Abstract
Bioprinting that can synchronously deposit cells and biomaterials has lent fresh impetus to the field of tissue regeneration. However, the unavoidable occurrence of cell damage during fabrication process and intrinsically poor mechanical stability of bioprinted cell-laden scaffolds severely restrict their utilization. As such, on basis of heart-inspired hollow hydrogel-based scaffolds (HHSs), a mechanical-assisted post-bioprinting strategy is proposed to load cells into HHSs in a rapid, uniform, precise and friendly manner. HHSs show mechanical responsiveness to load cells within 4 s, a 13-fold increase in cell number, and partitioned loading of two types of cells compared with those under static conditions. As a proof of concept, HHSs with the loading cells show an enhanced regenerative capability in repair of the critical-sized segmental and osteoporotic bone defects in vivo. We expect that this post-bioprinting strategy can provide a universal, efficient, and promising way to promote cell-based regenerative therapy.
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Introduction
Reconstruction of challenging bone defects that cannot spontaneously heal, e.g., large-sized segmental and osteoporotic bone defects1,2, remains a thorny problem in clinic. The difficulty is mainly attributed to the limited migration or weak regenerative potential of resident cells3,4. Notably, multiple cell types such as mesenchymal stem cells (MSCs) and endothelial cells (ECs), are involved in specific regenerative biofunctions e.g., osteogenesis and angiogenesis, playing vital roles in bone regeneration5,6,7,8. Regarding large-sized segmental bone defects, severely damaged surrounding tissues cause a lack of key cells to migrate and reside in the defect site, leading to no bridging and nonunion9. Osteoporosis is a typical metabolic bone disease, in which cells are senescent with weak regenerative potential. Once osteoporotic bone defects occur, it is hard for the senescent cells to support self-healing of bone tissues10,11. Whereas direct injection of related cells into the defect site results in suboptimal therapeutic outcomes12, tissue-engineered bone substitutes consisted of biomaterial-based scaffolds and healthy cells bring a promising avenue for repair of challenging bone defects1,13.
Despite tremendous progress in bone tissue engineering in the past decades, there are still limitations to be concerned. For instance, cells are conventionally seeded on the surface of scaffolds and then cultured in vitro until they migrate entirely inside scaffolds. Obviously, it is extremely time-consuming, and difficult to control cell distribution14,15. The emergence of three-dimensional (3D) bioprinting technology that can directly customize cell-laden constructs via mixing cells and biomaterials as bioinks provides new possibilities16,17. Due to its cost-effectiveness and ease of operation, extrusion bioprinting has become the most popular modality for fabrication of cell-laden constructs, followed by inkjet and light-based bioprinting18,19. Recently, several reports from our team and others have demonstrated extrusion-bioprinted cell-laden constructs with an enhanced effect on repair of bone defects15,2.
Fabrication of HHSs
The HHSs were one-step printed through a coaxial needle using a 3D printer (BioScaffolder 3.1, GeSiM, Germany) at room temperature. The prepared GLN ink was loaded into a printhead and connected to the external nozzle of a coaxial needle, while the internal nozzle was connected to an empty printhead. During the printing process, the GLN ink was directly extruded from the coaxial nozzle to form hollow filaments with an air pressure of 300-500 KPa, and then constructs were fabricated at a printing speed of 6 mm/s. Hollow filaments with tunable inner and outer diameters were controlled by the size of coaxial needles. To further fabricate the constructs with complicated and irregular shapes, bone-shape constructs were generated by a 3D modeling software (3D Studio Max). Subsequently, the 3D models were uploaded onto GeSiM Robotics Software, which generated 2D and 3D views of structures and G-codes to print. A digital camera (Nikon, Japan) was used to acquire images of the printed constructs. Finally, the HHSs were exposed in a UV crosslinker for 80 min with glycerin moisturizing.
Characterization of HHSs
The morphologies of filaments and grids of HHSs were observed by a stereo-zoom microscope (Hirox RH-2000, USA). Hollow structures were displayed by immersing the HHSs in water, and the images were captured using a digital camera. For further characterization of the internal structures of HHSs, samples were sliced to ~200–300 µm in thickness and labeled with Rhodamine B. After staining for 1 min, slices were triply washed by deionized water and imaged with an inverted fluorescence microscope (Nikon, Japan). The average size of inner and outer diameters of one filament, and distances between two filaments in a layer were defined as d, D, and L respectively. Quantification was performed in ImageJ.
Mechanical properties of HHSs
The HHSs (10 × 10 × 10 mm) were immersed in PBS for 1 h to reach a swelling equilibrium before tests. Compression resilience of HHSs with various inner diameters (L0.4D0.6dz, z = 0, 0.2, 0.3, 0.4, n = 3) were determined using a mechanical testing instrument (Care, China) with a crosshead speed at 0.1 mm/s at room temperature. A strain of 80% was set for the compression-recovery tests, and the whole process was recorded by a digital camera. The compression modulus of HHSs was determined from the linear slope of the stress-strain curve at a strain of 5%. For characterization of resilient speed of HHS, luminous fluid was added onto the HHSs (L0.4D0.6d0.4) to visualize the compression-recovery process, which was recorded by a digital camera.
Fatigue resistance of HHSs was assessed by a cyclic compression test. The HHSs with tunable inner diameters L0.4D0.6dz (z = 0, 0.2, 0.3, 0.4, n = 3) were subject to 104 cycles at a frequency of 0.05 Hz with 40% compression strain. The HHSs were fixed in a water bath at room temperature during the test to avoid the influence of moisture evaporation from hydrogels. The test would be aborted if the HHSs were obviously fractured. The morphologies of HHSs before and after testing were respectively captured by a digital camera and stereo zoom microscope.
Tests of mechanical responsiveness
The mechanical responsiveness of HHSs (10 ×10 × 10 mm) to a solution was assessed as below. HHSs with tunable inner diameters (L0.4D0.6dz, z = 0, 0.2, 0.3, 0.4) were used and distances between two hollow filaments in same layer were also varied (LxD0.6d0.4, x = 0.2, 0.4, 0.6). Firstly, the HHSs were immersed in water at 37 °C for 8 h to reach a stable state under a static condition. Wet weight (W) was recorded at preset time points. The water uptake ratio was defined as (W-W0)/ρ/(l × w ×h ) × 100%, where W0 was the initial wet weight of the samples, ρ was the value of water density, and l, w, and h were length, width, and height of the HHS respectively. Subsequently, HHSs were handled with mechanical stimulation in a dynamic condition, and compression with different displacements (20%, 40%, 60%, and 80%) were performed on the HHSs. The wet weight (W′) was recorded after the shape recovery of HHSs to the initial state. In addition, the HHS (L0.4D0.6d0.4) was further subject to incrementally numbers of cycles of compression-recovery (1, 3, 5, and 10) with 20% and 40% compression strains. The water uptake ratio was defined as (W′-W)/ρ/(l×w×h) ×100%. All experiments were performed in triplicate to obtain the average data.
To further visualize the mechanical responsiveness of HHSs, HHSs with tunable inner diameter (L0.4D0.6dz, z = 0, 0.2, 0.4, 0.6) were chosen. After immersing HHSs in a solution containing 0.5 mg/mL fluorescent microspheres (Aladdin, M122063, China) for 5 min, HHSs were subsequently transferred to an IVIS imaging system (Caliper Life Sciences, USA), and the images before or after immersing were acquired at Ex = 605 nm, Em = 680 nm. Next, HHSs were transferred back to the solution and subject to incremental compression displacements (20%, 40%, 60%, and 80%) and number of cycles of compression-recovery (1, 3, 5, and 10) with 20% and 40% compression displacement. The images of HHSs were also captured using an IVIS imaging systems after each process of compression-recovery. All experiments were performed in triplicate to obtain the average data.
Cell loading to HHSs
Cell culture
Human bone marrow mesenchymal stem cells (hBMSCs, HUXMF-01001, Cyagen), hBMSCs-GFP (HUXMA-01101, Cyagen), and rat bone marrow mesenchymal stem cells (rBMSCs, were cultured in α-MEM (Hyclone)) containing 10% fetal bovine serum (FBS, Gibco) and 100 U/mL penicillin/streptomycin. Human umbilical vein endothelial cells (HUVECs, e005, iCell), HUVECs-RFP (h111, iCell) and rat umbilical vein endothelial cells (rECs, e004, iCell) were cultured in a special culture medium supplied from the iCell. All cells were cultured in a humidified atmosphere with 5% CO2 at 37 °C. rBMSCs were extracted from long bones of limbs of rats. Briefly, alpha-modified Eagle’s medium (α-MEM, HyClone) containing 20% fetal bovine serum (FBS, Gibco) and 100 U/mL penicillin/streptomycin (Hyclone) was injected using syringes to flush cells out of marrow cavities. After 24 h, the medium was replaced with fresh culture medium to remove nonadherent cells. The cells were sub-cultured in α-MEM containing 10% FBS and 100 U/mL penicillin/streptomycin. The sterilized HHSs (L0.4D0.6d0.4, length 10 mm, width 10 mm, height 10 mm) were immersed in α-MEM medium for 1 h at 37 °C before cell loading.
Rapid Loading
Cell loading into HHSs by mechanical assistance was investigated. Firstly, two approaches were used: The first one was direct immersion of HHSs in hBMSCs cell suspension (1 × 106 cells/mL) for 4 s under a static condition; while the second one utilized a compression-recovery cycle in hBMSCs cell suspension under 80% strain within 4 s (V1-mechanical response). The subsequent HHSs were immediately transferred from the cell suspension and further cultured in medium. After 4 h of culture, the samples were stained with 5 µg/mL fluorescein diacetate (FDA, Sigma), triply washed with PBS, and then visualized by confocal laser scanning microscopy (CLSM, Nikon C2, Japan). The number of cells loaded into HHSs were quantified by ImageJ. Subsequently, the influence of compression displacement (20%, 40%, 60%, and 80%) in hBMSCs cell suspension (1 × 106 cells/mL) and cycle times (1, 3, 5, and 10) under 40% compression displacement in hBMSCs cell suspension (5 × 105 cells/mL) on cell loading of HHSs were also evaluated respectively. All experiments were performed in triplicate at least.
Uniform distribution of cell loading
The cell loading into HHSs were handled in other two distinct means: One was directly seeding of 0.5 mL cell suspension (hBMSCs, 1 × 106 cells per mL) onto the HHSs; the other was removal of the medium in V2 under 80% compression displacement, and subsequent seeding of 0.5 mL hBMSCs cell suspension onto the HHSs (V2-mechanical response). After 4 h of culture, same characterization of cells was performed as above. Besides, the cells in the surface, middle, and bottom sections of HHSs were also quantified. All experiments were performed with five parallel samples.
Partitioned loading of multiple cell types
To achieve partitioned loading of multiple cell types into HHSs, V1 and V2 of HHSs were manipulated by combination of V1 + V2-mechanical response simultaneously. Firstly, HHSs were subject to a process of compression-recovery under 80% compression displacement in a HUVECs-RFP cell suspension within 4 s. Subsequently, samples were taken out and the grids were triply flushed (V2) with α-MEM medium. After culture for 4 h, the process of V2-mechanical response was similarly performed as described above, except that the hBMSCs-GFP were used instead of hBMSCs. The loading and distribution of HUVECs-RFP and hBMSCs-GFP in HHSs after 24 h of culture were visualized by CLSM.
Cell viability under mechanical stimulation
Cell damage is typically associated with a decrease in cell mitochondrial membrane potential and an increase in ROS generation besides a reduction in cell proliferation. To assess whether mechanical stimulation have negative influence on cell viability. Mitochondrial membrane potential, ROS and proliferation of the cells were examined. After a process of compression-recovery with 80% compression displacement and 10 cycles with 40% compression displacement in cell suspension, cells loaded in HHSs were collected by compression with 80% compression displacement again. Subsequently, the collected cells were seeded on 24-well cell culture plates at a density of 2 × 104 cells/well, and cells that did not undergo mechanical stimulation were used as control. The following measurements were performed: Mitochondrial membrane potential of cells cultured for 4 h and 24 h was detected by double fluorescence staining of mitochondria using JC-1 (C2006, Beyotime) according to the manufacturer’s instructions, either as green fluorescent J-monomers or as red fluorescent J-aggregates. ROS generated by cells was detected via a fluorescence probe DCFH-DA (35845, Sigma), and cells cultured with 200 μM H2O2 were used as positive control. After 4 h of culture, the cells were incubated in a 10 μM DCFH-DA solution at 37 °C for 30 min, and triply washed by PBS, and then stained with Hoechst 33342. Images of cells labeled by JC-1 and DCFH-DA were captured using a CLSM, and the intensity of red and green fluorescence was quantified by a gray value analysis in ImageJ. Cell proliferation was determined using a CCK8 assay (Do**do, Japan) according to the manufacturer’s instructions, and analyzed on day 1, 3, and 5 (n = 3).
In vitro functional examination
After 14 days of culture, the samples loaded with HUVECs were harvested to evaluate the maintenance of functions of HUVECs in HHSs for detecting CD31 and VWF immunofluorescence. Samples were triply washed with PBS and then fixed in 4% paraformaldehyde at 4 °C overnight. All samples were dehydrated, embedded in OCT, and sectioned at a thickness of 5 μm. The slices were permeabilized and blocked with 0.1% Triton-X and 10% goat serum, respectively. Then, the slices were incubated with the anti-CD31 antibody (ab28364, Abcam) and the anti-VWF antibody (SC-365712, Santa) at 1:100 dilution in 1% BSA-containing PBS solution at 4 °C overnight. Subsequently, the slices were washed 5 times by PBS and treated with a mixture of goat-anti-rabbit IgG Alexa Fluor 488 (ab150077, Abcam) and goat-anti-mouse IgG Alexa Fluor 647 (ab150115, Abcam) conjugated secondary antibody at 1:400 dilution for 1 h at room temperature, and washed 5 times by PBS. Finally, samples were counterstained with Hoechst 33342 and triply washed with PBS. Fluorescence images were captured via a CLSM.
Samples loaded with hBMSCs were cultured in osteogenic differentiation media. After 21 days of culture, the samples were harvested for alizarin red S (ARS) staining to evaluate the ability of calcification. The samples were handled as described above to acquired slices. Then, the slices were stained with ARS solution at a concentration of 1% (pH = 4.2–4.3) for 5 min, and triply washed by deionized water. The images were acquired by an optical microscope.
Establishment of challenging bone defect models
To assess the concept of HHS-cells for repair of the challenging bone defects, two typical challenging bone defect models were established: the rat femoral segmental and rat osteoporotic bone defects. The animal experiments were approved by the Institutional Animal Care and Use Committee of Shenzhen Institute of Advanced Technology, Chinese Academy of Science. HHS-cells samples were cultured for 3 days in vitro before implantation.
The rat femoral segmental defect model
32 male Sprague Dawley (SD) rats (12 weeks old) were divided into 4 groups randomly: Blank (no samples), HHS, HHS-M (HHS loaded with rBMSCs), and HHS-ME (HHS loaded with rBMSCs and rECs). A 5 mm critical-sized femoral segmental defect was created according to a previously published procedure51. Briefly, anesthesia was performed with 5% isoflurane and O2. Animals were placed in lateral position on a 37 °C warm heating pad, isoflurane was lowered to 2 to 2.5% to maintain anesthesia during the entire surgery. The hindlimb of the rat was shaved and disinfected (half of each left and right), and an ~2-cm-long longitudinal skin incision was made, and superficial fascia, biceps femoris and vastus lateralis muscles were separated, exposing the lateral aspect of the femoral bone. After thoroughly cleaning the soft tissue on femur, a seven-hole fixation plate (31 mm in length, 3.5 mm in width, 1.2 mm in thickness, titanium alloy; Baiortho, China) was applied to the lateral aspect of the femur and held in place using forceps and a clamp. At this point, the plate was permanently fixed to the bone using three proximal and three distal screws (screws, φ = 1.1 mm; outer screws, 8 mm in length; inner screws, 7 mm in length; Baiortho). Screw holes were created in the femur using drill sleeves and a drill bit (φ = 1 mm, Baiortho). A 5 mm critical-sized segmental defect was created using a reciprocating saw (Baiortho). Saline was continuously applied during the piercings and osteotomy, and pieces of bone were carefully removed with saline. A defect of ~5 mm was achieved in all animals. All samples used here were designed as cylinders (φ 5.5 mm × h 5 mm). The cylindrical samples were push-fitted, and then muscle, fascia and skin were successively closed with sutures. After wound closure, animals were injected with 8 U/mL penicillin and transferred back into their cage. To relieve the pain, all rats were orally administrated with ibuprofen (10 mg/kg, PHR1004, Merck) in drinking water for 24 h after surgery.
6 weeks after the defect surgery, randomly selected rats (a total of 16, n = 4 for each group) were anesthetized. Abdominal cavity was opened, and abdominal aorta and vein were exposed. Then a 1.2 mm angio-catheter was inserted in the aorta and a 1.6 mm angio-catheter was inserted in the vein. 100 mL of heparinized saline (1000 U/ml), 100 ml of 4% paraformaldehyde, and 15 ml of Microfil (Flow Tech, MV-120, USA) were perfused in turn. After being kept for 24 h at 4 °C, the femurs were collected from the rats, and imaged with microcomputed tomography (μCT, SCANCO MEDICAL μCT100, Switzerland) to observe formation of blood vessels.
6 and 12 weeks after the defect surgery, animals were euthanasia using CO2, and the femurs from each group (n = 4) were collected and fixed in 4% paraformaldehyde for 48 h. Bone healing in 4 groups were preliminary evaluated by X-ray. Subsequently, fixation plates and screws were removed from the femur before μCT examinations.
The rat osteoporotic bone defect model
A total of 12 female SD rats (3 months old) were all subject to ovariectomy surgeries and then raised for another 12 weeks to obtain osteoporotic rats. The osteoporotic rats were divided into 3 groups at random: Blank, HHS, and HHS-M. All samples used here were designed as cylinders (φ 3 mm × h 4 mm). A part of surgical procedures was described above. Two distal femoral metaphyseal critical-size defects (φ 3 mm × h 4 mm) were established using a drill in left and right of an osteoporotic rat. The eight samples in each group were implanted in the femoral defects. 4 and 8 weeks after of the defect surgery, animals were sacrificed and the distal femurs from each group (n = 4) were collected and fixed in 4% paraformaldehyde. BMSCs and OVX-BMSCs were respectively isolated from long bones of limbs of healthy and osteogenic rats. Then, alpha-modified Eagle’s medium (α-MEM, HyClone) containing 20% fetal bovine serum (FBS, Gibco) and 100 U/mL penicillin/streptomycin (Hyclone) was injected using syringes to flush cells out of marrow cavities. The cells were cultured in a humidified atmosphere with 5% CO2 at 37 °C. After 24 h, the medium was replaced with fresh culture medium to remove nonadherent cells. The cells were sub-cultured in α-MEM containing 10% FBS and 100 U/mL penicillin/streptomycin until passage 2 (P2). The P2 cells were then harvested for further study. BMSCs and OVX-BMSCs were respectively seeded on the 24-well plates at a cell density of 2 × 104/well. Cell senescence was assessed by the senescence βgalactosidase (SAβgal) staining kit (Beyotime) and qRT-PCR for p16 gene expression. Cell proliferation was analyzed on 1 and 3 days by CCK8. In addition, the ability of osteogenic differentiation was evaluated by ALP staining on 4 and 7 days and alizarin red (ARS) staining on 14 and 21 days, and qRT-PCR for Runx2, Alp, Bmp2, Bsp, Col 1a1, and Ocn genes expression on 7 days.
The rat subcutaneous implantation and repair of distal femoral metaphyseal defect
To evaluate inflammation and foreign body response in vivo experiments, three groups: sham, HHSs (length 8 mm, width 8 mm, height 4 mm), and HHSs-M were subcutaneously implanted on the backs of rats (12 weeks old) under aseptic and anesthetic conditions. Anesthesia was performed with 5% isoflurane and O2. Animals were placed on a 37 °C warm heating pad, isoflurane was lowered to 2–2.5% to maintain anesthesia during the entire surgery. After 7 days of surgery, animals were sacrificed using CO2, and all specimens were collected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 5 μm thickness. The related assessments were conducted including complete blood count, blood biochemistry analysis (liver and kidney function indicators), and histology staining of the heart, liver, spleen, and kidney, as well as immunohistochemical staining of inflammation-related markers IL-6 and TNF-α, and macrophage phenotype related markers CD80 and CD 206. The procedure for immunohistochemical staining was the same as mentioned above. The slices were respectively incubated with an anti-IL6 antibody (bs-6309r) at anti-TNF-α antibody (bs-10802r), anti-CD80 antibody (A001961) and anti-CD206 antibody (AB64693) at 1:200 dilution in 1% BSA-containing PBS solution at 4 °C overnight.
To assess the survival of seeded cells in the HHSs implantation, HHSs-M (length 8 mm, width 8 mm, height 4 mm) with luciferase-overexpressed rBMSCs were subcutaneously implanted in rats. Subsequently, bioluminescence images were captured in rats and a quantitative analysis of bioluminescence was conducted at 1, 4, 7, and 10 days. The images of HHSs were also captured using an IVIS imaging systems.
To assess the hollow structures of HHSs in promoting blood vessel growth and bone formation, two groups of HHSs (L0.4D0.6d0 and L0.4D0.6d0.4) were used. Subcutaneous implantation and a distal femoral metaphyseal defect model were established. All animals were 3 months old in the experiments. For subcutaneous implantation, two subcutaneous pockets for implantation of the HHSs (length 8 mm, width 8 mm, height 4 mm) were made on the backs of rats under aseptic and anesthetic conditions. Two groups were respectively transplanted into the left and right (n = 4 for each group). After 2 weeks of surgery, animals were sacrificed, and all specimens were collected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 5 μm thickness, and stained with H&E. The surgical procedure for distal femoral metaphyseal defect was the same as the rat osteoporotic bone defect model. A distal femoral metaphyseal critical-size defect (φ 3 mm × h 4 mm) was established in normal rats. Two groups were respectively transplanted into the left and right of the rat’s defects at random (n = 3 for each group). All samples used here were designed as a cylinder (φ 3 mm × h 4 mm). After 8 weeks of surgery, all animals were sacrificed and the distal femurs for each group were collected, and fixed in 4% paraformaldehyde for 48 h. All samples were scanned using a μCT, then decalcified with EDTA, embedded in paraffin, sectioned at 5 μm thickness, and processed according to standard histological staining procedures. The sections were stained with H&E and Masson’s trichrome.
μCT analysis
All samples harvested from animals were scanned using a μCT with 18 μm resolution and a maximum voltage of 70 KV, to evaluate new bone formation within the defect region. The obtained grayscale images were further reconstructed and analyzed using the Scanco software. During the reconstruction, a global threshold was used to segment the newly formed bone from each implant. The bone volume (BV), bone volume fractions (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were analyzed to evaluate the formation of new bone.
Histological staining
After the μCT imaging, all samples were decalcified with EDTA, embedded in paraffin, sectioned at 5 μm thickness, and processed according to standard histological staining procedures. The sections were then stained with haemotoxylin and eosin (H&E) to assess general tissue morphology, and Masson’s trichrome to observe collagen fibers and visualize bone formation.
Statistical analysis
All data are expressed as the means ± standard deviation. Statistical analyses were performed using GraphPad Prism 8 software. Statistically significant differences between two experimental groups were assessed using Student’s t tests and comparisons among more than two groups were assessed using a Two-way ANOVA. Differences were considered statistically significant when *p < 0.05. **p < 0.01, ***p < 0.001, and ****p < 0.0001 were considered highly significant, ns referred to no significant difference.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
All the data supporting the results in this study are available within the paper and its Supplementary Information files. Any additional requests for information can be directed to, and will be fulfilled by, the corresponding authors. Source data are provided with this paper.
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Acknowledgements
The authors gratefully acknowledge the support for this work from the National Key R&D Program [Grant No. 2022YFB3804403 and 2018YFA0703100]; the National Natural Science Foundation of China [Grant Nos. 32122046, 82072082, 32000959, and 32201097]; the Shenzhen Medical Research Fund [Grant Nos. A2303016]; and the Shenzhen Fundamental Research Foundation [Grant Nos. JCYJ20200109114006014, JCYJ20210324115814040 and JCYJ20210324113001005].
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J.Y. and C.R. conceived and designed the study. J.Y, Z.C, X.P, X.W. and H.S. performed in vivo experiments. J.Y. performed the chemical synthesis. J.Y., Z.C., C.G. and J.L. performed in vitro characterization and cell experiments. J.Y. and C.R. analyzed the data. J.Y., K.L., G.W., W.L., and C.R. drafted the manuscript. H.P. and C.R. supervised the entire project. All authors discussed the results and commented on the manuscript.
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Yang, J., Chen, Z., Gao, C. et al. A mechanical-assisted post-bioprinting strategy for challenging bone defects repair. Nat Commun 15, 3565 (2024). https://doi.org/10.1038/s41467-024-48023-8
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DOI: https://doi.org/10.1038/s41467-024-48023-8
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