This study was approved by the Ethics Committee of Beijing Stomatological Hospital, Capital Medical University (Approval No.: CMUSH-IRB-KJ-YJ-2023-07), with informed consent from the patients. All participants were recruited by a surgeon from the dental implant center. All enrolled T2DM patients in this study were diagnosed with T2DM by endocrinologists, and all patients had HbA1c below 8%. Over the past 3 years, 15 patients who met blood glucose control standards and were preparing for implantation surgery were included in the study. Compared with those in the T2DM group, participants in the non-T2DM group were selected at a 1:1 ratio, and all basic information, including age, sex, general health status, DM type, and implant placement, was matched to avoid the influence of other factors. Patients with contraindications for implantation surgery, such as cardiovascular disease, kidney disease, or uncontrolled periodontitis, were excluded. The STROBE guidelines were followed to ensure the rigor of our study (Table S1).
Cell culture
The primary culture method of human jawbone-derived BMSCs was based on a previous report from the research group.48 In short, implantation surgery was performed by a surgeon. Discarded bone fragments were collected during the preparation process of implantation surgery and stored in phosphate-buffered saline (Gibco, Grand Island, NY, USA) containing antibiotics (10 000 U/mL penicillin and 10 mg/mL streptomycin; Gibco). After centrifugation at 1 100 r/min, the bone fragments were resuspended in mesenchymal stem cell culture medium (MSCM; ScienCell, Carlsbad, CA, USA), inoculated into culture dishes, and cultured in a moist cell culture incubator containing 5% CO2 at 37 °C for 7 days without movement. A single-cell suspension was obtained using filters with a 70 μm pore size (Falcon, BD Labware, Wilmington, DE, USA) and then cultured in MSCM. The culture medium was changed every 3 days. Fourth-generation BSCMs were used in subsequent experiments. Primary cells obtained through low-speed drilling technology and cultured using the above methods exhibit BMSC characteristics.49 Human bone marrow-derived primary macrophages were purchased from Punosai Biotechnology Co., Ltd.
Osteogenic induction and detection of BMSCs
P4 BMSCs were seeded at a rate of 5 × 105 cells per well in a 6-well culture dish and cultured in osteogenic medium using the StemPro osteogenic differentiation kit (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions for 14 days. Osteogenic differentiation was induced after the cells reached 70%–80% confluence. According to the manufacturer’s instructions, on the seventh day after osteogenic induction, the intracellular ALP activity in BMSCs was evaluated using an ALP activity assay kit (Nanjing Jiancheng Institute of Biotechnology, China) and standardized based on protein concentration. The optical density (OD) was recorded at 520 nm. After 14 days of osteogenic induction, the cells were fixed in 70% ethanol and stained with 2% ARS (Sigma-Aldrich, St. Louis, MO, USA) for 5 min. Then, 1 ml of isopropanol was added to each well to dissolve the red perylene quinone derivative in the calcium nodules, and the OD value was measured at 550 nm using a specific gravity meter (5111970DPC, Thermo Fisher Scientific, Waltham, USA). For the blood glucose fluctuation group, culture medium containing 25 mmol/L or 5 mmol/L glucose was exchanged every 12 h.
Cell transfection
The Smpd3 knockdown lentivirus control and experimental lentivirus were fused with green fluorescent protein (GFP), namely, Scramsh and Smpd3 sh1-3, and the Smpd3 overexpression lentivirus control and experimental lentivirus, namely, vector and Smpd3, were purchased from Shanghai Jima Gene Biotechnology Co., Ltd. (Shanghai, China). These lentiviral constructs were stably transfected into BMSCs with a multiplicity of infection (MOI) of 50 using 5 μg/ml puromycin (Shanghai Jima Gene Biotechnology Co., Ltd.). After 18 h, the lentivirus medium was replaced with a fresh culture medium.
Western blotting
Cell lysis was performed using radioimmunoprecipitation analysis (RIPA) buffer (Sigma-Aldrich). The protein was separated by 15% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA) using a semidry transfer device (Bio-Rad). The membrane was sealed with 5% dehydrated milk in TBS-T (20 mmol/L Tris HCl, pH 7.6, 150 mmol/L NaCl, 0.05% Tween 20) for 1 h and then incubated with the primary antibody at 4 °C for 24 h. The primary antibodies used were anti-Smpd3 (A10197; 1:1 000, ABclonal), anti-Runx2 (A2851; 1:1 000, ABclonal), anti-ALP (ab307726; 1:1 000, Abcam), anti-OCN (ab133612; 1:1 000, Abcam), anti- β-actin (AC038; 1:10 000, ABclonal), anti-P62 (sc-28359; 1:1 000, Santa Cruz), anti-LC3A/LC3B (A5618; 1:1 000, ABclonal), anti-p-p38 MAPK (AP0526,1:1 000, ABclonal), anti-p38 MAPK (A14401; 1:1 000, ABclonal), anti-p-mTOR (AP0115; 1:1 000, ABclonal), anti-mTOR (A2445; 1:1 000, ABclonal), anti-GAPDH (AC002); 1:10 000, ABclonal, anti-CD63 (A19023; 1:1 000, ABclonal), anti-CD9 (A19027; 1:1 000, ABclonal), anti-β- tubulin (AC008; 1:1 000, ABclonal), and anti-histone H3 (A2348; 1:1 000, ABclonal). The secondary antibodies were HRP goat anti-rabbit IgG (AS014; 1:1 000, ABclonal) and HRP goat anti-mouse IgG (AS003; 1:1 000, ABclonal). The protein bands were visualized using Super Signal reagent (Bio-Rad).
Reverse transcription and real-time quantitative PCR
TRIzol (Invitrogen) was used for total RNA isolation, and first-strand cDNA was synthesized using a reverse transcription system (TaKaRa, Kusatsu, Japan). The Power SYBR Green PCR Master Mix (Roche, Basel, Switzerland) and 7500 real-time PCR detection systems (Applied Biosystems, Foster City, CA, USA) were used to quantify all gene transcripts using quantitative real-time PCR. The following are the thermal cycling conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The primers used are listed in Table S2. Data were analyzed using the 2−ΔΔCT method.
Immunofluorescence
After fixation and infiltration, BMSCs were incubated overnight with primary antibodies under humid conditions at 4 °C. After PBS washes, the corresponding secondary antibody was added to the primary antibody for 1 h. The primary antibodies used in this study are listed in Table S1. After PBS washes, the cells were incubated with DAPI for 10 min to delineate the nucleus. Immunofluorescence images were captured using a fluorescence microscope (Leica, Germany). The cells were fixed in PBS with 4% formaldehyde for 20 min. The cells were washed with 0.3% Triton X-100 (TX-100) in PBS three times and incubated with 5% BSA and 0.3% Triton 100 at RT for 1 h. Then, the cells were incubated in PBST for 24 h using the following primary antibodies: anti-LC3A/LC3B (A5618, 1:100, ABclonal), anti-iNOS (A3774, 1:100, ABclonal), anti-CD206 (24595, 1:100, Cell Signaling), Alexa Fluor 488-conjugated goat anti-rabbit (AS053, 1:200, ABclonal), and Alexa Fluor 594-conjugated goat anti-rabbit (AS039, 1:200, ABclonal). After three washes, the cells were incubated with secondary antibodies at RT for 1 h. DAPI was used for nuclear staining, and after three washes, the fluorescence quenching agent was removed, and a cover glass was added. The cell images were obtained using confocal microscopy (LSM 710; Zeiss, Leica, Germany).
Preparation of Exos-Smpd3@Ns
BMSCs were inoculated into T-75 culture bottles and passaged near confluence after trypsin digestion. The cells were incubated in MSCM containing 10% exosome-deficient FBS for another 3 days, and conditioned medium was collected, centrifuged at 3 000×g for 15 min, and passed through a 0.22 μm Sigma-Aldrich (USA) filtration unit to remove dead cells and debris. For concentration of the extracellular vesicles and remobilization of the proteins in the culture medium, the medium was centrifuged through an Ultra15 centrifugation filtration unit (100 kD, UFC910024) (Millipore, USA) according to the manufacturer’s protocol. Extracellular vesicles were directly used for downstream experiments. Next, we prepared a blood glucose fluctuation intelligent hydrogel delivery system, Exos-Smpd3@Ns. TSPBA-PVA, gelatin nanospheres, and silver nitrate, which were provided by Xi’an Qiyue Biotechnology Co., Ltd., were used to prepare phenylboronic acid-based polyvinyl alcohol crosslinkers using the method described in the literature.24 Specifically, a phenylboronic acid-based crosslinking agent, N1-(4-borobenzyl)-N3-(4-borophenyl)-N1, N1, N3, N3 tetramethylpropane-1,3-diamine (TSPBA), was synthesized by selecting PVA with a molecular weight of 145 kDa and reacting it with PVA. The phenylboronic acid (PBA) group of TSPBA can form a phenylboronic acid ester bond with the diol group of PVA. Moreover, a PBA/OH ratio of 0.5 was used, and silver nitrate was added. With a sodium borohydride reducing agent, silver nitrate was reduced to nanosilver ions, forming a complex nanosilver ion phenylboronic acid-based polyvinyl alcohol crosslinking agent (TSPBA-PVA), which contained a colloid volume fraction of 0.5 < φ < 0.64. Preparation of a high-concentration gelatin nanosphere suspension with a value of 0.64 was performed by direct mixing 1 µg/µl exosomes and TSPBA-PVA solution, yielding Exos-Smpd3@Ns.
Analysis of Exos-Smpd3@Ns
SEM (Zeiss, Sigma 300, Germany) was used to observe the microstructure of Exos-Smpd3@Ns. Specifically, Exos-Smpd3@Ns were frozen and fractured at −80 °C and then freeze-dried to remove moisture. The cross section of the hydrogel sample was then coated with gold to improve conductivity. NanoBrook 90plus PALS (Brookhaven, USA) was used to determine the particle size of extracellular vesicles and prepare dumbbell-shaped samples for tensile testing. The quantified fracture energy (kJ/m3) obtained by calculating the area under the tensile stress‒strain curve was used to characterize the work needed to break the sample per unit volume. The elastic modulus was defined as the initial slope (10%) value of the stress‒strain curve. Rheology and adhesion testing are described in the Supplementary Information. The self-healing behavior of the hydrogel was evaluated by tensile testing. The hydrogel sample was cut into two separate blocks and then brought into direct contact. After 1 min of contact, we evaluated the sample using a tensile test. The adhesive strength (n = 5) characterized by the lap shear stress of the hydrogel was recorded.
Detection of inflammatory factors
We used a TNF-α/IL-1β Quantikine ELISA kit (R&D, USA) to evaluate TNF-α and IL-1β. Analysis of 41 inflammatory factors in macrophages was performed using an AAH-INF-3 antibody membrane chip (Raybiotech, USA). All test kits were used according to the manufacturer’s instructions.
Animals and surgical procedures
The nude mouse experiment was approved by the Animal Care and Use Committee of Beijing Stomatological Hospital (KQYY-202211-007). The animal experiments on beagle dogs were approved by the Animal Care and Use Committee of Beijing Stomatological Hospital (KQYY-202211-007). This study complies with the guidelines of Animal Research: In vivo Experimental Reports of Preclinical Animal Research (Appendix), and approval was obtained. All animal experiments were conducted in accordance with institutional guidelines.
Forty-five-week-old male BALB/C homozygous nude mice (nu/nu) (SPF Biotechnology Co., Ltd., Beijing, China) were randomly divided into four groups with 10 mice in each group. All mice were treated with STZ (Sigma, USA) dissolved in citric acid trisodium citrate buffer solution (0.1 mol/L pH 4.5) to prepare a 1% injection. The sample was filtered by a 0.22 µm filter and intraperitoneally injected at a dose of 150 mg/kg, and a diabetic diet was given. Blood glucose levels were measured every 3 days after injection through the tail vein. When the average blood glucose level was greater than 16.7 mmol/L, the modeling standard was reached. Then, insulin was used for the blood glucose fluctuation group. Insulin (0.5 U/mL) was injected the next day, with a subcutaneous injection rate of 0.054 mL per 10 g. Blood glucose levels were measured for 2 h. When blood glucose significantly decreased, blood glucose fluctuations were considered to have occurred. In 1 month, the blood glucose dynamics in the blood glucose fluctuation group of nude mice were determined and are shown in Fig. 5g. The steps for transplanting nude mice were the same as described previously.49 In short, a single-cell suspension of P4 BMSCs was obtained after trypsin digestion, and the cell density was adjusted to 4 × 106 cells per mL. The cells were incubated with 40 mg of hydroxyapatite bone powder at 37 °C for 1 h and centrifuged at 150×g for 5 min at 37 °C. The processed cells and control cells were implanted into the subcutaneous space on the back of 5-week-old female BALB/C homozygous nude mice (nu/nu) (SPF Biotechnology Co., Ltd., Beijing, China), and the mice were placed in cages (6 per cage) in a controlled environment (20–25 °C, 40%–60% relative humidity). Animals were fed a sterile diet (SPF Biotechnology Co., Ltd.), and they could freely access water; each group was implanted in two symmetrical positions (n = 10).
Six 18-month-old male beagles (Beijing Fangyuanyuan Animal Farm) were used to establish three diabetic beagles. Each beagle had three loci on one side as the experimental group, so N = 9. In addition, three beagles with the same age, sex, weight and normal blood glucose were selected as the negative controls. The process of establishing diabetic dogs was as follows. First, all beagle dogs were fed high-fat and high-calorie feed (5 100 kcal/d: 53% fat, 19% protein, 28% carbohydrate, purchased from Beijing Huabu Biotechnology Company). STZ was injected, and the animals were fasted for 12 h before injection and allowed to drink water freely. Then, intravenous anesthesia with 3% pentobarbital sodium (1 mL/kg, Shanghai National Pharmaceutical Group Chemical Reagent Company) was administered. During the experiment, anesthesia was maintained by intravenous injection of 3% pentobarbital sodium (1 mL/kg). The total dosage of pentobarbital sodium used as a supplement should not exceed 40% of the standard dose (experimental dog weight * 1 mL/kg). After anesthesia, the animal was fixed on the operating table in a back position and disinfected with iodine and 75% alcohol. A sterile sheet was laid out, and 62.5 mg/mL of STZ citric acid solution was administered intravenously to the right saphenous vein of a beagle at a dose of 18.5 mg/kg for 2–3 min. After the injection, 0.9% normal saline was slowly intravenously administered for approximately 30 min. Fasting blood glucose was measured at 2, 4, 6, and 8 weeks after the injection. When the fasting blood glucose of the beagles was 7.0 mmol/L or the blood glucose at 2 h after a meal was >11.1 mmol/L, the diabetes model was successfully established. Then, 0.5 U/kg insulin was subcutaneously injected every other day, and blood glucose was measured 2 h later. When the blood glucose dropped significantly, the blood glucose was stated to have fluctuated. The dynamic changes in blood glucose in dogs within one month are shown in Fig. S6.50 Eight weeks before the jawbone defect, the first to fourth premolars on both sides of the beagle’s mandible were surgically extracted. The anesthesia method was the same as before, and attention was given to protecting the cheek, tongue, and lateral walls of the alveolar fossa. Teeth were carefully extracted without damaging the extraction socket. After tooth extraction, 0.1 mg/kg Metacam (PO; Boehringer Ingelheim Co., Ridgefield, CT, USA), 1 mg/kg ketorolac (Toradol 30 mg, Shanghai Roche Pharmaceuticals Co., Ltd., Shanghai, China), 1.7 mg/kg tramadol (Adolonta injectable, Grünenthal, Huayou Medical Group, Beijing, China), and 0.01 mg/kg buprenorphine (Buprex, Reckitt Benckiser Pharmaceuticals Limited, Berkshire, UK) were used to reduce pain. To prevent postoperative infection, we administered amoxicillin (20 mg/kg PO; The United Laboratories Co., Ltd., Hong Kong, China) for 6 days. Each dog was fed a liquid diet for 2 weeks, followed by a soft diet in a single cage. The extraction sites were allowed to heal for 8 weeks.51 In the next stage, following the standard procedure, with the same anesthesia and postoperative care methods as previously described, a drilling machine with a diameter and depth of 5 mm was used to drill holes in the mandible of a beagle dog using a minimally invasive method with water cooling. Three cylindrical bone defects with a diameter and depth of 5 mm were made on each side, and an injection was made on the side of the blood glucose fluctuation dog with 100 µl of Exos-Smpd3@Ns. On the other side, 100 µL of PBS was injected as a control, and the wound was sutured layer by layer (Fig. S7).
Micro-CT evaluation
Prior to mechanical and histological evaluations, a micro-CT system (Siemens, Germany, Invaeon; 80 kV, 500 μ) was used. The specimen was scanned with a 1 500 ms exposure time. The area of interest was selected, mainly a cylinder with a diameter of 5x5x5 mm3. The Tb. Th, Tb. N, Tb. Sp, BS/BV, and BV/TV were measured.
Organizational observation
Nude mouse replanted samples were decalcified in 10% ethylenediaminetetraacetic acid (EDTA; pH 7.4) for 8 weeks and then embedded in paraffin. The slices (5 μm thick) were stained with hematoxylin and eosin (H&E). Images were obtained using an optical microscope (Olympus BX43F, Osaka, Japan) and the accompanying Olympus DP72 digital camera imaging system. The bone defect samples of beagle dogs were fixed with 4% formalin, decalcified with 10% EDTA (pH 7.0), and dehydrated with a series of graded ethanol solutions and 100% acetone. Then, the sample was embedded in methyl methacrylate, and tissue slides (25) were prepared in the buccal lingual direction parallel to the bone defect axis using an EXAKT 400CS grinder (Leica, Wetzlar, Germany). Then, the slides were stained with toluidine blue and Goldner trichrome. Images were captured and analyzed using an optical microscope (Olympus BH2 with S Plan FL2 lens, Tokyo, Japan) and a computerized digital image analysis system (Leica Imaging system, Cambridge, England). Then, Tb. Th, Tb. N, Tb. Sp, BS/BV, and BV/TV were measured.
Statistical analysis
All statistical calculations were conducted using SPSS 16 statistical software (SPSS, Cary, North Carolina, USA). Statistical significance was determined through independent sample t tests or analysis of variance. A P-value less than 0.05 was considered statistically significant: *P < 0.05, **P < 0.01, **P < 0.001, ****P < 0.000 1.
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- Source: https://www.nature.com/articles/s41368-024-00328-6