forcing patients to undergo additional surgeries. In addition, 40% of repeat surgeries occur within 5 years due to postimplantation complications such as infection, malposition, implant rupture, or foreign body immune response [in situ, surgeons are still limited by the donor site availability and morbidity. Moreover, the surgical procedure is also costly and requires a high level of surgical skill, with failure still an occasional outcome (up to 2% rate) [Current approaches are therefore now focused on the use of autologous fat transplantation using the patient's own adipose tissue from other body sites. However, these procedures involve several surgical interventions and are associated with a high risk of complications and donor site morbidity. They lead to large-volume resorption due to graft contracture or necrosis, sometimes up to 90% in long-term studies –7. The m2% rate) . At thisin vivo-like structure with a natural degradation where shrinkage of the implant will be associated by its remodeling through the encapsulated autologous cells.It is here that 3D bioprinting appears as a promising solution, garnering immense interest over the past decade. Bioprinting enables the replacement of damaged tissues by facilitating the production of biosimilar constructs suitable for implantation, closing the gap between artificially engineered tissue constructs and native ones . 3D prinin vitro of mature adipocytes, whose identity and functions are comparable to native tissue [in vitro model of adipose tissue displaying fully mature functional adipocytes as in native adipose tissue is urgently needed.So far, few studies have been published to address the clinical need for a bioprinted restoration of functional adipose tissues. They generally used natural biomaterials rather than synthetic ones whose structure and composition are often associated with low biocompatibility or limited bioinductibility. Moreover, their additive content, such as unreacted monomers or catalysts, can induce immune body reactions, limiting their clinical uses. Of the natural bioprintable biomaterials, the best-performing ones directly use all the adipose extracellular matrix (ECM) components from the decellularization of native human adipose tissue in the bioink . Howevere tissue , 24. Thein vitro handling [For this purpose, using directly mature adipocytes has the benefit of improved efficiency and functionality with potentially less immunogenicity . As theyhandling . In a prhandling , known fThe next challenge is to overcome the current lack of a vascular network within the bioprinted adipose tissue which currently limits their transplantation as tissue transplant for patients. The aim is to get a final porous structure which allows cell infiltration and tissue ingrowth, while guaranteeing nutrient exchange by a promoted vascularization. Generally, oxygen, nutrients, metabolites, and catabolites have limited diffusion of less than a few millimeters through a scaffold . DespiteUsing our previous findings, the purpose of this study was thus to show for the first time the possibility of bioprinting viable and functional vascularized adipose tissues, including the fragile mature adipocytes, for a ready-to-use, high-benefit soft tissue regeneration. Overall, 3D bioprinters are available in three different forms: inkjet, extrusion-based, and laser-assisted , of varyin vivo-like way, with a high cell survival where mature adipocytes were able to maintain their functionality for at least 7 days in in vitro culture following the bioprinting.We firstly demonstrated the advantages of bioprinting using the gellan gum (GG) supporting bath for this purpose as compared to the classic patterning bioprinting on a surface. Then, higher size tissues showed the possibility of obtaining a fully vascularized structure throughout the bioprinted adipose tissue in an Clostridium histolyticum , and Triton X-100 (T8787) were purchased from Sigma-Aldrich . Fetal bovine serum (35010CV) was purchased from Corning . Penicillin, streptomycin, BODIPY™ 500/510 C1, C12 ), goat anti-mouse secondary antibody Alexa Fluor® 647, and Hoechst 33324 (H3570) were obtained from Thermo Fisher Scientific . The 4% paraformaldehyde  and mouse anti-human CD31 antibody (M0823) came from Wako Pure Chemical Industries . The human umbilical vein endothelial cell  and endothelial growth medium  were purchased from Lonza . The Nile Red compound was purchased from Tokyo Chemical Industry . Dulbecco's Modified Eagle's Medium (DMEM) came from Nacalai Tesque Inc. . Cell-Based Propidium Iodide Solution (10011234) came from Cayman Chemical .Porcine type I collagen was kindly donated from Nippon Ham . Gellan gum (GG) (Mw = 500 kDa) was obtained from Sansho . Fibrinogen , thrombin , bovine serum albumin , phosphate-buffered saline powder , collagenase from μm filter, microsyringe 25 mm filter holder, Merck, Darmstadt, Germany), before being freeze-dried for 48 h . The obtained CMF was kept in a desiccator at room temperature.Based on our previous studies –38, the 3 in size using autoclaved scissors and tweeters, directly in the collagenase solution at 2 mg/mL in DMEM 0% FBS, 5% BSA, and 1% antibodies (sterilized by filtration). After one hour of incubation at 37°C at 250 rpm, DMEM was added and the lysate was filtrated using a sterilized 500 μm iron mesh filter, before being centrifuged 3 minutes at 80g. The lysate was then washed two times in PBS with 5% BSA and 1% antibiotics and once in complete DMEM, by centrifugation between each wash. For the washing steps, the liquid fraction between the top layer (mature adipocytes) and the pellet (ADSC) was aspirated and discarded. Then, the pellet was resuspended in DMEM for ADSC expansion by changing the medium every two days and then by passaging the cells when they reach 80% of confluency. The mature adipocyte layer was moved to a new tube, and cells were counted in a 10 μL isolated volume, by staining the nuclei for 15 minutes with Hoechst in DMEM and using a Turker Burk hematocytometer on a fluorescence microscope.The 3 different patients' human liposuctioned adipose tissues were isolated at Kyoto Prefectural University of Medicine Hospital. After a PBS wash, 8-10 g of tissue were minced to get fragments of around 1 mm6 ADSC/mL and 1.25 × 106 HUVEC/mL, as already reported in our previous study [μm filter). Finally, the mature adipocytes were added at a final concentration of 3 × 106 cells/mL and the tissues were bioprinted.To construct the fat tissues, the CMF were first weighted and washed in DMEM without FBS before being centrifuged for 1 minute at 10,000 rpm to adjust the final concentration depending on the volume added to resuspend it . When needed, the ADSC and the HUVEC were added after trypsin detachment  and centrifuged for 1 minute at 3500 rpm  to get a final cell concentration of 2.5 × 10us study . The pelus study , then fiμm filter) just before printing. The solution was then transferred to a glass syringe  with standard metal needles . The syringe was set on a dispenser  using an adapter, and the dispensing system was kept at room temperature. The dispensing programs were designed on MuCADV  and completed debugging before dispensing. Two lines were printed on a 35 mm diameter glass-bottom dish, and the syringe and bed parts of the instrument were maintained at 4°C during the printing to defer the gelation of fibrinogen in the syringe. Then, a mixture containing the mature adipocytes, the fibrinogen, and the CMF in DMEM was prepared to bioprint an additional line between the 2 vascular lines. For the vascular and adipose lines, 20G and 15G needles were used to print the line structures, respectively. The printing parameters were at a dispensing speed of 1.28 mm/sec, with a moving speed at 10 mm/sec for the vascular lines and 2 mm/sec for the adipose line. The dish was then maintained at 37°C in the incubator to let the gelation occur for 15 minutes. Finally, the EGM-2 culture medium with 1 μg/mL of insulin was added and renewed every 2-3 days, up to 7 days.For all the bioprinting steps see , syringeμg/mL of insulin for the vascularized adipose tissues) with the renewal of the medium every 2-3 days.For the supporting bath bioprinting, first, gellan gum (GG) solution was produced by dissolving a 0.05-0.3 wt% gellan gum powder in PBS 1x solution and stirring at 85°C for 2 hours . The sol–1 and the time of each step. The shear rate was changed at 10 s–1 for 5, 10, 20, or 30 sec followed by a shear step at 0.01 s–1 for 10, 60, or 120 sec.Viscoelastic testing was performed using the oscillating rheometer RheoStress 6000  with a 0.052 mm gap using a 35 mm, 1° cone plate geometry (C35/1 TiL) at 20°C. Bioinks were prepared immediately prior to the measurement of the viscosity, as mentioned above, without or with cells  and with different CMF concentrations , but constant fibrinogen concentration at 6 mg/mL. They were loaded into the gap between the parallel upper and lower plates of the rheometer. These bioinks were also compared to the native fat tissue from which was extracted the mature adipocytes and the ADSC. GG supporting baths were prepared with various concentrations. The rheological behavior of GG solutions was studied using the same instrument as for bioink characterization. To determine the yield stress quantitatively, steady rate sweeps were conducted by varying the shear rate from 0.01 to 10 sμm polyester membrane, Costar 3470, Corning, Corning, NY, USA) at room temperature for measuring and compared to in vivo real human adipose tissues cut at the same scale. A spherical mold (5 mm in diameter) was used to measure the elastic modulus at a head moving speed of 1.0 mm/min. The compressive test protocol was employed increasing the engineering strain until the testing stress to 200 mN. The modulus is automatically calculated by the EZ test in the elastic range (1-2 mN).The elastic modulus of the cross-linked vascularized adipose tissues was measured with the EZ test . After mixing the adipose cells, the CMF (1.2%), the fibrinogen (6 mg/mL), and the thrombin (3 U/mL) in DMEM, the gelation was performed for 15 min. The gelated tissues were then put in a 24-well Transwell plate . After one PBS wash, the tissues were stained with calcein and ethidium homodimer-1 for 45 min at 37°C in the dark and then imaged using epifluorescence Confocal Quantitative Image Cytometer CQ1. Z-stack with the same steps and using the maximum intensity projection was performed keeping the same exposition time and excitation power for each sample. ImageJ software was used for the analysis of the projections, calculating the percentage of each staining.μL using the same mixture of CMF, fibrinogen, mature adipocytes, HUVEC, and ADSC were seeded in a 24-well Transwell plate  and cultured for 7 days before being fixed, rinsed 3 times in PBS, and sent to the Applied Medical Research company  for paraffin embedding and CD31 immunostaining. The images were captured using an FL EVOS Auto microscope .Tissues were fixed with 4% paraformaldehyde solution in PBS overnight at 4°C. They were then permeabilized in 0.05% Triton X-100 in PBS for 15 minutes and incubated for one hour at room temperature in 1% BSA in PBS. The first antibody mouse anti-human CD31 was added in BSA 1% and incubated overnight at 4°C. Finally, samples were incubated with the secondary antibody goat anti-mouse Alexa Fluor® 647 at room temperature in the dark for 2 hours and nuclei were counterstained with Hoechst as well as the Nile Red™ compound for intracellular lipid accumulation. The samples were rinsed in PBS and observed using epifluorescence microscopes . The phase contrast images were taken using Olympus IX71 . Images of the 3D reconstructed capillary and from inside one lumen were made using surface reconstruction on 3D volume images with the Imaris software . For histology staining, 3D tissues of 60 μM of the fluorescently labeled fatty acid analog  was then added to the culture medium for a duration of 60 minutes with 10 μg/mL of insulin to induce the fatty acid uptake, following Rogal et al.'s method [For the fatty acid uptake monitoring by the adipocytes, first, the tissues were starved for 6 hours in DMEM without glucose and FBS, containing only 1% of BSA. 4 s method , counterThe adipose tissues were collected from Kyoto Prefectural University of Medicine Hospital  from liposuction isolation of different female donors at the ages of 38, 71, and 72 and with BMI at 24.01, 22.6, and 26, respectively. All use was approved by the Human Ethics Committee  of Kyoto Prefectural University of Medicine Institutional Review Board and conformed to the principles outlined in the Declaration of Helsinki.Statistical analyses were performed using ezANOVA software  by Tukey's multiple comparison test (two-way ANOVA). When no marks are shown on the graphs, it means that the differences are not significant.μm length, included in a fibrin gel, also allowed the bioprinting of vascular structures [in vitro vascularized adipose tissue regeneration. First, similar line patterns, containing the cells required for the angiogenesis induction (ADSC and HUVEC) [In our previous studies, we emphasized the importance of collagen microfibers (CMF) to provide stable and functional human engineered tissues –38. Thesructures  from seld HUVEC) , were prd HUVEC) .Therefore, it was decided to proceed with the 3D bioprinting in suspension media, using a supporting bath, allowing the extrusion-based 3D printer device method to be used to its full potential. The bioprinting of tissue in a bath allows the possibility of printing non-self-supporting large-sized structures from low-viscosity bioinks into complex and well-defined structures, preventing their settlement and collapse . This suFor this purpose, the aim was first to use a suspension media which displays solid-like characteristics when no or low stress is applied and becomes liquid-like media when the bioprinting starts. Second, following the disturbance of the suspension media by the passing nozzle, the microstructure needs to spontaneously recover, permitting the transition from a fluidized state back to a solid-like state, thereby encapsulating the printed materials (cells and bioink). This property is called thixotropy. From this concept, we aimed to explore the potential of gellan gum as a support material for bath-enabled extrusion 3D printing of adipose tissues. Gellan is an extracellular microbial polysaccharide which can form tunable hydrogels, since its mechanical properties can be adjusted according to its concentration or by cross-linking multivalent cations, such as calcium ions . GranulaRheological measurements were performed to determine the most suitable GG concentration for the supporting bath during the bioprinting of 3D adipose tissues. Mature adipocytes are fairly deformable but require moderate shear stress to maintain their viability and functionality after bioprinting. As very few data are available concerning the possible acceptable range for the adipose cells, it was difficult to determine a suitable concentration for the GG bath solution.From a cytometry study of mature adipocytes, Majka et al.  showed tG′) and loss (G″) moduli of the GG were measured in a shear stress sweep test  and thus seemed more suitable for the embedded bioprinting of fragile mature adipocytes. The 0.2 and 0.3% concentrated GG thixotropic behavior required high shear stress that may damage the cells during printing or might even hinder a smooth printing process. Additional assays performed on these two concentrations highlighted the higher bioink diffusion through the GG bath at 0.1 wt% compared to 0.15 wt% , and the gel reassembly after strain release was instantaneous, recovering when the shear rate decreased (0.01 s−1). Moreover, this behavior did not change throughout the 2 cycles, and the GG bath was able to maintain its original viscosity after cycling. This property allowed us to print multilayered structures on the same path in the supporting bath. The 0.15 wt% GG bath was therefore deemed to be a suitable supporting bath for bioprinting adipose tissue.The rheological characteristics of this 0.15 wt% concentrated GG supporting bath were further evaluated, as it influences the printing performance . After tin vitro reconstruction in an optimized bioink.Rheological measurements were then taken on the bioink formulations to characterize their rheological properties, as the viscosity will also influence their printability. The viscosity of different bioinks was investigated, using CMF at different weight percentages  in the same fibrinogen concentration (6 mg/mL), regarding the shear rate. Initial viscosity values increased along with CMF concentration . All thein vitro tissues  being already reported in our previous study as not optimal for the vascular lumen formation with HUVEC cells, which is of importance for the implantation application to allow anastomosis occurrence   was added to the culture medium, with insulin to induce its uptake by stimulating the translocation of fatty acid transporter 1 from intracellular vesicles to the plasma membrane [in vitro culture. Propidium Iodide (PI) counterstaining again confirmed the low mortality rate inside the printed tissue. This further reinforces the fact that the angiogenesis induction in the vascularized bioprinted model did not alter the specific mature adipocyte function or affect the maintenance of their viability.Finally, not only the viability but also the maintenance of adipose tissue functionality after bioprinting are important. Therefore, the specific ability of mature adipocytes to take up fatty acids from the culture medium and to store them inside their unilocular lipid vesicles was monitored , as theymembrane . The fatin vitro adipose tissue regeneration. For the first time, we demonstrated the successful fabrication of an in vivo-like adipose tissue using directly the high-potential mature adipocytes, with a simple and cost-effective printing process. The obtained bioprinted tissues reproduced the desired structure of native adipose tissue, displaying mature adipocytes surrounded by a dense vascularized blood network, which should allow each cell to obtain efficient diffusion of nutrients and oxygen in the grafted tissue after host anastomosis thanks to their lumen structures. Thus, due to this vasculature network, in addition to the inner porous structure created by the assembly of the collagen microfibers [in vivo structure (seen in We conclude that our method using the CMF-based bioink to encapsulate mature adipocytes, ADSC, and HUVEC during their supporting bath bioprinting to generate multilayered constructs is a promising method for rofibers , this me seen in .The nondestructive printing method reported in this work maintains not only the viability but also the functionality of the encapsulated mature adipocytes. Adipose tissue is now widely recognized as an important organ for the whole body, as it participates in the regulation of a broad range of biological functions . Thus, tz-axis. To our knowledge, the current biggest size bioprinted tissue in the supporting bath is already in the centimeter scale [While fully customized tissues can be printed by this method, enabling the reconstruction of the personalized shapes for the patients, the final current limitation now relies on the printing working space availability. Usually, the bioprinters are limited in their vertical printing more than the horizontal space area. But recently, additional motion stages were developed, expanding the range of the motorized table for the movement in the er scale , so it ser scale , even ifTo further address the significance of this research in terms of medical matters, while this model allowed us to mimic human physiology in a customized shape depending on the patient's needs, we can wonder about the implantation method for these high-scale bioprinted tissues. Regarding their elasticity, they should be flexible enough to be inserted even in a small incision, in a similar way to the current artificial silicone breast implants in which Young's modulus elasticity is in a range of 14-1500 kPa, so even stiffer –63. The


P = 0.015) and long head of biceps (LHB) tendon rupture (P = 0.007) were associated with superior migration of humeral head. Superior subscapularis tear (P = 0.003) and LHB tendon rupture (P < 0.001) were associated with narrowing of glenohumeral joint. Female sex (P = 0.006) and superior subscapularis tear (P = 0.006) were associated with humeral head collapse. This study identified the rupture of infraspinatus and LHB as risk factors of superior migration of humeral head, and the rupture of subscapularis and LHB and female sex as risk factors of cuff tear arthropathy.As massive rotator cuff tears progress, various radiographic changes occur; however, the factors associated with radiographic changes remain largely unknown. This study aimed to determine the factors that affect radiographic severity in massive rotator cuff tears using multivariate analyses. We retrospectively reviewed 210 shoulders with chronic massive rotator cuff tears. The dependent variables were superior migration of the humeral head (Hamada grades 2–3), narrowing of the glenohumeral joint (grade 4), and humeral head collapse (grade 5). Baseline variables that were significant in univariate analyses were included in multivariate models. There were 91, 59, 43, and 17 shoulders classified as Hamada grades 1, 2–3, 4, and 5, respectively. Multivariate analysis showed that infraspinatus tear ( Hamada classification is widely used for radiographic classification of massive rotator cuff tears that reflect these changes5. This classification assigns massive rotator cuff tears to five radiographic grades: preserved acromiohumeral interval (grade 1); narrowing of acromiohumeral interval (grade 2); subacromial acetabulization in addition to grade 2 features (grade 3); narrowing of the glenohumeral joint in addition to grade 3 features (grade 4); and collapse of the humeral head (grade 5)  and teres minor (TM) tears than grade 1, which suggests that the status of SSC and TM contributes to the progression of massive rotator cuff tears. However, Walch et al.13 identified age at the time of surgery, delay of surgery, duration of follow-up, status of TM, and fatty infiltration of the infraspinatus (ISP) and SSC as factors that affected the progression of Hamada arthritis stage in patients who underwent only arthroscopic tenotomy of the long head of biceps (LHB) for rotator cuff tears. Although these recent advances have led to a better understanding of the pathogenesis of cuff tear arthropathy, multiple factors are considered to affect the progression to a higher grade in the Hamada classification system, and these factors have not been fully elucidated. This study aimed to use multivariate analyses to identify the predictive factors affecting radiographic severity in massive rotator cuff tears.However, the mechanism of radiographic progression of massive rotator cuff tears has not been fully elucidated, and few studiesThis study was approved by the Institutional Review Board of the Keio University School of Medicine (Reference study number: 20130147). All methods were conducted in accordance with relevant guidelines and regulations. Opt-out consent method was performed for each patient on our hospital’s bulletin board and web site. Opt-out consent relies on implicit consent, where willingness to participate is tacit or presumed and can be retracted by active objection.14, we defined massive rotator cuff tears as a complete tear of two or more tendons in the rotator cuff as diagnosed by magnetic resonance imaging (MRI). The inclusion criterion was a chronic massive rotator cuff tear of more than six months after the onset of symptoms. In addition, patients whose plain radiography and MRI were performed within three months from the evaluation of physical findings and medical history was obtained at the initial visit were included in the study. The exclusion criteria were patients with limited passive range of shoulder motion, septic shoulder arthritis, or previous shoulder surgery.The present study was a retrospective study and involved patients who presented with massive rotator cuff tears between April 2011 and April 2021. Based on previous reportsIn this study, we identified 210 shoulders that fulfilled the inclusion criteria. The mean age of the patients was 72.9 ± 8.3 years (range: 45–89 years). The mean time from symptom onset was 2.9 ± 3.7 years (range: 0.5–20 years). Eighty shoulders (38.1%) had a history of trauma.5 grade 1, 2, 3, 4, or 5 based on plain radiographic findings. One examiner evaluated the involved tendon using MRI, with the results of plain radiographic findings blinded. Plain radiographs were taken with the patient in a standing position, and MRI was performed in the supine position. In SSC tears, the upper 2/3 of the SSC attaches to the lesser tuberosity as a tendon, while the lower 1/3 attaches to the muscle16. Therefore, superior SSC and inferior SSC are considered to have different functions18; we evaluated SSC tears separately for superior and inferior SSC in this study. In addition, since the inferior SSC and TM attach to the humerus as muscles and not tendons19, it is difficult to determine the presence of tears; therefore, we defined inferior SSC tears or TM tears as fatty infiltration into the muscles as Goutallier classification20 grade 3 or higher17.We classified the patients who fulfilled the eligibility criterion as having Hamada classification21, we defined the active elevation of the shoulder that is limited to 90° as pseudoparalysis in this study. Two orthopedic surgeons with over 10 years of experience in shoulder surgery assessed the range of shoulder motion using a goniometer. In addition, we evaluated fatty infiltration into each rotator cuff muscle with T1 sagittal oblique MRI immediately lateral to the scapular spine’s attachment to the body of the scapula and defined the presense of fatty infiltration as Goutallier grade 3 or higher, because a previous report22 has shown a significant association between fatty infiltration into these muscles of Goutallier's grade 3 or higher and superior migration of humeral head.We investigated the risk factors of migration of the humeral head, narrowing of the glenohumeral joint, and humeral head collapse in patients with massive rotator cuff tears. The dependent variables were Hamada grades 2–3 for superior migration of the humeral head in shoulders without glenohumeral arthritis (Hamada grades 1–3), Hamada grade 4 for narrowing of the glenohumeral joint in shoulders without humeral head collapse (Hamada grades 1–4), and Hamada grade 5 for humeral head collapse. The Hamada classification is the radiographic grading of massive rotator cuff tear, but it remains unclear whether it progresses accordingly. However, it is unlikely to drop from a higher grade to a lower grade; therefore, we compared Hamada grade 1 versus grades 2–3, grades 1–3 versus grade 4, and grades 1–4 versus grade 5 in this study. Explanatory variables included age, sex, duration of symptoms, trauma, smoking, diabetes, hypertension, rheumatoid arthritis, pseudoparalysis, ISP tear, superior SSC tear, fatty infiltration into supraspinatus (SSP), ISP, superior SSC, inferior SSC, and TM, and LHB tendon rupture. Medical history was evaluated based on the clinical notes. Based on the previous definition of pseudoparalysisAll statistical analyses were conducted using the SPSS software . In univariate analysis, we used Student's t-tests to compare the average of continuous values, such as age and duration of symptoms. In contrast, chi-squared tests were used to compare the proportion of discrete variables, such as sex, history of trauma, smoking, medical history of diabetes, hypertension, rheumatoid arthritis, involved tendons, and LHB tendon rupture. Baseline variables, which were statistically significant in the univariate analysis, were included in the multivariable models. Multivariate analyses were performed using logistic regression analysis to identify the independent predictors of superior migration of the humeral head, narrowing of the glenohumeral joint, and humeral head collapse. The Cox and Snell R square and the Nagelkerke R square values were calculated to evaluate the variability explained by the regression model. Regression model fit was estimated using the Hosmer–Lemeshow goodness-of-fit test. Accuracy of this model was calculated as follows; (true positive) + (true negative)/(true positive) +  + (true negative) + . Furthermore, we developed a logistic regression equation for independent predictors derived from logistic regression analyses. We create the receiver operating characteristic (ROC) curve of the regression equation and calculated the area under the curve (AUC) to evaluate the equation’s predictive value. In this study, we compared Hamada grade 1 versus grades 2–3, grades 1–3 versus grade 4, and grades 1–4 versus grade 5 to clarify the association with each radiographic findings; however, repeating statistical tests in the same population may have created an ɑ error. Therefore, the statistical significance level was set at 0.017, divided by 3, rather than 0.05, based on the Bonferroni’s correction.Hamada grade 1 included 91 shoulders (43.3%), grade 2 included 26 (12.4%), grade 3 included 33 (15.7%), grade 4 included 43 (20.5%), and grade 5 included 17 (8.1%). SSP tears were observed in 210 shoulders: ISP tears in 160 (76.2%), superior SSC tears in 100 (47.6%), inferior SSC tears in 21 (10.0%), and TM tears in 17 (8.1%). Figure P = 0.005), fatty infiltration into SSP (P = 0.006), and LHB tendon rupture (P = 0.003).In the univariate analyses, superior migration of the humeral head was significantly associated with ISP tear (P = 0.015) and LHB tendon rupture . The Cox and Snell R square and the Nagelkerke R square values were 0.139 and 0.189, respectively. The Hosmer–Lemeshow goodness-of-fit test showed no significant difference from good model fit (P = 0.906). Accuracy of this model was 67.3%. The AUC of ROC curve was 0.715 (95% CI 0.633–0.798), implying that the equation may be used to classify 71.5% of superior migration of humeral head , and LHB tendon rupture (P < 0.001).In the univariate analyses, narrowing of the glenohumeral joint was significantly associated with superior SSC tear (P = 0.003) and LHB tendon rupture . The Cox and Snell R square and the Nagelkerke R square values were 0.155 and 0.237, respectively. The Hosmer–Lemeshow goodness-of-fit test showed no significant difference from the good model fit (P = 0.445). Accuracy of this model was 79.3%. The AUC of ROC curve was 0.767 (95% CI 0.688–0.847), implying that the equation may be used to classify 76.7% of narrowing of glenohumeral joint , pseudoparalysis (P < 0.001), superior SSC tear (P = 0.001), infiltration into superior SSC (P = 0.005), fatty infiltration into inferior SSC (P < 0.001), and fatty infiltration into TM (P = 0.002).In the univariate analyses, the collapse of the humeral head was significantly associated with female sex (P = 0.006), superior SSC tear , and fatty infiltration into inferior SSC tear . The Cox and Snell R square and the Nagelkerke R square values were 0.175 and 0.406, respectively. The Hosmer–Lemeshow goodness-of-fit test showed no significant difference from the good model fit (P = 0.962). Accuracy of this model was 91.9%. The AUC of ROC curve was 0.838 (95% CI 0.739–0.936), implying that the equation may be used to classify 83.8% of humeral head collapse (Table Multivariate analysis showed that risk factors of collapse of the humeral head were female sex (odds ratio 10.30, 95% CI 1.98–54.43; In this study, we examined the relationship between radiographic findings and ruptured tendons in a massive rotator cuff tear. Furthermore, we conducted multivariate analyses to identify the predictive factors that affect the presence of superior migration of the humeral head, narrowing of the glenohumeral joint, and collapse of the humeral head in massive rotator cuff tears. As a result, this study showed that as the radiographic grade progressed to Hamada grades 4–5, the ratio of SSC and TM tears increased, whereas the ratio of ISP tears was high in grades 2–3. In addition, we identified ISP tear and LHB tendon rupture as risk factors of superior migration of the humeral head, superior SSC tear and LHB tendon rupture as risk factors of narrowing of the glenohumeral joint, and female sex and superior and inferior SSC tears as risk factors of humeral head collapse.6. Specifically, since ISP plays a major role in regulating the humeral head inferiorly when the SSP is torn, superior migration of the humeral head reflects the presence of rotator cuff tears, especially multiple-tendon rotator cuff tears involving the ISP23. This study indicated that ISP tears, in addition to SSP tears, are a significant risk factor of superior migration of the humeral head; our results were consistent with those in the past reports23. In another clinical study, as fatty infiltration of SSP and ISP progressed, the ratio of patients with superior migration of the humeral head increased22, suggesting that the degeneration of the SSP and ISP is associated with the onset of superior migration of the humeral head. However, in this study, fatty infiltration of SSP was significantly associated with superior migration of the humeral head only in univariate analysis, but not in multivariate analysis, which suggests that the presence of ISP tendon tear may be more predictive for superior migration of the humeral head than the change in volume of fatty infiltration of SSP or ISP. In addition, this study suggested a significant association between LHB tendon rupture and superior migration of the humeral head; however, there has been some debate as to whether LHB tendon rupture is a cause or a consequence of superior migration of the humeral head26. LHB tendon acts as a humeral head depressor and shoulder stabilizer, and its rupture causes superior migration of the humeral head26. Hamada et al.5 reported that LHB tendon rupture was significantly more common in Hamada grades 3–5 cases than in cases of grade 1 or 2, which is consistent with the results of the present study. However, arthroscopic biceps tenotomy without rotator cuff repair was reported to have no mid- to long-term influence on progressive radiographic changes13, and LHB tendon rupture in patients with rotator cuff tears did not significantly reduce the acromiohumeral interval22. These studies22 raised the possibility that LHB tendon rupture was not the specific cause of superior migration of the humeral head, but the result of grade progression. Although this study was able to demonstrate an association between LHB tendon rupture and superior migration of the humeral head, the causal association remains unclear.Superior migration of the humeral head, which is a characteristic finding of massive rotator cuff tears, is assumed to occur by the breakdown of the force couples in the coronal plane, consisting of the superiorly directed force vector of the deltoid and the inferiorly directed force vector of the rotator cuff muscle4. The SSC is known to form the anterior portion of the transverse force couple, contributing to dynamic glenohumeral stability29. Specifically, the intra-articular component of the superior SSC tendon acts as the primary restraint to anterior glenohumeral translation in the glenohumeral motion from neutral to midrange30. Therefore, disruption of the SSC is assumed to cause muscular imbalance and subsequent glenohumeral microinstability32. In a recent clinical study, SSC repair failure was reported to significantly increase the risk of developing secondary glenohumeral osteoarthritis33, indicating that dysfunction of the SSC contributes to the progression of glenohumeral osteoarthritis. These findings support the view that the tear of the superior SSC caused glenohumeral anterior micro-instability, leading to the onset of cuff tear arthropathy. Furthermore, because biomechanical studies have shown that LHB also contributes to glenohumeral joint stability in all directions in addition to the stability in the coronal plane36, glenohumeral micro-instability associated with LHB tendon rupture might explain the association between LHB tendon rupture and glenohumeral osteoarthritis in this study.In the mechanism of osteoarthritis of the glenohumeral joint in patients with massive rotator cuff tears, imbalance in the strength of the internal and external rotators associated with massive rotator cuff tears is considered to cause transverse and axial instability in the glenohumeral joint, resulting in narrowing of the glenohumeral joint and anterior subluxation and medialization of the humeral head7 proposed mechanical and nutritional factors for the onset of humeral head collapse. Mechanical factors include anteroposterior instability of the glenohumeral joint caused by a rotator cuff tear7. Neer et al.7 reported that repetitive trauma from the altered biomechanics associated with glenohumeral joint stabilizers causes glenohumeral articular wear. Nutritional factors include leakage of synovial fluid, which reduces the perfusion of nutrients into the articular cartilage by a loss of closed joint space, and joint inactivity, leading to structural alteration in articular cartilage7. These factors cause histological atrophy of the articular cartilage and osteoporosis of the subchondral bone of the humeral head, ultimately leading to humeral head collapse7. In this study, superior SSC tear contributed to the onset of humeral head collapse. Inferior SSC status was also strongly associated with humeral head collapse, although not statistically significant. Inferior SSC has been reported to contribute to the stability of the glenohumeral joint for anterior translation by positioning anterosuperior to the humeral head in mid-range abduction37. In a cadaveric study18, the radius of the trajectory of the glenohumeral joint was significantly greater only in the model without traction force on SSP and superior and inferior SSC compared to the model with static loading on all rotator cuff muscles. Therefore, if dysfunction of inferior SSC occur in addition to superior SSC tears, further anteroposterior instability develops in the glenohumeral joint, thereby increasing the risk for humeral head collapse. In addition, the anterior humeral circumflex vessels, which provide the blood supply to the humeral head39, run through the dorsal margin of the SSC40. Thus, vessel rupture with SSC tear may be another cause of humeral head collapse. TM also increases the compression force across the glenohumeral joint in end-range motion and stabilizes the glenohumeral joint especially in the anterior direction41; however, fatty infiltration into TM was significantly associated with humeral head collapse only in univariate analysis and not in multivariate analysis. These results suggest that SSC has more influence on humeral head collapse than TM. The mechanism of association between female sex and humeral head collapse has not been clarified; however, the lower bone mineral density of the proximal humerus in women42 may contribute to the onset of subchondral collapse. Pseudoparalysis was significantly associated with humeral head collapse in univariate analysis, but not in multivariate analysis. This discrepancy may be explained by the possible confounding relationship between the status of inferior SSC and pseudoparalysis, as these factors have been shown to be associated43.Humeral head collapse is considered an end-stage change in plain radiographs associated with massive rotator cuff tears. Neer et al.26. Further longitudinal observational studies will be needed to clarify the progression of the radiographic changes in cases with massive rotator cuff tears.This study has several major limitations. First, explanatory variables in this study do not fully explain the objective variable and there are factors not measured in this study that may be residual confounding, because R square values in the logistic regression analysis of this study are small. For example, patients’ activity, deltoid function, and strength of each patient’s bone might affect the severity of radiographic findings. Second, patients more often presented to our institution for the purpose of surgery because the institutions participating in this study were general hospitals where surgery could be performed. Few patients with asymptomatic massive rotator cuff tears were included in this study. This may have caused a selection bias in this study. Third, ruptures of inferior subscapularis and teres minor in this study were determined from fatty infiltration into these muscles on MRI, which may not accurately reflect rupture. Fourth, although internal validity of logistic regression models in this study may be relatively high, these results may not be as applicable externally. Further studies will be needed to examine a test set of additional massive rotator cuff tears. Finally, the present study was a cross-sectional study of patients with massive rotator cuff tears. The duration of symptoms was not the same among the groups. Although the duration of symptoms had no impact on the radiographic changes of the shoulder, their onset periods did not always correspond with the occurrence of rotator cuff tears; thus, the duration could possibly be inaccurate. Additionally, the causal association between the radiographic findings and each of the factors identified as being associated with radiographic change in this study remains unclear, due to the nature of a cross-sectional study. For example, this study suggested an association between LHB tendon rupture and superior migration of the humeral head, but whether this is a cause or a consequence of superior migration of the humeral head remains unsolved, as has been discussed in previous literaturesIn conclusion, this study indicated rupture of the infraspinatus and biceps long head tendon as risk factors of superior migration of the humeral head and that rupture of the subscapularis and biceps long head tendon and female sex are risk factors of cuff tear arthropathy.


All above results suggest that differences of rhizospheric and endophytic bacteria are exactly recruited as “workers” by different watermelon phenotypes relating to rind color formations.To elucidate the biological mechanism of yellow rind formation on watermelon, the characteristics of soil bacterial community structure in rhizosphere and endophytic bacteria in stem of yellow rind watermelon were analyzed. Based on high-throughput sequencing technology, plant stem and rhizosphere soil samples, which collected from yellow and green rind watermelons were used in this paper, respectively. The structural characteristics of the endophytic bacteria in stems and soil bacterial communities in rhizospheres of yellow and green rind watermelons were comparative studied. Firstly, significant different proportions of some dominant bacteria and abundances could be detected between yellow and rind watermelons. Meanwhile, although different abundances of endophytic bacteria could be found, but no significant differences were observed between yellow and green rind watermelons. Moreover, Gemmatimonadota, Myxococcota, WPS-2,  Citrullus lanatus (Thunb.) Matsum. and Nakai var. lanatus (2n = 2x = 22)] is a Cucurbitaceae family horticulture crop that is also one of the top ten most eaten fresh fruits on the planet1. Watermelons have become one of the model crops for fruit-quality study due to their wide range of characteristics, including form, size, rind thickness and color, flesh texture and color, and sugar and carotenoids content2.Watermelon [5. The rind color of watermelon is an important commodity in modern society for its gorgeous appearance is significance improving the quality, which is considered as an important trait for influencing consumer preference and focusing of breeders7.Watermelon exhibits a wide range of rind colors: dark or light green, light green-gray, and yellow are the common colors1. Chlorophyll plays a crucial role in photosynthesis, as everyone knows8. Carotenoids are 40-carbon isoprenoids that serve important functions in photosynthesis and photoprotection in photosynthetic organisms, as well as providing evolutionarily adaptive colorations in plants, fungi, and animals10. Various taxa, including bacteria, archaea, fungus, algae, land plants, and mammals, have been shown to contain over 750 structurally distinct carotenoids11.The key pigments that impact watermelon rind colors are chlorophyll and carotenoids, which play crucial roles in gathering light energy and converting it to chemical energy14, such as the mitigation of environmental stresses, drought and cold or heat damages, etc.17. Endophytic bacteria are distributed in all internal parts of the plant and have functions in nitrogen fixation19, phosphorus solubilization21, production of plant growth regulating substances23, enhancement of host resistance25 and bioremediation26, which play an important role in regulating the microecological balance for enhancing resistance and promoting the healthy growth of the host plant27.At present, studies have also confirmed that soil microbes in rhizospheres of crops contribute to sustainable crop production28, cytokinin29, abscisic acid30, gibberellin31, and ethylene32. Endophytic bacteria also could increase the chlorophyll content in tomato33 and P. edulis34.Studies also have found that endophytic bacteria are closely associated with the production of plant endogenous hormones, such as growth auxin35. However, the genes how to regulate forming yellow or green rind and their molecular mechanisms are still unknown in watermelon. And whether the rhizospheric and endophytic bacteria contributes to rind color formation or not are also still unclear. In this study, to explore the compositions and functions of the rhizospheric and endophytic bacteria whether relates to rind color formation of watermelon differences of rhizospheric and endophytic bacterial compositions between yellow and green rinds watermelon were comparative analyzed.Recently, watermelon with yellow rind has gained increasing popularity among consumersTo evaluate the extent of the similarity of the rhizospheric and endophytic bacterial communities, unweighted UniFrac Principal Component Analysis (PCA) at OTU level was performed. The results suggested that soil bacterial compositions in rhizosphere of yellow and green rinds of watermelons were quite significant differences with the CK Fig. a, but it36 , Actinobacteriota (45.07%), Bacteroidota (2.36%) and others (1.54%), respectively. By contrast, Proteobacteria (47.61%), Actinobacteriota (36.70%), Bacteroidota (13.88%) and others (1.82%) were also the dominant endophytic bacteria in stems of green rind watermelons. That is, the proportions of dominant endophytic bacterial phyla in stems between yellow and green rinds watermelon were also different.p < 0.05, p < 0.01).The Wilcoxon rank-sum test was used to analyze the significant differences at phylum level for the top 15 soil bacteria in terms of relative abundance percentage. As seen in Fig. Cupriavidus, Nocardioides, Ensifer and Pseudomonas were the unique soil dominant bacterial genera in rhizosphere of yellow rind watermelon. On the contrary, norank_f__JG30-KF-CM45, Bacillus, Devosia, Rhodanobacter and Luteimonas were the special soil dominant bacterial genera in rhizosphere of green rind watermelon .The Wilcoxon rank-sum test was used to analyze the significant differences at genus level for the top 15 soil bacteria in terms of relative abundance percentage. As seen in Fig.  CK Fig. a. norank CK Fig. b. norankons Fig. c .However, there was no significant difference of endophytic bacteria at genus level between yellow and green rind watermelons Fig.   algorithm was employed to predict bacterial functions in the three groups. Among them, only the pathway which related to organismal systems of soil bacteria in rhizosphere of yellow rind watermelon could be found significantly higher than those of green rind watermelon Fig. a. In add5. The inheritance of the watermelon rind color is a qualitative trait, but the genetic pattern and developmental mechanisms are complex37. In general, the color of leaves and fruit rinds are mainly depended on the contents and the formation of pigments38. As is well-known, chlorophyll and carotenoid are the main pigments which affecting watermelon rind color1. Among them, chlorophyll content is the primary factor for the green rind in Cucurbitaceae, the dark green rind watermelon contains a much higher level of chlorophyll than that of the light green rind watermelon40. It is well documented that chlorophyll degradation is positively correlated with ethylene signaling41. A recent study by Han et al. found that the key gene responsible for apple peel greening is the ethylene-inducible factor ERF17. Furthermore, the number of serine repeats in ERF17 affects the binding activity of ERF17 to the promoter sequences of chlorophyll degradation-related genes, and an increase in the number of serine repeats enhances the activity of chlorophyll degradation-related enzymes, and the expression of ERF17 was positively correlated with PPH (pheophytin pheophorbide hydro-lase)42.As is an important horticultural crop all over the world, watermelon rind colors are mainly shown at dark green or light green, light green-gray or yellow44. Li et al. found that exogenous auxin delayed tomato color change mainly by inhibiting the expression of major genes in the β-carotene metabolic pathway45. Wisutiamonkul et al. also found that the ethylene inhibitor 1-MCP (1-methylcyclopropene) inhibited the production of carotenoids and delayed fruit color change by suppressing the expression of genes such as ZDS (zeta-carotene desaturase), LCY-B (lycopene beta-cyclase) and BCH (beta-carotene hydroxylase) through the inhibition of endogenous ethylene production46. Liu et al. had found a dramatic reduction in chlorophyll a and chlorophyll b and an increase in carotenoids in yellow rind watermelon1. Moreover, Zhu et al. also found that ethylene accelerated carotenoid accumulation by up-regulating the expression of carotenoid biosynthetic genes47. In this paper, we found that norank_f_Gemmatimonadaceae and Bradyrhizobium were significantly different in rhizospheres between yellow and green rind watermelons. As Bradyrhizobium, was reported for producing indole-3-acetic acid (IAA)48. Boiero et al. also confirmed that Bradyrhizobium could increase the content of plant endogenous ethylene49. It indicated that the enrichment of Bradyrhizobium in rhizosphere of yellow rind watermelon could induce higher contents of carotenoid formation. Meanwhile, we also found that Cupriavidus, Nocardioides, Ensifer and Pseudomonas were the unique soil dominant bacteria in rhizosphere of yellow rind watermelons. Among them, Pseudomonas can produce salicylic acid (SA) as a siderophore50. Previous study also confirmed that the application salicylic acid (SA), total carotenoid content, size of xanthophyll pool, and de-epoxidation rate increased significantly with an increase in SA concentration in both plant species51. Moreover, Pseudokineococcus, Stenotrophomonas, Marmoricola, Ralstonia and Pantoea were the special dominant endophytic bacteria in stem of yellow rind watermelons. Flavobacterium and Falsirhodobacter were the unique dominant endophytic bacteria in stem of green rind watermelons. However, their functions relate to rind color formation between yellow and green rind watermelons are still unknown, further researches need to be conducted.In addition, carotenoids are synthesized via the isoprenoid pathway, a very large secondary metabolic pathway that synthesizes chlorophyll, gibberellin, abscisic acid, and cytokinins in addition to carotenoids52. Even though many soil bacterial functions are similar in rhizosphere between yellow and green rind watermelons, such as Cellular processes, Environmental information processing, Genetic information processing, human diseases, Metabolism and Unclassified. However, the organismal systems of soil bacteria in rhizosphere of yellow rind watermelons were significantly higher than those of green rind watermelons. It indicated that soil bacteria in rhizospheres of yellow and green rind watermelons were carried out different functions. In addition, similarity, some PICRUSt functions of endophytic bacteria were also quite similar between yellow and green rind watermelons, such as Cellular processes, Environmental information processing, Genetic information processing, Human diseases, Organismal systems. However, the metabolism and unclassified functions of endophytic bacteria in stems of yellow rind watermelons were significantly higher than those of green rind watermelon. It also suggested that different functions of the endophytic bacteria enriched in different phenotype watermelons (yellow and green rind watermelons). From the discussion above, it may conclude that differences of rhizospheric and endophytic bacteria are exactly recruited in different phenotype watermelons. Moreover, different functions of soil bacteria in rhizosphere and endophytic bacteria in stems of watermelons, also can be speculated relating to different rind colors formation.PICRUSt function prediction analysis method has been widely applied in the study of bacterial functions in many plantsnorank_f_Gemmatimonadaceae and Bradyrhizobium enriched in rhizosphere of green rind watermelon; by contrast, Cupriavidus, Nocardioides, Ensifer and Pseudomonas enriched in rhizosphere of yellow rind watermelon. In addition, in comparison to the green rind watermelons, Pseudokineococcus, Stenotrophomonas, Marmoricola, Ralstonia and Pantoea were the special dominant endophytic bacterial genera in stems of yellow rind watermelons. Meanwhile, the organismal systems of soil bacteria in rhizosphere and the metabolism and unclassified functions of endophytic bacteria in stems of yellow rind watermelons were all significantly higher than those of green rind watermelons. All above results suggested that differences of rhizospheric and endophytic bacteria are exactly recruited as “workers” by different watermelon phenotypes relating to rind color formations.In conclusion, a field experiment was carried out to elucidate the compositions of soil bacteria in rhizosphere and endophytic bacteria in stems of yellow and green rind watermelons. The results revealed that Gemmatimonadota, Myxococcota, WPS-2, −1, 0.74 g kg−1, 17.0 mg kg−1 and 78.0 mg kg−1, respectively.The experiment was conducted in the experimental base of Suxu town , Nanning city, Guangxi Zhuang Autonomous Region, southwest of China. The physical and chemical properties of the soil in field of the experimental base are as follows: pH 4.45, the contents of organic matter, total nitrogen, available phosphorus and potassium contents were 9.96 g kgThe yellow rind watermelon varieties, Gui Jin Bao (JB), Gui Jin Guan (JG) and the green rind watermelon varieties Gui Ya (GY), Gui Hong Yu (HY), which all provided by Horticultural Research Institute, Guangxi Academy of Agricultural Sciences Fig.  were use53. Briefly, a circle with a radius of about 25 cm was shoveled loose with a sterilized shovel at the center of the plant, the whole watermelon plant was pulled out by hand holding the base of stem. The soil attached to the roots was forcefully shaken off and carefully collected as the rhizosphere soil samples. And the three replicated soil samples from no watermelon growing area were also collected randomly as background (CK). The soil samples were passed through a 2-mm sieve and stored at − 80 ℃ for DNA extraction. Meanwhile, stem samples were collected separately from the plant using sterilized scissors and placed in sealed sterile bags and labeled. i.e., soil and appendages were rinsed from the surface of the samples with sterile water and blotted dry with sterile filter paper. The stems were placed in sterile bags and stored at − 80 ℃ for endophytic bacterial compositions analysis.Soil and plant samples were collected at May 20, 2021 Rhizosphere soils from every watermelon variety were collected randomly according to the shake method described by Riley and BarberThe extraction, PCR amplification and sequencing of the total DNA from stem and soil samples were all completed by Majorbio Bio-Pharm Technology Co. Ltd. . The specific sequencing types and primer sequences were shown in Table ® DNA Kit  according to the manufacturer’s instructions. The DNA extract was checked on a 1% agarose gel, and the DNA concentrations and purity were determined with a NanoDrop 2000 UV–Vis spectrophotometer . PCR amplification and sequencing of the total DNA extracted from the plant stem samples were performed by Shanghai Majorbio Bio-pharm Technology Co., Ltd, the hypervariable region V3–V4 of the bacterial 16S rRNA gene were amplified with primer pairs 338F and 806R, the primers 799F and 1192R were selected for the first round of PCR amplification of the V5–V7 variable region, and the primers 799F and 1193R were selected for the second round of PCR amplification of the V5-V7 variable region. PCR amplification was performed on an ABI GeneAmp® 9700 PCR thermocycler , and the PCR products were recovered using 2% agar-gel electrophoresis. The products were purified by using an AxyPrep DNA Gel Extraction Kit  and quantified using a Quantus Fluorometer 55. The purified amplicons were pooled in equimolar quantities and were paired-end sequenced (2 × 300) on the Illumina MiSeq platform  according to the standard protocols of the Majorbio Bio-Pharm Technology Co. Ltd. . Raw reads were deposited in the NCBI Sequence Read Archive (SRA) database. (Accession Number: SRP341161).Microbial community genomic DNA was extracted from samples using the E.Z.N.A.56.The raw 16S rRNA gene sequencing reads were demultiplexed, quality-filtered by Trimmomatic and merged by FLASH with the following criteria: (1) the 300 bp reads were truncated at any site receiving an average quality score of < 20 over a 50 bp sliding window, and the truncated reads shorter than 50 bp were discarded, reads containing ambiguous characters were also discarded; (2) only overlapping sequences longer than 10 bp were assembled according to their overlapped sequence. The maximum mismatch ratio of overlap region is 0.2. Reads that could not be assembled were discarded; (3) Samples were distinguished according to the barcode and primers, and the sequence direction was adjusted, exact barcode matching, 2 nucleotide mismatch in primer matchinghttp://drive5.com/uparse/), and chimeric sequences were identified and removed. The taxonomy of each OTU representative sequence was analyzed by RDP Classifier (http://rdp.cme.msu.edu/) against the 16S rRNA database using confidence threshold of 0.757.Operational taxonomic units (OTUs) with 97% similarity cutoff were clustered using UPARSE  was performed using unweighted UniFrac distance metrics. Meanwhile, to evaluate the bacterial composition between samples, Partial Least Squares Aiscriminant Analysis (PLS-DA) and Analysis of Similarities (ANOSIM) were also used. In addition, PICRUSt was used to estimate the functional components of bacterial communities using the Kyoto Encyclopedia of Genes and Genomes (KEGG) datasetwww.majorbio.com) of the Majorbio Bio-Pharm Technology Co. Ltd. .The experimental data were analyzed using Excel 2019 and IBM SPSS Statistics 21. And the results are shown as means with their standard deviations (mean ± SD). Online data analysis was conducted by using the free online platform of the Majorbio Cloud Platform (


High physical work demands are believed to be partly responsible for the high sickness absence among home care workers, but no studies have assessed their physical work demands using precise device-based measurements. Hence, the objective of this observational study was to assess physical work demands in home care, using wearable sensors.From six home care units in a large municipality in Norway, 114 of 195 eligible home care workers filled in a questionnaire, a diary about work hours, and wore five accelerometers, and a heart rate sensor for up to six consecutive workdays.On average, the homecare workers spent 50% of the working hours sitting, 25.2% standing, 11.4% moving, 8.3% walking fast, 1.9% walking slow, 1.2% stair-climbing, 0.3% cycling, and 0.05% running. We found the following exposures to demanding postures: arm-elevation in an upright body position ≥30° was 36.7%, ≥60° was 4.1%, and ≥90°was 0.5%; forward trunk inclination in an upright body position ≥30° was 9.9%, ≥60° was 4%, and ≥90° was 1%; and for kneeling it was 0.8%. We found the average cardiovascular load (%heart rate reserve) during work to be 28%. There was considerable individual variation in these physical exposures at work.This study presents precise information on various physical work demands of home care workers in Norway. Home care workers spent on average half the workday sitting and the remaining time in various occupational physical activities. Presently, few device-based exposure limits have been proposed for acceptable amounts of occupational physical exposures, but the level of arm-elevation, forward trunk inclination, and the considerable variation of physical workloads among home care workers, indicate that preventive measures should be taken. This observational study presents detailed information on objectively measured physical work demands of home care workers in Norway. The findings extend our understanding of occupational ergonomic risk factors and can contribute to improved occupational health and safety measures and interventions in home care.Home care workers assist individuals that need help with activities of daily living due to a disability, impaired health, or disease. This requires the home care worker to perform various work tasks in caregiving to patients that can be physically demanding, such as prolonged standing and walking, lifting, carrying, pushing, and pulling. These work tasks can also often involve challenging work postures, such as bending forward, elevated arms, and kneeling .Due to the increasing number of older adults in need of care and the lack of qualified personnel, it is of increasing importance that home care workers have working conditions that are not detrimental to health. To identify how it is feasible to improve the physical working conditions, it is necessary to first generate precise knowledge of the current physical work demands in this vulnerable occupational group.Self-reported methods, and visual- and video-based observations have been the primary tools for assessing physical work demands, but these methods are either highly inaccurate, less cost-effective, or time consuming  were recruited from six of a total of 13 home care service units in Trondheim, the third largest city in Norway. Only workers that had direct contact with patients were included. All workers in these home care units were provided written and oral information about the research project and gave written consent before the study. Exclusion criteria were: (1) physical disability not allowing normal behavior, (2) office work, (3) bandage band aid and adhesives allergy, and (4) pregnancy. The study was conducted according to the Declaration of Helsinki and approved by the Regional Committees for Medical Research Ethics—Central Norway (No.: 64541).Baseline measurements of height and weight were collected using a standardized digital body weight scale and a wall-mounted SECA 206 measuring tape .Five triaxial AX3 accelerometers  were mounted on the skin of the home care workers, using adhesive double-sided tape  and secured with waterproof medical tape . They were worn 24 h per day for up to six consecutive workdays at a sampling frequency of 25 Hz and a range of ±8 G. The accelerometers were attached to the following anatomical locations: (1) below the head of the fibula, on the proximal and lateral aspect of the calf, (2) on the distal, anterior and medial aspect of the femur (approximately 10 cm above the crest of the patella), (3) below the iliac crest of the hip, (4) the upper back approximately 5 cm to the side of the processus spinosus at the level of Th1–Th2 vertebrae, and (5) on the upper arm, approximately at the insertion of the deltoid muscle   and was calculated using the following equation. The data was manually downloaded and processed using a modified version of the custom-made MATLAB software   was used to derive working hours from the dataset. A minimum of two working days was considered valid, and a cutoff of ≥4 h was set for all workdays for each worker to be eligible for inclusion in further analysis , and percentages. The statistical processing of the data was conducted in Python using the code editor Spyder for data analysis. Furthermore, the statistical libraries Pandas version 1.2.4, Seaborn version 0.11.1. NumPy, Matplotlib.pyplot, and the OS module were imported, allowing scientific computing.For an average of 3.8 workdays, 2913 h of accelerometer data and 2826 h of heart rate data were recorded in 114 home care workers. The flow of participants can be seen in The home care workers consisted of nurses, nursing assistants, learning disability nurses, and occupational therapists, having home care as their main employer, and worked an average of 38.5 h a week. According to the self-reported measurements, 46 (40.4%) of the home care workers reported that they persistently felt fatigued.Meantime in percent and minutes spent in physical exposures and demanding postures at work, standard deviations, mean %HRR and the relation between HRR and physical exposures at work are depicted in Home care workers had an average aerobic workday load of 28% HRR, spent half the workday sitting, and the other half in the following activities: 25% standing, 11% moving, 8% walking fast, 2% walking slow and very little in stair-climbing, cycling, and running. Regarding demanding postures: arm-elevation in an upright body position ≥30° occurred 31% of the workday, ≥60° was 4%, and ≥90° was 0.5%; forward trunk inclination in an upright body position ≥30° was 10%, ≥60° was 4% and ≥90° was 1%; and for kneeling it was 0.8%. Furthermore, our results indicate an uneven distribution of physical exposures among home care workers.Most studies of physical work demands have used self-reported assessment methods, known for being imprecise and prone to bias  than childcare (≥60° of 5% and ≥90° of 1.1%) (On an average home care workday, about half was conducted sitting and the other half in occupational physical activity, whereof one-fourth was spent standing and one-fourth doing various movements. Recent research indicates that physical exposures and their impact on health is domain specific due to different characteristics (The high amount of arm elevation ≥30° we observed in home care workers, has been associated with a more than two-fold risk of sickness absence in a recent study (The considerable variation in work demands depicted in In line with the ‘Goldilocks Work principle’ (Some limitations should be acknowledged in this study: First, 58% of eligible home care workers completed the study. Thus, we cannot exclude the possibility of selection bias, as it is plausible that workers not willing to participate had poorer health and different demographics. Second, the participants were only recruited from Trondheim municipality, meaning that our results are not necessarily generalizable to the rest of Norway. However, the organization of home care services is rather homogenous. Third, currently available technical measurement systems feasible for long-term workplace investigations are not capable of detecting demands related to muscle torque and trunk rotation. Fourth, accelerometers were only mounted on one side of the body which could lead to cases of demanding postures not being classified but adding several accelerometers would be quite inconvenient for participants.This observational study presents detailed information on device-based measured physical work demands of home care workers in Norway. Home care workers spent on average half the workday sitting and the remaining time in various occupational physical activities and demanding postures. Currently, few device-based exposure limits have been proposed for acceptable amounts of occupational physical exposures, but the level of arm-elevation, forward trunk inclination, and the considerable variation of physical workloads among home care workers, indicate that preventive measures are required. These findings extend our understanding of occupational ergonomic risk factors and can contribute to improved and more tailored occupational health and safety measures in home care, ultimately reducing long-term sickness absence and early retirement, allowing home care workers to stay productive longer through the design of a safer and better workplace. The results can also inform targeted interventions aiming to reduce awkward work positions or evening out workloads between home care workers.