Scaffold preparation
3D mannequin design
The Rhinoceros® 7.0 modeling software program and its parametric modeling plug-in Grasshopper have been employed to design the assist construction of the helical icosahedral Gyroid minimal floor, which was mathematically expressed via a separate formulation:
$$eqalign{:textual content{cos}left(frac{2{uppi:}}{U}xright)textual content{sin}left(frac{2{uppi:}}{U}yright)&+textual content{cos}left(frac{2{uppi:}}{U}yright)textual content{sin}left(frac{2{uppi:}}{U}zright) cr & + textual content{cos}left(frac{2{uppi:}}{U}zright)textual content{sin}left(frac{2{uppi:}}{U}xright)=textual content{t}cr}$$
The place U denotes the dimensions of the minimal structural unit of the TPMS, t is the structural parameter that determines the form of the minimal structural unit. Right here, t = 0 is about right here, the minimal structural unit measurement is 2 mm, the wall thickness is 0.45 mm, and the mannequin file is saved in STL format.
Manufacturing of the scaffold
The gear used within the laser powder mattress fusion (LPBF) experiment was the ProX DMP 320 from 3D Methods. The scale of the fashioned substrate aircraft was 275 mm × 275 mm × 420 mm, with the machining accuracy of the elements being ± 0.1% of the present printing vary, and the oxygen content material throughout printing was decrease than 25 ppm. The LPBF parameters have been as follows: laser energy of 80 W, scanning pace of 400 mm/s, layer thickness of 30 μm, hatch area of 80 μm, and spot measurement of 80 μm. To scale back thermal stress, when scanning the adjoining layers above and beneath, the laser scanning course was rotated by 73° successively. After printing was accomplished, the substrate and samples have been taken out, and wire-electrode reducing was used to acquire the printed samples.
Dynamic electrolytic sprucing
To take away the adhesive powder on the floor of the scaffold, anhydrous ethanol answer containing 10% quantity fraction perchloric acid was utilized to carry out dynamic electrochemical sprucing. A magnetic rotor was employed to stir on the pace of 600 rpm/min, with a sprucing time of 10 min. After the sprucing, anhydrous ethanol was used for ultrasonic cleansing and dried in a vacuum drying oven.
Floor fluorination remedy
The polished assist was soaked in a fume hood with 40% hydrofluoric acid and shaken on a shaker for six h till a dense magnesium fluoride coating of about 1.5 μm thickness was fashioned on the floor of the scaffold. After fluorination, it was cleaned with deionized water and anhydrous ethanol.
Dicalcium phosphate dihydrate (DCPD) coating
Sodium nitrate(NaNO3), Calcium dihydrogen phosphate (Ca(H2PO4)2.H2O) bought from Sinopharm Chemical Reagent Co., LTD., analytical pure, purity ≥ 98.0%.The porous scaffold floor was coated with DCPD utilizing a chemical deposition methodology. The fluorinated scaffold was immersed in a supersaturated answer, and after 12 h of static response, a uniform white DCPD coating fashioned on the scaffold floor. The composition of the DCPD answer is detailed in Desk 2 beneath.
5% Sr2+ octacalcium ohosphate (OCP) coating remedy
Sodium dihydrogen phosphate (NaH2PO4), Strontium nitrate (Sr(NO3)2), Sodium hydroxide (NaOH) bought from Sinopharm Chemical Reagent Co., LTD., analytical pure, purity ≥ 96.0%.To enhance the corrosion resistance and promote osteogenesis properties of the scaffold, a hydrothermal response answer containing Sr2+ with a molar focus of 5% of the sum of the molar concentrations of Sr and Ca parts was ready utilizing a hydrothermal transformation methodology. The pH was adjusted to six.5 utilizing NaOH. The pattern and answer have been positioned in a 500 mL hydrothermal response kettle. The reactor was positioned within the oven to react at 90℃ for 12 h. The DCPD coating on the floor of the pattern was reworked into 5Sr-OCP coating, and the looks of the pattern was gray-white matte. The formulation of hydrothermal response liquid is proven in Desk 3.
Characterization and mechanical properties of 3D printed porous scaffolds
Characterization of 3D printed scaffolds
The general circumstances of JDBM and JDBM/SrOCP scaffolds have been examined utilizing stereomicroscopy. SEM was employed to look at the scaffold floor morphology, whereas EDS was employed to watch the aspect distribution on the scaffold floor. The nanomorphology and floor roughness of the scaffolds have been assessed utilizing AFM evaluation. The hydrophilicity of the scaffolds was decided utilizing contact angle (CA) measurements. Porosity of the porous scaffolds was calculated utilizing the ethanol substitute methodology in response to the next formulation [ASTM F2150–17 (2022) e1] [46]: Porosity (%)=(V1-V3)/(V2-V3) × 100%.
The amount V1 signifies the overall quantity of the graduated cylinder after addition of absolute ethanol and immersion within the scaffold pattern. The amount V2 denotes the overall quantity recorded after removing of air from the fabric’s pores utilizing a vacuum desiccator and filling them with ethanol. Quantity V3 signifies the overall quantity of fabric remaining after removing from the graduated cylinder.
In vitro degradation assessments
In keeping with ASTM-G31-21, the scaffolds have been immersed in ’Hank’s answer (37℃, pH 7.4) at a ratio of 20 mL/cm2. The extracts have been collected on days 1, 4, 7, and 14 for evaluation utilizing ICP to measure Mg2+, Sr2+, Ca2+, and P concentrations. Furthermore, the residual weight of the magnesium steel scaffold was calculated to judge its degradation conduct. SEM was employed to look at the floor morphology of the scaffolds after immersion for 7 days, and EDS was utilized to watch the distribution of parts. XRD evaluation was carried out to find out the composition of floor merchandise post-degradation.
Mechanical property check
The cylindrical porous magnesium scaffold assist was securely positioned on the tray of the digital common testing machine and compressed in each the X-axis and Z-axis instructions, respectively. The displacement fee was set at 1 mm/min to precisely set up the management. The load-displacement curve was mechanically recorded till a displacement of three.0 mm was reached. Lastly, the stress-strain curve was plotted.
In vitro experiments
Biocompatibility of JDBM/SrOCP scaffolds
HBMSCs have been collected from Wuhan Punosai whereas HUVECs have been bought from Zhejiang Meisen. HBMSCs have been cultured in MSCM (full medium supplemented with 5% fetal bovine serum, MSCGS mesenchymal stem cell progress complement, streptomycin 100 ug/ml, penicillin 100 U/ml). HUVECs have been cultured in ECM (full medium supplemented with 5% fetal bovine serum, ECGS endothelial cell progress complement, streptomycin 100 µg/ml, penicillin 100 U/ml). All cells have been incubated at 37 °C below a CO2 focus of 5% and humidity of 95%. Subcultures have been carried out at roughly 80–90% confluence. All samples have been sterilized via Co-60 irradiation and scaffold extracts have been obtained following ISO 10993-5 tips. Contemplating the decrease degradation fee of Mg-based supplies in vivo in comparison with in vitro circumstances, the scaffold extracts have been diluted following protocols described within the literature.
The biocompatibility of porous magnesium steel scaffolds was examined on HBMSCs and HUVEC cells utilizing the Cell Counting Equipment-8 (CCK-8, Dojindo, Japan). HBMSCs have been seeded at 2 × 103 cells/nicely in 96-well plates and HUVEC cells have been seeded at 2.5 × 103 cells/nicely. Scaffold extract was used to exchange the tradition medium and incubated for 1, 3, and 5 days. At particular time intervals, a ten% quantity of CCK-8 answer was added and incubated for additional 1.5 h within the cell incubator. Subsequently, 100 µl of the supernatant was collected and its absorbance (OD) at 450 nm was recorded utilizing a microplate reader (Bio-Tek, USA).
Cell viability was consider utilizing the stay/lifeless staining equipment (Beyotime, China). HBMSCs and HUVECs cells have been seeded in 24-well plates at a density of 4 × 103 cells/nicely and 4.5 × 103 cells/nicely, respectively. After washing 3 times with PBS answer, the cells have been incubated with 500 ul of stay/lifeless staining reagent for 30 min and noticed below a fluorescence microscope.
Cell proliferation was evaluated utilizing Ki67 immunostaining. HBMSCs and HUVEC cells have been seeded at a density of three × 103 cells/nicely in a 24-well plate. After cell adherence, the medium was changed with scaffold extract and cultured for 3 days. Samples have been then washed with PBS, mounted with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 15 min, blocked with goat serum, and incubated in a single day at 4 °C with Ki67 antibody (ab15580, Abcam), adopted by incubation with fluorescently labeled secondary antibody for 1 h. The nuclei have been stained with DAPI (Solarbio, C0065) and analyzed utilizing a fluorescence microscope. All experiments have been carried out in triplicate.
Cell morphology on the floor of JDBM/SrOCP scaffolds
Because the JDBM scaffold exhibited fuel manufacturing when immersed in vitro, it hindered cell adhesion on its floor. Subsequently, HBMSCs have been seeded onto the floor of the JDBM/SrOCP scaffold to research its morphological traits. The seeding density of HBMSCs was 2 × 105 cells/ml. Following a direct co-culture for five days, the cells have been washed twice with PBS after which mounted in 2.5% glutaraldehyde at 4℃ for over 4 h. Subsequently, they have been washed twice with PBS for 20 min, every time. The cells have been sequentially subjected to ethanol dehydration with every step lasting between 20 and 30 min. Lastly, samples underwent supercritical drying and have been ready by way of gold sputtering for two min earlier than SEM pictures have been captured to watch cell morphology on the scaffold floor.
Alkaline phosphatase and alizarin crimson staining
HBMSCs have been employed to evaluate the osteogenic properties of the scaffolds in vitro. HBMSCs have been seeded into 24-well plates at a density of 6 × 103 cells per nicely. As soon as the cells had totally adhered to the floor, the entire medium was changed with scaffold extracts ready utilizing an osteogenic induction medium (OriCell, HUXMX-90021). The tradition was continued for 7 and 14 days. The alkaline phosphatase staining was carried out utilizing a BCIP/NBT staining equipment (C3206, Beyotime). After co-culturing for 21 days, calcium nodules have been stained utilizing alizarin crimson (ALIR-10001, OriCell). The staining outcomes have been examined below a stereomicroscope (DS-Ri2, Nikon).
RT-qPCR
Actual-time quantitative polymerase chain response (RT-qPCR) was carried out to find out the expression of osteogenic genes alkaline phosphatase (ALP), collagen kind I (COL1), Runt-related transcription issue 2 (RUNX2), osteocalcin (OCN), and angiogenic genes platelet endothelial cell adhesion molecule (CD31) and vascular endothelial progress issue (VEGF). GAPDH served because the reference gene. Particularly, HBMSCs and HUVECs have been seeded into 6-well plates at a density of two × 105 cells/nicely. As soon as the cells adhered totally, the HBMSCs have been changed with scaffold extract ready utilizing an osteogenic induction medium, whereas the HUVECs have been changed with the identical scaffold extract. HBMSCs have been cultured for 7 and 14 days, whereas HUVEC cells have been cultured for an extra 3 days. Subsequent, whole RNA was extracted with the RNA extraction equipment (Toyobo, FSQ-201). RT-qPCR evaluation was carried out utilizing the Takara SYBR® Premix Ex TaqII equipment (GenStar, A303) and CFX96 real-time detection system. Every pattern was replicated 3 times to enhance accuracy. The primer sequences for all genes are introduced in Supplementary Desk S1.
Immunofluorescence staining
The HBMSCs have been cultured utilizing the oblique contact methodology. They have been seeded in 24-well plates at a density of two × 103 cells/nicely. As soon as the cells had totally adhered, they have been changed with the scaffold extract collected from the osteogenic induction medium. The cells have been mounted with 4% paraformaldehyde for 20 min at particular time factors, washed with PBS answer, handled with 0.1% Triton X-100 for 15 min, and washed with PBS. They have been then blocked with goat serum, and incubated with the Runt-related transcription issue 2 (RUNX2, ab192256, Abcam,1: 200), kind I collagen (COL1, ab138492, Abcam,1:100), osteopontin (Opn, ab63856, Abcam,1:100), osteocalcin (OCN,23418-1-AP, Proteintech,1:100), in a single day at 4 °C. All samples have been incubated with fluorescently labeled secondary antibodies for 1 h, nuclei have been labeled with DAPI (Solarbio, C0065), washed 3 times with PBS, and examined below a fluorescence microscope in triplicate.
Scratch check
The tradition of HUVECs was carried out utilizing the oblique contact methodology. The cells have been subsequently seeded on a 6-well plate at a density of 4 × 105 cells per nicely. As soon as the HUVEC reached the goal confluence, a vertical scratch was made within the heart of every nicely utilizing a 200 uL pipet tip. After thorough washing with sterile PBS, any indifferent cells have been eliminated, and ECM endothelial cell medium with out serum was added. Pictures have been taken at 0 and 24 h post-scratching, and the extent of scratch closure was quantified utilizing ImageJ software program.
Transwell assay
The transwell chambers have been positioned in 24-well plates and divided into three teams, every with three wells. Chambers for the clean group have been added with 600 µl of ECM endothelial cell low serum medium, whereas the JDBM and JDBM/SrOCP teams have been handled with 600 µl of ECM endothelial cell low serum medium containing the extract, respectively. The higher chamber was supplemented with 200 µl suspension containing 2 × 104 HUVEC cells and cultured for twenty-four h. After gently scraping off any remaining cells from the higher layer, the decrease layer of the chamber was mounted with 4% paraformaldehyde for 20 min. Subsequently, staining with a 0.1% crystal violet answer (Solarbio, G1063) was carried out for 30 min. Cell migration occasions have been noticed utilizing a lightweight microscope (Ni-U, Nikon, Japan), and the variety of migrated HUVEC cells was quantified utilizing ImageJ software program.
Tube formation investigation of scaffolds
Angiogenesis assays have been carried out utilizing Matrigel (Corning, 354234) in ibidi µslide Angiogenesis plates. Particularly, the ibidi angiogenesis particular nicely plates have been coated with Matrigel and HUVECs have been suspended in ECM endothelial cell medium, JDBM scaffold extract, and JDBM/SrOCP scaffold extract. A 50 ul cell suspension containing 1 × 104 HUVECs was seeded on the ibidi plates. After a cultivation interval of 6 h, tube formation was noticed and captured utilizing a lightweight microscope (Ni-U, Nikon, Japan). The tube formation was quantitatively analyzed utilizing ImageJ software program. Key parameters measured comprised whole tube size, variety of nodes, and variety of junctions.
Transcriptomic assay
In abstract, HBMSCs have been lysed utilizing TRIzol reagent, and the overall RNA was extracted for library building, transcriptome knowledge assortment, and evaluation by Novogene Co., Ltd (Beijing, China). The standard and amount of the RNA have been assessed with an Agilent 2100 Bioanalyzer. It was then sequenced on the Illumina NovaSeq 6000 platform. DESeq2 software program (model 1.20.0) was used for gene expression quantification and differential gene expression evaluation between the 2 comparability teams. To research purposeful annotations of differentially expressed genes, cluster profile software program (model 3.8.1) was employed for GO (Gene Ontology) and KEGG pathway enrichment analyses.
In vivo experiments
Bone restore evaluation in vivo
The experimental protocol was permitted by the Experimental Animal Ethics Committee of the Chinese language PLA Basic Hospital (approval quantity: S2020-169-01). A rat femoral condyle defect mannequin was established to validate the scaffold’s in vivo osteogenic and angiogenic capabilities. Eight-week-old male Sprague Dawley (SD) rats (n = 54, 280–320 g) have been intraperitoneally anesthetized with sodium pentobarbital at a dosage of 30 mg/kg. In a sterile surroundings, a longitudinal incision was created within the medial distal femur to show the medial femoral condyle. Subsequent, a cylindrical bone defect with dimensions of two.6 mm in diameter and a couple of.8 mm in depth was generated by drilling into the medial femoral condyle utilizing a 2.6 mm drill. The scaffolds have been implanted into the defect, and after attaining adequate hemostasis, the surgical web site was meticulously sutured layer by layer. Subsequently, the wound was sterilized utilizing iodophor. The rats have been randomly divided into three teams: clean group (n = 18), JDBM group (n = 18), and JDBM/SrOCP group (n = 18).
Vascular perfusion
To watch early revascularization after scaffold implantation, we carried out microvascular perfusion at 4 and eight weeks after scaffold implantation. Three rats have been randomly chosen from every group and at every time level. After anesthesia, the rats have been positioned supine on a plate and a midline incision was made via the stomach’s dermis and muscle layer. The xiphoid course of was elevated to entry the thoracic cavity by reducing via the diaphragm and sternum. Subsequently, an infusion needle was inserted roughly 2 mm deep into the left ventricle whereas concurrently severing the inferior vena cava. Following euthanasia, the rats have been perfused with regular saline (containing 500 U/L heparin) till the venous outflow modified from crimson to colorless regular saline. Subsequently, paraformaldehyde fixative was constantly perfused till the decrease limbs and tail of the rats trembled. Following the MICROFIL® perfusion answer directions, the syringe was manually pushed till the blood vessels of important organs such because the liver, kidneys, and mesentery within the rat’s physique step by step turned yellow, indicating profitable perfusion. The samples have been saved in a fridge in a single day at 4° C to permit full solidification and decalcification after sampling.
Micro-CT evaluation
The samples have been collected at 4, 8, and 12 weeks post-surgery. Instantly after assortment, they have been totally mounted utilizing a 4% paraformaldehyde answer after which underwent MicroCT scanning (Belgium, Bruker, Skyscan1276) at a spatial decision of 13 μm for three-dimensional reconstruction. A ray supply voltage of 85 kV, present of 160 µA, and scan rotation angle of 360° have been used. The area of curiosity for all samples was outlined as a cylindrical quantity with a diameter of two.6 mm and a peak of two.8 mm. Bone morphometric parameters, together with bone quantity/whole tissue quantity (Bv/Television), trabecular quantity (Tb. N), trabecular spacing (Tb.Sp), trabecular thickness (Tb. Th), Bone mineral density measurements (BMD), and vascular quantity fraction (BVV/TV) have been calculated. All samples have been collected in triplicate.
Laborious tissue part
The EXAKT system was employed to arrange onerous tissue sections. Rat femora have been subjected to gradient ethanol dehydration after which embedded in light-cured resin. Sections with a thickness of 200 μm have been collected utilizing the E300CP onerous tissue slicing machine (EXAKT, Germany) after which polished on a grinder to acquire a thickness vary of 25 to 35 μm. Microscopic statement was carried out utilizing Goldner trichrome staining (Solarbio, G3550).
Histological assessments and immunohistochemical evaluation
Histology and immunohistochemistry strategies have been employed to evaluate early vascularized bone regeneration skill of the scaffold after 4 weeks of implantation. Bone tissue samples have been mounted utilizing 4% paraformaldehyde, decalcified in 10% ethylenediamine tetraacetic acid (EDTA), embedded in paraffin, sectioned at a thickness of 5 μm, and stained with hematoxylin and eosin (HE). To carry out immunohistochemical staining, sections have been deparaffinized and subjected to antigen retrieval utilizing 3% hydrogen peroxide (H2O2) and pepsin (Abcam, ab64201). Subsequent, the sections have been handled with 0.1% Triton X-100 for 15 min and blocked with 10% goat serum. They have been then incubated with Osteopontin (Opn, ab63856,Abcam,1:100), osteocalcin (OCN,23418-1-AP, Proteintech, 1:100), CD31 (ab182981,abcam,1:100) and EMCN (sc-65495,Santa Cruz,1:200) in a single day at 4° C and at room temperature for 1 h with the secondary antibody. Lastly, they have been analyzed with a lightweight microscope utilizing the DBA chromogenic substrate system. Immunofluorescence staining was carried out utilizing a protocol just like that of immunohistochemistry. The first antibody was incubated in a single day at 4 °C, after which washed 3 times with PBS. Subsequently, the samples have been incubated with the secondary antibody (AB150077, Abcam, 1:200) at room temperature. Nuclear staining was carried out utilizing DAPI. Pictures have been captured with a fluorescence microscope, and the immunofluorescence depth was quantified utilizing ImageJ software program. All experiments have been carried out in triplicate.
Statistical evaluation
All experiments have been repeated 3 times. Statistical evaluation and graphs have been carried out utilizing GraphPad Prism 9.4 software program. All knowledge are introduced because the imply ± customary deviation (SD). Scholar’s t-test was employed to match two teams, and one-way evaluation of variance (ANOVA) was used to match a number of teams. The distinction was thought-about to be statistically vital at P < 0.05.