Application of dental pulp stem cell-conditioned medium combined with deep crypreservation of autologous cranial flaps

Application of dental pulp stem cell-conditioned medium combined with deep crypreservation of autologous cranial flaps | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Application of dental pulp stem cell-conditioned medium combined with deep crypreservation of autologous cranial flaps Ye Liu, Yonghao Liu, Zhifei Ye, Xinxin Wang, Junyi Li, Peter L Mei, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5909455/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jun, 2025 Read the published version in Stem Cell Research & Therapy → Version 1 posted 5 You are reading this latest preprint version Abstract Background Autologous cranial flaps preservation after decompressive craniectomy (DC) is crucial for cranioplasty, yet standard cryopreservation carries high complication rates (15-35%), primarily infections and bone resorption. These complications frequently necessitate surgical revisions and increase morbidity risks. Current methods lack standardized preservation solutions that simultaneously ensure osteocyte survival and prevent microbial growth. Developing integrated bacteriostatic and osteoprotective storage media remains an urgent unmet need to enhance patient outcomes. Objectives This study investigates optimized preservation protocols for autologous cranial flaps to mitigate post-cranioplasty complications, while evaluating the preservative efficacy and clinical translation potential of dental pulp stem cell-conditioned medium (DPSC-CM) as a novel osteogenic storage solution. Methods Dental pulp stem cells (DPSCs) were cultured in serum-free medium to generate DPSC-CM. To evaluate preservation efficacy of DPSC-CM, first, DPSC-CM was preliminarily evaluated by examining the cell viability after freezing and resuscitation. Second, a murine critical-size calvarial defect model was surgically established. Autologous cranial flaps underwent 4-week storage in experimental preservation solutions (DPSC-CM versus conventional cryoprotectants) were reimplanted. Postoperative bone regeneration was systematically quantified through high-resolution micro-CT analysis and histomorphometric evaluation of bone regeneration capacity. Given DPSC-CM's osteopreservative potential, in vitro analyses confirmed DPSC-CM's osteogenic/angiogenic capacity through proliferation/migration assays, osteogenic differentiation, and biomarker quantification. Results DPSC-CM demonstrated superior efficacy in cell preservation. Studies in a mouse model of cranial defects showed that the cranial flaps preserved with DPSC-CM in combination with deep cryopreservation (-196°C) showed significantly better bone healing after cranioplasty than the other groups, and their neoangiogenic and anti-inflammatory abilities were also significantly better than those of the other groups. DPSC-CM was found to be superior to DPSCs in the osteogenesis of mouse embryonic osteoblast cells (MC3T3-E1 cells) and the angiogenesis of human umbilical vein endothelial cells (HUVECs). Conclusions Considering the superiority of osteogenesis and vascularization in vivo and in vitro, as well as the modulating of the local inflammatory microenvironment, DPSC-CM synergistic combination deep cryopreservation emerges as a novel strategy of preserving cranial flaps after DC. This multidisciplinary approach establishes a transformative framework for advancing autologous cranial flaps storage technologies, demonstrating translational promise through biological optimization of traditional cryopreservation protocols. Decompressive craniectomy Autologous cranial flaps Deep cryopreservation Dental pulp stem cells Dental pulp stem cell-conditioned medium Bone regeneration Vascular regeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Decompressive craniectomy (DC) serves as a critical neurosurgical intervention for intracranial pressure management in traumatic brain injury, osteopathic disorders, congenital cranial anomalies, and post-tumor resection scenarios 1 – 3 . During secondary reconstruction phases, cranial defect repair through cranioplasty utilizes either autologous cranial flaps or biocompatible synthetic materials 4 – 6 . While evolving reconstructive modalities now encompass allogeneic/xenogeneic grafts, tissue-engineered constructs, and synthetic biomaterials (e.g., polyetheretherketone, titanium alloys) 7 – 10 , longitudinal clinical evaluations consistently identify autologous bone transplantation as the gold-standard modality due to its inherent biocompatibility and structural congruence 11 . The clinical practice of autologous cranial flaps preservation post-DC emerged in the mid-20th century, with cryopreservation and subcutaneous pouch (SP) storage establishing themselves as standard preservation modalities 12 . While empirical cryoprotectant formulations (e.g., 0.9 percent NaCl solution (NS), dimethyl sulfoxide (DMSO), glycerol, and alcohol solutions) have been clinically trialed, their suboptimal biocompatibility profiles frequently manifest as postoperative complications including aseptic resorption and microbial colonization 13 , 14 . Notably, the absence of international consensus guidelines governing optimal preservation protocols remains a persistent challenge in neurosurgical practice. Recent studies have shown that paracrine effects of implanted mesenchymal stem cells can promote bone regeneration in vivo. And conditioned media containing most of the paracrine factors from cultured MSCs (mesenchymal stem cells-conditioned medium, MSC-CM) can induce tissue regeneration as efficiently as MSCs 15 , 16 . In addition, the use of MSC-CM has many advantages over MSCs themselves, including a lower risk of tumorigenesis, easier preservation and handling, and negligible immunogenicity, which reduces barriers associated with xenografts or xenotransplantation and preservation-related barriers 17 . Among MSCs subtypes, dental pulp stem cells not only have a wide range of sources and low immunogenicity but also exhibit significant regenerative advantages over bone marrow mesenchymal stem cells (BMMSCs), including: enhanced proliferative kinetics; marked osteoinductive capacity; dual angiogenic/immunomodulatory paracrine secretion profiles 18 – 21 . Studies have shown that dental pulp stem cell-conditioned medium (DPSC-CM) can stimulate the migration of endogenous osteocytes, promote osteoblast differentiation, stimulate angiogenesis, and ultimately achieve bone repair and regeneration 22 – 25 . It is inferred from previous research results that DPSC-CM may be an effective preservation solution for preserving cranial flaps. There is no research report on the use of mesenchymal stem cell-conditioned medium (MSC-CM) for the preservation of cranial flaps, so we used DPSC-CM as a cryopreservation solution for the preservation of autologous cranial flaps to investigate its effect on the regenerative capacity of bone after cranioplasty.In this study, we used NS, DMSO, α modified eagle's medium (α-MEM), and DPSC-CM to preserve mouse cranial flaps at -196℃, and a group of SP preservation as a clinical control, to compare their effects on bone regeneration and angiogenesis in a mouse cranial defect model after cranioplasty. The results showed that the cranial flaps preserved by DPSC-CM, after cranioplasty, was able to promote the rapid growth and migration of osteoblasts and blood vessels at cranial suture, while no complications such as bone resorption and infection were observed. In addition, we also validated the dual role of DPSC-CM bone regeneration and angiogenesis in in vitro experiments. We demonstrated that DPSC-CM can be used as a cryopreservation agent to preserve autologous cranial flaps, which can better promote bone healing, restore brain blood supply, and reduce complications such as infection and bone resorption after cranioplasty. 2. Materials and methods 2.1 Culture and characterization of DPSCs The dental pulp stem cells (DPSCs) were extracted and identified according to our previously established protocol. DPSCs were isolated from third molars obtained after routine surgery in healthy adolescent donors. With the approval of the Ethics Committee of Wuhan University People's Hospital and the informed consent of the donor (Approval Number: WDRY-2022-K025, Wuhan, China). DPSCs were isolated using a previously described method. Briefly, pulp tissue was briefly extracted from obstructed third molars, digested with 3 mg/mL of type I collagenase and 4 mg/mL of dispase for 30 min in an incubator, centrifuged at 1000 rpm for 5 min, and the pellet was resuspended and seeded into culture flasks containing α-MEM (Gibco, USA), which was supplemented with 20% fetal bovine serum (FBS; Gibco, USA) and a final concentration of 1% penicillin/streptomycin (Gibco, USA). The culture flasks were incubated at 37°C under 5% CO 2 . The medium was changed every three days until the stem cells reached 70% fusion. 2.2 Analysis of DPSC surface markers by flow cytometry Third-passage DPSCs were harvested at 70–80% confluence using 0.25% trypsin-EDTA solution (Gibco, USA), followed by centrifugation at 1000 ×g for 5 min. Cells were washed twice and resuspended in PBS supplemented with 2% FBS for subsequent staining. For immunophenotypic characterization, cell suspensions were incubated with fluorochrome-conjugated antibodies targeting human surface markers: CD44-FITC, CD73-PE, CD90-APC, CD105-PerCP (positive markers), CD31-FITC, and HLA-DR-PE (negative markers), along with isotype-matched controls (BD Biosciences, USA). All incubations were performed in the dark at 4°C for 30 min. After antibody staining, cells were washed and resuspended in 200 µL PBS for analysis. A minimum of 10,000 events per sample were acquired on a CytoFLEX LX flow cytometer (Beckman Coulter, USA), with fluorescence compensation adjusted using single-stained controls. Data analysis was performed using FlowJo v10.8 software (TreeStar Inc., USA), with gating strategies based on isotype control thresholds (< 1% background staining). 2.3 Multilineage differentiation of DPSCs To assess the ability of DPSCs to differentiate in multiple directions, third-generation DPSCs were differentiated into adipocytes, osteoblasts, or chondrocytes using adipogenic, osteogenic, or chondrogenic differentiation-inducing medium (OriCell, China) according to the manufacturer's instructions after reaching 80% fusion. For osteogenic differentiation, DPSCs were cultured in an osteogenic induction medium (OriCell, China), which was changed every 3 days for 21 days. To observe mineralization deposition, cells were fixed with 4% paraformaldehyde for staining with alizarin red S (ARS) solution. For adipogenic differentiation, DPSCs were cultured in an adipogenic induction medium (OriCell, China). After 21 days, they were stained with fresh oil red O solution. For chondrogenic differentiation, DPSCs were cultured in a complete chondrogenic differentiation induction medium (OriCell, China) for 21 days and then stained with alcian blue. 2.4 Conditioned medium (CM) preparation from DPSCs (DPSC-CM) For DPSC-CM preparation, cells were plated at a density of 5 × 10⁵ cells per 10 cm culture dish. Upon reaching 80–90% confluence, complete culture medium was replaced with serum-free medium to initiate CM production. Following 48-hour incubation, the conditioned supernatant was collected and subjected to sequential processing: initial centrifugation at 1,000 rpm for 5 min to remove cellular debris, followed by sterile filtration through a 0.22 µm membrane. The clarified CM was subsequently concentrated using centrifugal filtration devices (Millipore UFC900324, Germany) and cryopreserved at -80°C for downstream experimental applications in both in vitro and in vivo systems. 2.5 Characterization of DPSC-CM For transmission electron microscopy (TEM) analysis of DPSC-CM microstructure, 20 µL of DPSC-CM sample was deposited onto carbon-coated copper grids and allowed to adsorb for 3–5 minutes. Excess liquid was carefully removed by filter paper blotting, followed by air-drying at room temperature for 10 minutes. The grids were then washed with DPBS and negatively stained with 2% (w/v) uranyl oxalate for 1–2 minutes, after which excess stain was blotted and the grids were air-dried again. Samples were imaged with TEM (HITACHI, HT7700, Japan). Particle size distribution was determined using a Nanoparticle tracking analysis (NTA) instrument (Zetasizer Nano ZS, Malvern Panalytical, UK). 2.6 Cell culture and osteogenic differentiation of mouse embryonic osteoblasts cells (MC3T3-E1 cells) For osteogenic differentiation, cells were seeded in 6-well plates at a density of 1 × 10⁵ cells/well in complete growth medium. Upon reaching 70% confluence, the culture medium was replaced with osteogenic induction medium consisting of: α-MEM base supplemented with 10% FBS; 50 µg/mL ascorbic acid (Merck, USA); 4 mM β-glycerophosphate (Merck, USA).Cells were maintained in this differentiation cocktail for 14 days with medium changes every third day to ensure consistent nutrient availability and metabolic waste removal. 2.7 Cell freezing and resuscitation Cell freezing and resuscitation experiments were performed using MC3T3-E1 cells and human umbilical vein endothelial cells (HUVECs), both of which were purchased from Pricella Biotechnology Company(Procell, China). Three cryopreservation groups were established: 1. DMSO-only group: 100%DMSO; 2. DMSO + FBS group: 10% DMSO + 90% FBS; 3. DMSO + DPSC-CM group: 10% DMSO + 90% DPSC-CM. An equal number of cells (1×10⁵ cells/cryopreservation tube) were cryopreserved using a gradient freezing protocol (-1°C/min) at -80°C, and cell recovery was initiated 24 hours later. To allow cells to fully adhere and spread, cell digestion and counting were performed 24 hours post-thaw. 2.8 Ki67 fluorescent staining assay Resuscitated cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature and then rinsed with PBS (3 × 5 min). 0.1% Triton X-100 was osmotically stabilized in PBS for 10 min (room temperature). Treat with blocking buffer (5% normal goat serum + 1% BSA, PBS) for 1 hour at room temperature to reduce nonspecific binding. Primary antibody incubation: Incubate with anti-Ki-67 antibody (1:200, ABclone, China) at 4°C overnight. Rinse with PBS (3 × 5 min). Add ABflo® 488-conjugated Goat anti-Rabbit IgG (1:200, ABclone, China) and incubate for 1 hour at room temperature away from light. The nuclei were stained with DAPI (1:1000, Beyotime, China). The stained sections were observed using a microscope (IX71, Olympus, Japan) and photographed at 100× magnification. 2.9 Animal and moral statement Male C57BL/6 mice (8-week-old, 30 ± 2 g body weight) were used in this study, and random allocation into 5 experimental groups (n = 6/group). The experiments were conducted in the animal house of Wuhan University People's Hospital, and the mice were housed in separate cages at a temperature of 25°C ± 2°C, relative humidity of 50% ± 15%, and a light/dark cycle of 12 h. All mice were allowed to drink and eat freely. All animal studies complied with the Wuhan University Guidelines for the Care and Use of Laboratory Animals (Institutional Animal Care and Use Committee No. 20231102B) and internationally recognized principles. The work has been reported in line with the ARRIVE guidelines 2.0. 2.10 Surgery and treatment All surgeries were performed under isoflurane (4%) anesthesia. In the cranial defect model, mice were anesthetized using isoflurane (4%) by inhalation, and a median cranial defect of 5 mm in diameter was constructed using an electric dental drill at a low drilling speed under continuous saline perfusion. The harvested cranial flaps were preserved in cryoprotective solutions (1 mL per tube) containing respective components: 0.9 percent NaCl solution (NS), dimethyl sulfoxide (DMSO), α-MEM, or DPSC-CM, and the cryopreservation tubes were stored in liquid nitrogen at -196°C in a gradient freezing mode. In the subcutaneous preservation (SP) group, a 1.5 cm longitudinal incision was surgically created in the femoral region of C57BL/6 mice under aseptic conditions, followed by meticulous dissection to establish a lateral subcutaneous pocket for cranial flaps implantation. Postoperative protocols included intramuscular penicillin prophylaxis and topical povidone-iodine (10%) wound disinfection for 3 days. After 4 weeks of cranial flaps preservation, the cryopreservation tubes containing the cranial flaps were removed from liquid nitrogen, warmed to room temperature in a gradient, and the flap was implanted in situ in the defect site. The wound was closed in layers (periosteum, skin) by absorbable sutures. Postoperative care included analgesia with buprenorphine and antibiotic treatment with penicillin. After surgery, the animals were kept individually under constant conditions. No animal deaths were noted during or after surgery. According to the American Veterinary Medical Association (AVMA) 2020 edition of Guidelines for Euthanasia of Animals, animals were euthanized at weeks 4 and 8 after implantation of the cranial flaps, and all animals were euthanized using carbon dioxide gas. Regenerated tissue was harvested from the defect area for further evaluation. 2.11 Micro-CT analysis of mouse cranial flaps Following in situ cranial flaps implantation, samples were scanned using a Skyscan 1176 micro-computed tomography scanner (Bruker microCT, Germany) at a resolution of 6.5 µm at weeks 0, 2, 4, and 8. Raw data were reconstructed using NRecon software (Bruker, Germany), followed by 3D model generation with CTVox (Bruker, Germany). For quantitative analysis, a cylindrical volume of interest (VOI; 5 mm diameter × 1 mm height) was aligned with the original defect boundary using CTAn software (Bruker, Germany). Key parameters calculated included: Bone volume (BV), bone surface area (BS), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and bone mineral density (BMD). 2.12 Western blot The co-cultured MC3T3-E1 cells and HUVECs were lysed in lysis buffer (Beyotime, China) with a mixture of protease and phosphatase inhibitors (MCE, USA). Following in situ cranial flaps implantation, animals were euthanized at 4 and 8 weeks, and fresh bone tissue samples were homogenized in lysis buffer (Beyotime, China) containing a protease/phosphatase inhibitor cocktail (HY-K0013, MCE, USA) using a 3D cryo-mill (Servicebio, China) for 45 s at high speed to extract total proteins, whose concentrations were subsequently quantified with the Enhanced BCA Protein Detection Kit (Thermo, USA). A total of 10 µg of protein from each sample was electrophoresed in SDS-PAGE gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Germany), which were blocked with 5% nonfat milk and incubated with primary antibodies at 4°C overnight. The primary antibodies used to detect protein expression were runt-related transcription factor 2 (RUNX2) (1:500, Proteintech, China), osteocalcin (OCN) (1:500, ABclone, China), platelet endothelial cell adhesion molecule-1 (CD31) (1:500, Proteintech, China), tumor necrosis factor-α (TNF-α) (1:500, Proteintech, China), interleukin 6 (IL-6) (1:500, Proteintech) and interleukin 10 (IL-10) (1:500, Proteintech, China). After washing, membranes were incubated with HRP-conjugated goat anti-rabbit or mouse IgG secondary antibodies (1:1000, Proteintech, China) for 1 h at 37°C, followed by protein visualization using an Enhanced Chemiluminescence Kit (Biology, China). Relative protein expression levels were quantified via densitometric analysis of immunoreactive bands using ImageJ software. 2.13 Histological analysis for bone regeneration Histological evaluation of bone regeneration was performed through hematoxylin and eosin (H&E) and Masson trichrome staining, wherein cranial specimens harvested at 4 and 8 weeks post-cranioplasty were fixed in 4% paraformaldehyde, decalcified in 0.5 M EDTA for 8 weeks at room temperature, and paraffin-embedded before being sectioned into 5 µm slices using a tissue microtome. Deparaffinized sections underwent sequential xylene immersion (Shanghai Huawei Pharmaceutical Co., Ltd., China) for 15 min and ethanol gradient dehydration (anhydrous, 90%, and 75% ethanol, 10 min each), followed by H&E staining (Beyotime, China) to evaluate bone architecture and pathological alterations, and Masson trichrome staining (Reckitt Benckiser This Biotech, China) to visualize collagen fiber distribution in regenerated bone. Stained sections were imaged under a light microscope (Olympus, Japan) for qualitative assessment of osteogenic activity and matrix remodeling. 2.14 Immunofluorescent staining After being dewaxed and dewatered, the prepared cranial sections (5 µm) were softened by incubation with 0.1% Triton X-100 for 15 min and blocked with 3% bovine serum albumin for 30 min. Sections were then incubated with primary antibodies RUNX2 (1:500, Proteintech, China), OCN (1:500, Proteintech, China), and CD31 (1:500, Proteintech, China) overnight at 4°C, followed by incubation with secondary antibodies for 1 h at room temperature. The nuclei were stained with DAPI (1:1000, Beyotime, China). The stained sections were observed using a microscope (Olympus, Japan) and photographed at 100× magnification. 2.15 Cell co-culture A Transwell co-culture system (0.4 µm pore membrane; BD Biosciences, Franklin Lakes, NJ, USA) was employed to investigate cell-cell interactions. The experimental setup included: Lower chamber (6-well plate): MC3T3-E1 cells seeded at 1 × 10⁶ cells/well in α-MEM medium. Upper chamber (3 experimental groups): Control (CON) group: 2 mL α-MEM basal medium; DPSCs group: 1 × 10⁵ DPSCs in 2 mL α-MEM; DPSC-CM group: 2 mL DPSC-CM. The co-culture system was maintained for 14 days under standard conditions (37°C, 5% CO₂), with medium replacement every 3 days to ensure metabolic homeostasis. 2.16 Cell viability assay MC3T3-E1 cell viability was quantified using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). The MC3T3-E1 cells treated with α-MEM or DPSCs or DPSC-CM were seeded into 96-well plates at 1 × 10⁴ cells/well and cultured under standard conditions (37°C, 5% CO₂). Cellular metabolic activity was evaluated at five timepoints (0, 12, 24, 36, and 48 hours post-seeding) by adding 10 µL CCK-8 reagent to each well, followed by 1-hour incubation. Absorbance was measured at 450 nm using a microplate reader (Molecular Devices, USA). 2.17 Wound-healing and transwell assay In order to detect the effects of DPSCs and DPSC-CM on the migration ability of MC3T3-E1 cells, we performed wound-healing and transwell experiments. In the wound-healing experiment, MC3T3-E1 cells were seeded into six-well plates and cultured to a density of 90%; the original medium was discarded, and the cells were starved in serum-free medium for 12 h. A scratch wound was created in the middle of each well with a sterile 200 µL pipette tip, and the cellular debris was washed away with PBS, and then the cells were co-culture with α-MEM or DPSCs or DPSC-CM, respectively, for 24 h. Using an inverted microscope (Olympus, Japan) to take pictures at 0/12/24h, respectively, to monitor the migration rate of cells to the cell-free area. The area of cells migrating into the scratched area was calculated using ImageJ analysis software for quantitative analysis. The specific formula was: cell migration rate = (0 h scratch area − 12 h scratch area)/0 h scratch area. This procedure was repeated three times for each set of samples. Cell migration capacity was assessed using 24-well Transwell chambers with 6.5 µm pore membranes (Corning, NY, USA). MC3T3-E1 cells (1 × 10⁵ cells/mL in 100 µL serum-free α-MEM) were seeded in the upper chamber, while the lower chamber contained 600 µL of either α-MEM complete medium or DPSC-cultured α-MEM or DPSC-CM. After 24 h incubation under standard conditions (37°C, 5% CO₂), the transwell chamber were fixed with 4% paraformaldehyde (Merck, USA) for 15 min and stained with 0.1% crystal violet solution for 30 min. The stained cells were photographed using an inverted microscope (Olympus, Japan). Subsequently, the number of migrated cells was calculated using ImageJ software. 2.18 Tube formation, wound-healing and transwell assay Angiogenesis is essential for bone regeneration, so in order to detect the effects of DPSCs and DPSC-CM on the migration ability of HUVECs, We performed wound-healing and transwell assay experiments. The steps were consistent with the wound-healing and transwell assay experiments described above. To compare the in vitro angiogenic ability of each group, a tubule formation assay was performed. Matrigel (BD Biosciences, USA) was dissolved in a refrigerator at 4°C overnight, and Matrigel was rapidly added to 48-well plates in a volume of 100 µL per well and left at 37°C for 30 min to form a gel. HUVECs were seeded on Matrigel at a density of 2 × 10 4 cells/well, and HUVECs were co-cultured with α-MEM or DPSCs or DPSC-CM for 6 hours. The cells were imaged using phase contrast microscopy (Leica, German) to assess endothelial cell tubule formation. The total length of tubules, number of junctions, and reticulation were calculated using ImageJ. 2.19 Alkaline phosphatase (ALP) staining and activity assay To evaluate DPSCs/DPSC-CM effects on osteogenic differentiation, MC3T3-E1 cells were analyzed through ALP histochemical staining and enzymatic activity quantification. MC3T3-E1 cells were cultured in 6-well plates at a density of 2 × 10 5 cells/well, and MC3T3-E1 cells were incubated in the differentiation process with α-MEM or DPSCs or DPSC-CM and incubated at 37°C under 5% CO 2 , and the differentiation medium was changed every three days. After 14 days of culture, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature, rinsed with PBS, and stained for ALP using the BCIP/NBT Alkaline Phosphatase Chromogenic Kit (Beyotime, China). To determine ALP activity, proteins were extracted after treatment of MC3T3-E1 cells as described above. ALP activity was measured using an Alkaline Phosphatase Assay Kit (Beyotime, China) according to the manufacturer's instructions. The absorbance of alkaline phosphatase activity was measured at 405 nm using a microplate reader. 2.20 Alizarin Red S (ARS) staining and quantification for mineralization measurement To evaluate extracellular matrix mineralization, MC3T3-E1 cells (2 × 10⁵ cells/well) were co-cultured with α-MEM or DPSCs or DPSC-CM under osteogenic conditions for 14 days. MC3T3-E1 cells were rinsed with PBS and fixed with 4% paraformaldehyde for 30 min. ARS (1 mL) (Beyotime, China) was added to the culture wells and incubated for 30 min at room temperature, then rinsed 5 times with distilled water to remove excess dye. Images of the stained cells were taken under an inverted fluorescence microscope (Olympus, Japan). For quantitative analysis, the stained cells were lysed with 10% cetylpyridinium chloride (Solarbio, China) for 1 h at room temperature, then transferred to a 96-well plate and quantified by measuring the absorbance at 570 nm with a microplate reader (Biotek Instruments Inc., USA). 2.21 Statistical analysis Data from three independent experiments are presented as mean ± standard deviation (SD). The significance between the two groups was analyzed using two-tailed Student's t-test or one-way analysis of variance (ANOVA). For multiple comparisons, Tukey post-hoc tests were used. All experimental data were statistically analyzed using GraphPad Prism (9.5.1) software. p < 0.05 was considered significant; *p < 0.05, **p < 0.01, ***p < 0.001. 3. Results 3.1 Characterization of dental pulp stem cells (DPSCs) and dental pulp stem cell-conditioned medium (DPSC-CM) Systematic characterization confirmed that dental pulp stem cells (DPSCs) retained typical morphological features (spindle-shaped fibroblast-like morphology) (Figure S1A), MSCs-specific surface markers (CD44/CD73/CD90/CD105) (Figure S1C), and trilineage differentiation potential (osteogenic/chondrogenic/adipogenic) (Figure S1B), ensuring homogenization of cell extraction. Numerous studies have demonstrated the significant advantages of mesenchymal stem cells-conditioned medium (MSC-CM) in bone repair and regeneration 26-28 . To further explore the therapeutic potential of MSC-CM, we selected dental pulp stem cells (DPSCs) – a widely accessible cell source with minimal ethical concerns – to prepare dental pulp stem cell-conditioned medium (DPSC-CM). Chouaib et al. previously characterized DPSC-CM composition using human growth factor antibody arrays, revealing that DPSC-CM contains a complex mixture of bioactive factors responsible for its biological effects 29 . In our study, transmission electron microscopy (TEM) analysis identified abundant extracellular vesicles within DPSC-CM (Figure 1A). Nanoparticle tracking analysis (NTA) further demonstrated that these vesicles exhibited a size distribution ranging from 58 nm to 290 nm, with an average diameter of approximately 79 nm (Figure 1B) – characteristics consistent with exosome. These findings suggest that extracellular vesicles and growth factors in DPSC-CM may serve as critical mediators of bone repair and regeneration. Based on this evidence, we subsequently investigated the potential preserving capacity of DPSC-CM. 3.2 DPSC-CM can reduce mechanical damage caused by ice crystals during cell cryopreservation and provide nutritional support Dimethyl sulfoxide (DMSO) is widely recognized as a potent cryoprotectant in cell preservation, functioning by permeating cell membranes to reduce intracellular water freezing points and inhibit ice crystal formation during cryopreservation. However, its cytotoxicity at concentrations exceeding 10% necessitates cautious formulation design 30,31 . Standard cryopreservation protocols typically combine 10% DMSO with either a mixture of basal medium (40-70%) and fetal bovine serum (FBS, 20-50%) or 90% FBS alone. The basal medium supplies essential nutrients and pH buffering, while FBS acts as an extracellular protective matrix and metabolic support through its rich repertoire of growth factors and proteins 32 . To validate the preservation and nutritional support effects of DPSC-CM on cells, we conducted cell experiments by replacing serum with DPSC-CM for cell preservation and observed the post-thaw cell state and proliferation efficiency. In the context of bone repair and regeneration, bone regeneration and vascular network repair are particularly critical. Therefore, we selected two cell types for cryopreservation experiments: mouse embryonic osteoblast cells (MC3T3-E1 cells) and human umbilical vein endothelial cells (HUVECs). It was found that the DMSO + DPSC-CM group exhibited a higher cell count compared to the DMSO-only and DMSO + FBS groups, with statistical significance. CCK-8 assay results demonstrated that the proliferation rates of both MC3T3-E1 cells and HUVECs in the DMSO + DPSC-CM group were faster than those in the other two groups, with significant differences (P＜0.05) (Figure 1C-D). Additionally, Ki67 fluorescence staining was performed, and the fluorescence statistical data showed similar results(Figure 1E-H). These experimental findings indicate that DPSC-CM can replace serum by providing extracellular molecular barriers and nutritional support, reducing mechanical damage and metabolic stress on cells, making it an excellent alternative cryopreservation solution. 3.3 Cranial flaps preserved by DPSC-CM have greater bone repair capacity Based on the in vitro findings, we applied DPSC-CM to the cryopreservation of calvarial cranial flaps to validate its efficacy as a cryoprotectant solution. In this section, we applied DPSC-CM as a bone preservation fluid in the deep cryopreservation of mouse cranial flaps. We established a mouse cranial defect model in which surgically excised cranial flaps were preserved in designated solutions. The experimental setup included clinically standard solutions: 0.9% sodium chloride (normal saline, NS), dimethyl sulfoxide (DMSO), and subcutaneous pouch preservation. DPSC-CM served as the experimental group. Additionally, an α-MEM control group was established to account for potential confounding effects of culture medium nutrients on bone preservation. Subsequently reimplanted via cranioplasty after 4 weeks (Figure 2A-B). Longitudinal micro-CT imaging and analysis at 0, 2, 4, and 8 weeks post-cranioplasty revealed distinct outcomes across groups. Figure 2C displays 3D reconstructed models of cranial flaps at 4 and 8 weeks, demonstrating significant bone resorption in the NS and SP groups, while the remaining three groups exhibited favorable healing outcomes. However, due to incomplete fixation of the bone flaps during cranioplasty, minor displacement was observed in all groups. Longitudinal micro-CT quantitative analysis demonstrated that, with the accumulation of time, bone surface (BS), bone volume (BV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) gradually increased compared to baseline measurements on postoperative day 1, while trabecular separation (Tb.Sp) decreased (Figure 2E). Notably, the DPSC-CM group demonstrated superior osteogenic outcomes, with significantly elevated BS, BV, Tb.N, and Tb.Th values (p < 0.05) and reduced Tb.Sp compared to control groups at 2-, 4-, and 8-week intervals. These morphological improvements were paralleled by enhanced bone mineral density (BMD) in the DPSC-CM treatment group (Figure 2D). Collectively, these findings indicate that calvarial cranial flaps cryopreserved with DPSC-CM exhibit accelerated and sustained maturation of cranial sutures following cranioplasty. To systematically evaluate the osteogenesis of cranial sutures, histological analyses were conducted at 4 and 8 weeks postoperatively. H&E staining revealed enhanced osteogenic activity in the DPSC-CM group, characterized by dense lamellar bone formation within connective tissue bridging the defect margins, with no evidence of bone resorption. In contrast, the DMSO and α-MEM groups exhibited localized resorption at cranial flap centers, while severe resorption was observed in NS and SP groups, including cranial flap degradation in some specimens (Figure 3A). Masson trichrome staining showed that the cranial flaps preserved with DPSC-CM formed a large amount of bone matrix and collagen with strong continuity at suture of the cranial flaps, whereas the other groups showed only a small amount of collagen fibers (Figure 3B). Notably, DPSC-CM-treated specimens displayed progressive osteogenic enhancement from week 4 to 8, evidenced by increased osteoblast density on H&E staining and expanded bone matrix and collagen production on trichrome staining (Figure 3A-B). 3.4 DPSC-CM promotes bone and vascular regeneration-related proteins expression and modulates the inflammatory microenvironment To substantiate the osteopreservative effects of DPSC-CM at the molecular level, we performed protein-level validation of its biological activity. Total protein extracts from fresh bone tissues harvested at 4 and 8 weeks post-cranioplasty underwent immunoblotting analysis. DPSC-CM-preserved cranial flaps demonstrated significantly elevated expression of osteogenic regulators RUNX2 and OCN, with quantitative densitometry revealing 2.3- and 3.1-fold increases respectively compared to other preservation groups at 4-week (Figure 4A-B). Concurrently, enhanced neovascularization was evidenced by upregulated CD31 expression (1.8-fold increase vs α-MEM groups, p＜0.05), indicating superior angiogenic potential relative to comparator groups. Protein expression profiles in the DPSC-CM group at week 8 exhibited comparable results, maintaining consistent molecular signatures with previous observations (Figure 4E-F). Notably, DPSC-CM treatment both exerted dual regenerative-immunomodulatory effects at week 4 and week 8, as reflected by: (1) reductions in pro-inflammatory mediators TNF-α and IL-6; (2) elevation of anti-inflammatory IL-10 (Figure 4C-D,G-H). This coordinated modulation of osteogenesis, vascularization, and inflammatory microenvironment underscores DPSC-CM's multifaceted therapeutic profile as a preservation solution for cranial flaps in cranial reconstruction. In addition, we also performed immunofluorescence staining on bone tissue sections from different groups at weeks 8 to evaluate the expression levels of important proteins in the bone formation process. In both NS and SP groups, substantial bone resorption following reimplantation resulted in incomplete visualization of the grafted cranial flaps, accompanied by markedly reduced expression of osteogenic markers (RUNX2 and OCN) and angiogenic marker CD31 (Figure 5). Multiplex immunofluorescence analysis revealed that DPSC-CM-preserved cranial flaps significantly enhanced the expression of osteogenic regulators (OCN and RUNX2) at cranial sutures, concomitant with upregulated CD31 expression, indicating robust neovascularization (Figure 5). These findings were fully consistent with complementary western blot data from both in vivo and in vitro experiments, thereby validating the reliability of our observations across multiple analytical platforms. 3.5 DPSC-CM promotes the proliferation and osteogenic differentiation of MC3T3-E1 cells Given the notable preservation effects of DPSC-CM observed in vivo, we sought to investigate its intrinsic regenerative potential in bone repair and angiogenesis. This premise prompted subsequent cellular-level investigations to systematically characterize DPSC-CM's mechanistic contributions to these biological processes. Research indicates that promoting bone repair and regeneration in vivo primarily involves enhancing the homing and proliferation of osteoblasts 33 . Therefore, we selected mouse embryonic osteoblast cells (MC3T3-E1 cells) to investigate the role of DPSC-CM in promoting cell proliferation and osteogenesis.To evaluate the effects of DPSC-CM on MC3T3-E1 cell proliferation and migration, we conducted CCK-8, wound-healing, and transwell assays. And we used untreated cells as a negative control and cells co-cultured with DPSCs as a positive control.CCK-8 analysis demonstrated that DPSC-CM co-culture induced a significant enhancement in MC3T3-E1 cell viability relative to both control and DPSCs-treated groups (Figure 6A). Migration assessments revealed that while DPSCs and DPSC-CM both stimulated directional movement of MC3T3-E1 cells, DPSC-CM exhibited superior migratory induction capacity. Quantitative analysis of wound closure rates (Figure 6C, F) and transwell membrane penetration (Figure 6D, G) confirmed statistically greater migration potential in DPSC-CM-treated cells compared to DPSCs-co-cultured counterparts (p<0.01). To investigate the osteogenic regulatory effects of DPSC-CM on MC3T3-E1 cells, we performed systematic biochemical characterization. Western blot analysis revealed significantly elevated expression levels of key osteogenic transcription factors RUNX2 and matrix protein OCN in DPSC-CM-treated cells compared to controls (Figure 6B, E). Subsequent evaluation of early osteogenic differentiation through ALP activity assays demonstrated that DPSC-CM exposure induced pronounced alkaline phosphatase activation. Quantitative analysis of ALP staining intensity showed statistically significant differences (p<0.01) between DPSC-CM-treated cells and both CON and DPSCs groups (Figure 6H, J), with enzymatic activity measurements corroborating these findings. To assess terminal differentiation capacity, mineralization potential was quantified using ARS staining. DPSC-CM-treated specimens exhibited markedly enhanced calcium deposition compared to CON and DPSCs groups, with quantitative spectrophotometric analysis confirming 4.2-fold and 1.3-fold increases in mineralization respectively (Figure 6I, K). This multi-parametric evaluation consistently demonstrated that DPSC-CM not only potentiates osteogenic differentiation of MC3T3-E1 cells but surprisingly exhibits mechanistically superior osteoinductive properties compared to direct DPSCs co-culture. 3. 6 DPSC-CM exhibits potent pro-angiogenic activity To investigate the angiogenic regulatory potential of DPSC-CM, we established a co-culture system with HUVECs to evaluate migratory and angiogenic capacities. Transwell assays demonstrated that both DPSCs and DPSC-CM stimulated HUVECs migration (Figure 7A, E), with pronounced cellular infiltration into lower chambers (Figure 7B, F). Notably, DPSC-CM-treated HUVECs exhibited statistically significant enhancement in migratory capacity compared to both CON and DPSCs groups (p＜ 0.001). Subsequently, angiogenic potential was assessed through tubulogenesis assays. DPSC-CM-treated HUVECs developed more complex tubular networks compared to controls (Figure 7C), with quantitative analysis revealing significant increases in total vascular structures, evidenced by elevated numbers of tubular segments and branching points relative to CON and DPSCs groups (Figure 7H, I). Furthermore, western blot analysis confirmed upregulated expression of CD31, a key endothelial junction marker, in DPSC-CM-exposed HUVECs (Figure 7D, G). These findings collectively demonstrate that DPSC-CM enhances both migratory competence and angiogenic functionality in endothelial cells. 4. Discussion Cranioplasty involving autologous cranial flaps transplantation or synthetic substitutes is critical for reestablishing physiological cerebrospinal fluid dynamics and restoring cerebral perfusion following decompressive craniectomy (DC) 34 , 35 . While autologous cranial flaps transplantation remains the clinical gold standard for cranial reconstruction 11 , the absence of standardized preservation protocols presents a significant clinical challenge. The standards for cryopreservation of cranial flaps may be summarized as follows: They must ensure non-toxicity (e.g., DMSO ≤ 10%), sterility, low endotoxin levels, programmed freezing (1°C/min to -150°C), and verification of cell viability ≥ 70% and bone structural integrity. The current preservation strategies—including cryopreservation using saline, DMSO, glycerol, or ethanol solutions, as well as subcutaneous storage—all have their respective drawbacks. For instance, DMSO exhibits cytotoxic effects on bone cells, ethanol solution storage leads to loss of bone cell viability, and subcutaneous implantation of bone flaps requires additional surgical procedures along with associated pain, often accompanied by bone resorption during preservation. Moreover, these methods frequently result in postoperative complications following cranioplasty, such as microbial colonization, vascular injury, peri-implant fibrosis, chronic pain, impaired osseointegration, and progressive bone resorption 36 – 38 . These limitations underscore the urgent need for optimized bone preservation methodologies in neurosurgical practice. Contemporary developments in regenerative medicine have validated the bone regenerative capacity of MSCs secretomes across multiple tissue origins. DPSCs emerge as particularly advantageous MSC sources given their abundant availability, minimally invasive harvest procedures, and immunologically favorable characteristics 39 . The DPSC-CM comprises various bioactive components - including signaling proteins, extracellular vesicles, and genetic regulators - that synergistically mediate tissue repair through immunomodulation and regeneration 20 , 40 . Our transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) of DPSC-CM further confirmed the presence of abundant vesicles with particle sizes consistent with exosomes. These distinctive properties position DPSC-CM as a promising candidate for cranial flaps preservation. Given these results, we employed a combination of DMSO and DPSC-CM for cryopreservation and subsequent revival of osteoblasts (MC3T3-E1) and HUVECs, preliminarily validating its protective effects at the cellular level. Post-thaw observations revealed that the DMSO + DPSC-CM group exhibited significantly accelerated cell proliferation rates. This suggests that DPSC-CM can functionally replace serum by providing essential nutrients (e.g., proteins and growth factors) to cryopreserved cells, thereby supporting viability and mitigating freeze-thaw stress.It has been reported that DPSC-CM contains several growth factors, and the biological effects of DPSC-CM in promoting bone regeneration have been investigated 41 , 42 . Chouaib et al. identified 16 distinct growth factors in DPSC-CM through human growth factor antibody array analysis, all of which exhibited expression levels above the detection limit. Surprisingly, the highly expressed bone morphogenetic proteins (BMP-4, BMP-5, and BMP-7), which belong to the transforming growth factor beta family, were found to play crucial roles in odontogenesis, osteogenesis, and chondrogenesis, as well as in fracture repair and osteoblast differentiation 29 .And in vivo, Ogata et al. observed the migration of rat MSCs and endothelial cells to the site of cranial defects using in vivo imaging methods and showed that MSC-CM induced early bone regeneration by accelerating the migration of endogenous MSCs 43 . Building upon these findings, we propose that these growth factors may critically contribute to the deep cryopreservation of cranial bone flaps. To test this hypothesis, we employed DPSC-CM as a preservation medium for cranial flaps, and to compare it with the existing 0.9 percent NaCl solution (NS), dimethyl sulfoxide (DMSO) and subcutaneous preservation (SP) in clinical practice, investigating its capacity to maintain the viability and structural integrity of autologous cranial flaps during deep cryopreservation (The α-MEM group served as an experimental control to account for potential confounding effects of culture medium nutrients on bone preservation). After 4 weeks of deep cryopreservation in designated preservation solutions, the calvarial bone flaps underwent cranioplasty. Micro-CT 3D reconstruction analysis of postoperative outcomes demonstrated successful osseointegration in the majority of cryopreserved autologous cranial flaps. These autologous cranial flaps showed extensive healing tissue formation at week 4, followed by moderate bone healing at week 8. DPSC-CM-preserved cranial flaps induced extensive bone healing, as evidenced by elevated CT indices such as BS, BV, and BMD. In contrast, some degree of bone resorption, deformation, and fracture occurred in the subcutaneously preserved and 0.9 percent NaCl solution-preserved groups. In fact, significant bone formation was observed in vivo at an early stage in the DPSC-CM group (about a 1.2-fold higher osteogenic ratio at week 2 compared to the other groups). Interestingly, in histological analysis, the DPSC-CM group had significantly more newly generated osteoblasts (blue) and mature osteocytes (red) at the bone defect site than the other groups, and a certain number of bone unit structures appeared. Using immunofluorescence staining of histological analysis, RUNX2 and OCN showed elevated expression at the bone defects, which was significantly different from other groups. Extensive research has established that DPSC-CM enhances cellular proliferation and migration, modulates endothelial behavior to stimulate vascular-like structure formation, and upregulates angiogenic gene expression 44 – 46 . Consistent with these findings, our immunohistochemical analysis of DPSC-CM-treated bone sections revealed CD31-positive vascular endothelial cells and neo-vasculature surrounding regenerated bone. Notably, DPSC-CM demonstrates multifaceted therapeutic potential, exhibiting anti-inflammatory properties evidenced by its capacity to mitigate diabetic polyneuropathy and aneurysmal subarachnoid hemorrhage-induced neuroinflammation 47 , 48 . Our study further revealed that DPSC-CM-preserved cranial flaps not only upregulated osteogenic markers (RUNX2/OCN) and angiogenic protein CD31 at surgical sites, but also significantly modulated inflammatory responses by suppressing pro-inflammatory cytokines (TNF-α, IL-6) while enhancing anti-inflammatory IL-10 expression, creating a favorable microenvironment for accelerated healing. These findings suggest DPSC-CM orchestrates bone regeneration through dual mechanisms: activating recipient cells to secrete osteogenic paracrine factors and vascular growth mediators, while simultaneously reshaping the cellular niche through localized immunomodulation. The coordinated regulation of angiogenic, osteogenic, and anti-inflammatory pathways collectively contributes to its enhanced regenerative efficacy in cranial repair.Given the remarkable advantages of DPSC-CM in cryopreserving cranial bone flaps, we sought to further investigate the intrinsic effects of DPSC-CM itself while conducting comparative analyses with DPSCs. We found that DPSC-CM enhanced cell proliferation, migration, osteogenesis, and the expression of osteogenic and angiogenic proteins, including RUNX2, OCN, and CD31. It also promoted capillary sprouting and tube formation in HUVECs. Interestingly, the addition of DPSC-CM had a more dramatic effect on bone regeneration compared to the transplantation of DPSCs themselves. Our findings demonstrate the multifaceted therapeutic potential of DPSC-CM in cranial flaps preservation, as DPSC-CM-treated cranial bones exhibit concurrent osteogenic, angiogenic, and anti-inflammatory activities following cranioplasty. In summary, DPSC-CM can be used as a new preservation fluid for autologous cranial flaps preservation with deep cryopreservation, which has the potential to promote angiogenesis and bone regeneration to a certain extent, helps to regulate the local microenvironment, and has certain advantages in comparison with relatively common preservation fluids and SP preservation. The underlying mechanisms by which DPSC-CM preserves calvarial flaps remain to be fully elucidated. However, the enhanced efficacy of DPSC-CM in flap preservation may be attributed to its unique bioactive composition and multimodal regulatory effects 49 , 50 . As a cell-free system, DPSC-CM contains a cocktail of trophic factors (e.g., VEGF, FGF-2, and IGF-1), anti-inflammatory cytokines (e.g., IL-10, TGF-β), and extracellular vesicles (EVs) carrying functional miRNAs (e.g., miR-21-5p, miR-146a). These components may collectively orchestrate a regenerative microenvironment through the following mechanisms: mitigation of ischemic and hypoxic damage, suppression of osteoclastic resorption, ECM remodeling and osteogenic activation, as well as immunomodulation and microbial defense 29 , 51 , 52 . Critically, DPSC-CM-based strategies eliminate the need for cytotoxic solvents (e.g., DMSO) and surgical trauma, thereby addressing the dual challenges of cellular toxicity and iatrogenic damage inherent in conventional preservation protocols. Future studies should focus on clarifying the specific mechanisms of DPSC-CM in calvarial flap preservation and explore lyophilization techniques to enhance clinical feasibility. However, there are several limitations that remain in this study. Firstly, in cranioplasty, we did not use a medium (e.g., bone cement) for fixation of the cranial flap, which would have led to the displacement of the autologous cranial flap after cranioplasty. Andrade et al. have shown that freezing leads to denaturation of the collagenous components in the bone cortex and that this qualitative change is exacerbated by decreasing temperatures and prolongation of freezing time 53 . We did not investigate the degree of degeneration associated with cranial flap freezing preservation and whether it affects further bone regeneration and angiogenesis. No biomechanical tests were also performed to determine the functional load-bearing capacity (e.g., strength and stiffness) of the bone after cranioplasty with autologous cranial flaps 54 , 55 . 5. Conclusion To our knowledge, this study pioneers the application of dental pulp stem cell-conditioned medium (DPSC-CM) as a biologically active cryopreservation solution for autologous cranial bone flaps. Our findings demonstrate that DPSC-CM-preserved cranial flaps exhibit enhanced osteogenic potential and angiogenic capacity post-cranioplasty, while significantly modulating local inflammatory responses at the implantation site. It provides a new vision and a mechanistic basis for determining its clinical application. Declarations Acknowledgments We thank all the members at the Center of Regenerative Medicine for providing technical support and valuable suggestions for this project. We declare that they have not use AI-generated work in this manuscript. Author contributions Conceptualization: QY, YL, YH, WX Methodology: YL, LYH, BW, XW, JL, PM Investigation: QY, WX, JL, LD, ZY Visualization: YL, LYH, BW, CY, XW, ZY Supervision: QY, YH Writing—original draft: YL, LYH, BW Writing—review & editing: YL, QY, YH, WX Funding This work was supported by the key Project of Ministry of Science and Technology China (YFXM2022000264 from QY), Chutian Researcher Project (X22020024 from YH) and the National Natural Science Foundation of China (U22A20314 from YH), Jiangxi"Ganpo Talents Program"(gpyc20240204 from WX). Data and materials availability All data needed to evaluate the conclusions in the paper are present in the paper and/or the supplementary information. The raw data supporting the findings of this study are available from the corresponding author on reasonable request. Ethics approval and consent to participate This study was conducted in accordance with the Declaration of Helsinki.The experimental method of dental pulp stem cell extraction was approved by the Clinical Research Ethics Committee of Wuhan University People's Hospital (protocol code: WDRY-2022-K025, approval date: December 1, 2021) Approved Project Name: Extraction of Human Dental Pulp Stem Cells and Basic Research. About animal testing, study entitled " Application of dental pulp stem cell conditioned medium combined with cryogenic preservation of autologous skull flap" has been approved by the Experimental Animal Ethics Committee of Wuhan University People's Hospital (date: November 4, 2023, No. 20231102B) and was conducted in accordance with AVMA guidelines. We had institutional approval for performing experiments using human cells. HUVECs were obtained from Pricella Biotechnology(Wuhan, China). Pricella Biotechnology Company has confirmed that there was initial ethical approval for collection of human cells, and that the donors had signed informed consent. The animal experiment protocol was approved by the Ethics Committee of Animal Experimentation of Wuhan University (protocol code: 20231102B, approval date: November 4, 2023). Title of the approved project: Application of dental pulp stem cell conditioned medium combined with cryogenic preservation of autologous skull flap. Consent for publication All authors consent for publication. Competing interests The authors declare no competing interests. Footnotes Publisher ’ s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Ye Liu, Yonghao Liu and Zhifei Ye contributed equally to this work. Contributor Information Qingsong Ye, Email: [email protected] . Yan He, Email: [email protected] . Wei Xiong, Email: [email protected] . References Hofmeijer, J. et al. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5909455","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":443144076,"identity":"30358df0-89d7-4e96-8470-1b4e6d4ddfa5","order_by":0,"name":"Ye Liu","email":"","orcid":"","institution":"center of regeneration medicine,Renmin Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Liu","suffix":""},{"id":443144077,"identity":"1bb5a53a-fa0f-4aca-9d31-b607072a8a65","order_by":1,"name":"Yonghao Liu","email":"","orcid":"","institution":"center of regeneration medicine,renmin hospital of wuhan university","correspondingAuthor":false,"prefix":"","firstName":"Yonghao","middleName":"","lastName":"Liu","suffix":""},{"id":443144078,"identity":"64edf49b-3ecb-4bf4-aeb2-ad131949fa6e","order_by":2,"name":"Zhifei Ye","email":"","orcid":"","institution":"School \u0026 Hospital of Stomatology Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhifei","middleName":"","lastName":"Ye","suffix":""},{"id":443144079,"identity":"ab679daa-2413-4b45-8de5-75296c7e2b14","order_by":3,"name":"Xinxin Wang","email":"","orcid":"","institution":"institute of regeneration and translational medicine,tianyou hospital of wuhan university of science and technology","correspondingAuthor":false,"prefix":"","firstName":"Xinxin","middleName":"","lastName":"Wang","suffix":""},{"id":443144080,"identity":"e4d17659-c3d2-4cbc-a193-4c5f372fd245","order_by":4,"name":"Junyi Li","email":"","orcid":"","institution":"center of regeneration medicine,renmin hospital of wuhan university","correspondingAuthor":false,"prefix":"","firstName":"Junyi","middleName":"","lastName":"Li","suffix":""},{"id":443144081,"identity":"d9d950d6-7708-43dc-ad16-4292db4b31e4","order_by":5,"name":"Peter L Mei","email":"","orcid":"","institution":"School \u0026 Hospital of Stomatology Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"L","lastName":"Mei","suffix":""},{"id":443144082,"identity":"588e7422-84e7-4309-bd1a-61ea67c00c6f","order_by":6,"name":"Li Duan","email":"","orcid":"","institution":"center of regeneration medicine,renmin hospital of wuhan university","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Duan","suffix":""},{"id":443144083,"identity":"ef2bda2c-f4fe-457e-9bbb-2c9bfb8e0a4d","order_by":7,"name":"Ben Wang","email":"","orcid":"","institution":"center of regeneration medicine,renmin hospital of wuhan university","correspondingAuthor":false,"prefix":"","firstName":"Ben","middleName":"","lastName":"Wang","suffix":""},{"id":443144084,"identity":"acfb9882-3eb7-4d2e-9893-11204780807b","order_by":8,"name":"Chun Xu","email":"","orcid":"","institution":"The University of Sydney School of Dentistry","correspondingAuthor":false,"prefix":"","firstName":"Chun","middleName":"","lastName":"Xu","suffix":""},{"id":443144085,"identity":"be594fd3-f9f9-4b4c-8fe1-4f4d7b8d8893","order_by":9,"name":"Wei Xiong","email":"","orcid":"","institution":"department of orthopedics,the school affiliated hospital of nanchang university","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Xiong","suffix":""},{"id":443144086,"identity":"3edf5d30-ce7b-424e-b463-ef76f221f1b3","order_by":10,"name":"Yan He","email":"","orcid":"https://orcid.org/0000-0002-0949-8525","institution":"institute of regenerative and translational medicine,tianyou hospital of wuhan university of science and technology","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"He","suffix":""},{"id":443144087,"identity":"7d74b4ba-ece1-409f-8f9a-4ee56425debd","order_by":11,"name":"Qingsong Ye","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYDACZgTrGJhiYydeC1saA0MCkGLGrRgd8JiBtTAQ0sJ3nPfwyx8Vd+w2HO/59uDjj23yfMwMjB8+5uDWInmYL82a58yz5A1nzm43nJFw27CNmYFZcuY23FoMDvOYGTO2HU42uJG7TZon4TYjUAsbMy8BLYY/QVruv3kG0mJPjBbjB7xth+0MbvCwgbQkEtQiCbSFmefM4QTJM2lmkjPSbie3MTM24/UL3/kzxh9/VBy25zt++JnEB5vbtvPbmw9++IhHC8MBBjYJIJW44ABciLEBj3qwFuYPQMpenoC6UTAKRsEoGMEAAGekUx4oXmI7AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-4233-4079","institution":"Center of Regenerative Medicine, Renmin Hospital of Wuhan University","correspondingAuthor":true,"prefix":"","firstName":"Qingsong","middleName":"","lastName":"Ye","suffix":""}],"badges":[],"createdAt":"2025-01-27 05:14:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5909455/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5909455/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13287-025-04407-1","type":"published","date":"2025-06-02T15:56:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80800569,"identity":"af247e4d-9b76-43b1-811a-0f2d64648f38","added_by":"auto","created_at":"2025-04-17 08:28:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1090205,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDPSC-CM can reduce mechanical damage caused by ice crystals during cell cryopreservation and provide nutritional support. \u003c/strong\u003e(A) Representative TEM image of DPSC-CM. Scale =500 nm. (B) Determination of the diameter of extracellular vesicles in DPSC-CM using nanoparticle tracking analysis. (C, D) Cell viability of MC3T3-E1 cells and HUVECs after freezing and resuscitation. (E, F, G, H) Representative cellular immunofluorescence staining images of Ki67 and quantitative statistical analysis. Scale =100 μm. The results are expressed as mean ± SD values of at least three independent experiments. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5909455/v1/c48376f055727e547e181bf6.png"},{"id":80801076,"identity":"48925b47-6c06-40ec-a643-6277ab294144","added_by":"auto","created_at":"2025-04-17 08:36:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1369175,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDPSC-CM promotes bone repair at cranial sutures in mice. \u003c/strong\u003e(A) The experimental design is illustrated schematically. (B) Illustration of surgical steps: Step 1: The head of the mouse was fixed, and the skin was cut after disinfection. Step 2: A 5mm cranial flap was removed using a skull trephine. Step 3: Cranioplasty was performed four weeks after surgery. Step 4: The skin of the head was sutured. (C) 3D reconstruction images of the cranial flaps of each group of mice at week-4 and -8. (D) Comparison of micro-CT results of BV, BS, Tb.Th, Tb.N, Tb.Sp, and BMD between groups. (E) Results of micro-CT analysis of changes in bone mineral condition (including BV, BS, Tb.Th, Tb.N, Tb.Sp, and BMD) over time in each group. The results are expressed as mean ±SD values of at least three independent experiments. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5909455/v1/53e250c15f3ae1c0bdd49fd2.png"},{"id":80800573,"identity":"ee059c1e-7fde-4c3e-92c7-8f66c1198307","added_by":"auto","created_at":"2025-04-17 08:28:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1665836,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDPSC-CM improves the histological appearance of cranial sutures in mice. \u003c/strong\u003e(A, B) Representative images and local zoom images of H\u0026amp;E staining and Masson staining of five groups of bone tissue. Scale =100 μm and 20 μm. The results are expressed as mean ± SD values of at least three independent experiments.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5909455/v1/a0af405f1e065e70213cc1cf.png"},{"id":80800571,"identity":"5fc24d55-50e5-496e-8eb6-31d80f1c4689","added_by":"auto","created_at":"2025-04-17 08:28:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":760394,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDPSC-CM promotes bone and vascular regeneration-related protein expression and modulates the inflammatory microenvironment. \u003c/strong\u003e(A-H) Representative images and quantification of western blot showed the expression of CD31, RUNX2, OCN, TNF-α, IL-6, and IL-10 in five different groups. The results are expressed as mean ±SD values of at least three independent experiments. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5909455/v1/87eaa3a2a45c1601bb65a904.png"},{"id":80800578,"identity":"e2d25f0c-3a8e-41a1-ab0f-2ed3d04ae73f","added_by":"auto","created_at":"2025-04-17 08:28:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1380345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDPSC-CM promotes bone and vascular regeneration-related protein expression at cranial sutures. \u003c/strong\u003e(A-D) Representative immunofluorescent staining images and quantification of CD31, RUNX2, and OCN in five different groups. Scale =1 μm. The results are expressed as mean ±SD values of at least three independent experiments. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5909455/v1/b0dbcd7b9b48aa18938f6a70.png"},{"id":80801080,"identity":"121738ae-dc74-4e2a-baaa-c8df9d8d9127","added_by":"auto","created_at":"2025-04-17 08:36:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2856175,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDPSC-CM promotes the proliferation and osteogenic differentiation of MC3T3-E1 cells. \u003c/strong\u003e(A) The viability of MC3T3-E1 cells. (B, E) Protein expression and quantification of key indicators of osteogenic function. (C, D, F, G) Cell migration was examined via wound-healing and transwell assay. Scale =100 μm. (H-K) ALP and ARS staining of MC3T3-E1 cells in the CON, DPSCs, and DPSC-CM groups. Scale =100 μm. The results are expressed as mean ± SD values of at least three independent experiments. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5909455/v1/f129790eaa6c234e1c4300d8.png"},{"id":80801942,"identity":"069614e7-debc-41bb-8932-6b643cdafeeb","added_by":"auto","created_at":"2025-04-17 08:44:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1819880,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDPSC-CM has a favorable angiogenic effect. HUVECs migration and invasion were analyzed by a wound-healing assay. \u003c/strong\u003e(A, E) and a transwell assay (B, F). Scale =100 μm. (D, G) Representative images and quantification of western blot showed the expression of CD31 in CON, DPSCs, and DPSC-CM groups. Scale =100 μm. (C, H, I) The tube formation assay showed tube formation ability in three groups. Scale =100 μm. The results are expressed as mean ± SD values of at least three independent experiments. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5909455/v1/641588952836f6ac08dd617c.png"},{"id":84242361,"identity":"60a99307-b732-46fe-b143-3d0453ca25ed","added_by":"auto","created_at":"2025-06-09 16:06:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11544499,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5909455/v1/ba990b62-5027-4464-b0d5-bed8ed8cae25.pdf"},{"id":80800576,"identity":"48d70a31-7cf8-4ee4-a849-002a67e0bf30","added_by":"auto","created_at":"2025-04-17 08:28:44","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":260268,"visible":true,"origin":"","legend":"","description":"","filename":"AuthorChecklistFull.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5909455/v1/dfe089e64f88d5695d22af10.pdf"},{"id":80800574,"identity":"d8a5a687-3d5c-4a27-a181-a98356f847aa","added_by":"auto","created_at":"2025-04-17 08:28:44","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":469846,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformationmodifiedversion.docx","url":"https://assets-eu.researchsquare.com/files/rs-5909455/v1/dc99d32b1aabf0d0a9e612d8.docx"},{"id":80801077,"identity":"0aac4a3f-f782-4de9-9ad1-7b0897c5412b","added_by":"auto","created_at":"2025-04-17 08:36:44","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":316765,"visible":true,"origin":"","legend":"","description":"","filename":"WB.docx","url":"https://assets-eu.researchsquare.com/files/rs-5909455/v1/2f891209a659cf9487cefb7f.docx"}],"financialInterests":"","formattedTitle":"Application of dental pulp stem cell-conditioned medium combined with deep crypreservation of autologous cranial flaps","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDecompressive craniectomy (DC) serves as a critical neurosurgical intervention for intracranial pressure management in traumatic brain injury, osteopathic disorders, congenital cranial anomalies, and post-tumor resection scenarios\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. During secondary reconstruction phases, cranial defect repair through cranioplasty utilizes either autologous cranial flaps or biocompatible synthetic materials\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. While evolving reconstructive modalities now encompass allogeneic/xenogeneic grafts, tissue-engineered constructs, and synthetic biomaterials (e.g., polyetheretherketone, titanium alloys)\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, longitudinal clinical evaluations consistently identify autologous bone transplantation as the gold-standard modality due to its inherent biocompatibility and structural congruence\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe clinical practice of autologous cranial flaps preservation post-DC emerged in the mid-20th century, with cryopreservation and subcutaneous pouch (SP) storage establishing themselves as standard preservation modalities\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. While empirical cryoprotectant formulations (e.g., 0.9 percent NaCl solution (NS), dimethyl sulfoxide (DMSO), glycerol, and alcohol solutions) have been clinically trialed, their suboptimal biocompatibility profiles frequently manifest as postoperative complications including aseptic resorption and microbial colonization\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Notably, the absence of international consensus guidelines governing optimal preservation protocols remains a persistent challenge in neurosurgical practice.\u003c/p\u003e \u003cp\u003eRecent studies have shown that paracrine effects of implanted mesenchymal stem cells can promote bone regeneration in vivo. And conditioned media containing most of the paracrine factors from cultured MSCs (mesenchymal stem cells-conditioned medium, MSC-CM) can induce tissue regeneration as efficiently as MSCs\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In addition, the use of MSC-CM has many advantages over MSCs themselves, including a lower risk of tumorigenesis, easier preservation and handling, and negligible immunogenicity, which reduces barriers associated with xenografts or xenotransplantation and preservation-related barriers\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAmong MSCs subtypes, dental pulp stem cells not only have a wide range of sources and low immunogenicity but also exhibit significant regenerative advantages over bone marrow mesenchymal stem cells (BMMSCs), including: enhanced proliferative kinetics; marked osteoinductive capacity; dual angiogenic/immunomodulatory paracrine secretion profiles\u003csup\u003e\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Studies have shown that dental pulp stem cell-conditioned medium (DPSC-CM) can stimulate the migration of endogenous osteocytes, promote osteoblast differentiation, stimulate angiogenesis, and ultimately achieve bone repair and regeneration\u003csup\u003e\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. It is inferred from previous research results that DPSC-CM may be an effective preservation solution for preserving cranial flaps. There is no research report on the use of mesenchymal stem cell-conditioned medium (MSC-CM) for the preservation of cranial flaps, so we used DPSC-CM as a cryopreservation solution for the preservation of autologous cranial flaps to investigate its effect on the regenerative capacity of bone after cranioplasty.In this study, we used NS, DMSO, α modified eagle's medium (α-MEM), and DPSC-CM to preserve mouse cranial flaps at -196℃, and a group of SP preservation as a clinical control, to compare their effects on bone regeneration and angiogenesis in a mouse cranial defect model after cranioplasty. The results showed that the cranial flaps preserved by DPSC-CM, after cranioplasty, was able to promote the rapid growth and migration of osteoblasts and blood vessels at cranial suture, while no complications such as bone resorption and infection were observed. In addition, we also validated the dual role of DPSC-CM bone regeneration and angiogenesis in in vitro experiments. We demonstrated that DPSC-CM can be used as a cryopreservation agent to preserve autologous cranial flaps, which can better promote bone healing, restore brain blood supply, and reduce complications such as infection and bone resorption after cranioplasty.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Culture and characterization of DPSCs\u003c/h2\u003e \u003cp\u003eThe dental pulp stem cells (DPSCs) were extracted and identified according to our previously established protocol. DPSCs were isolated from third molars obtained after routine surgery in healthy adolescent donors. With the approval of the Ethics Committee of Wuhan University People's Hospital and the informed consent of the donor (Approval Number: WDRY-2022-K025, Wuhan, China). DPSCs were isolated using a previously described method. Briefly, pulp tissue was briefly extracted from obstructed third molars, digested with 3 mg/mL of type I collagenase and 4 mg/mL of dispase for 30 min in an incubator, centrifuged at 1000 rpm for 5 min, and the pellet was resuspended and seeded into culture flasks containing α-MEM (Gibco, USA), which was supplemented with 20% fetal bovine serum (FBS; Gibco, USA) and a final concentration of 1% penicillin/streptomycin (Gibco, USA). The culture flasks were incubated at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e. The medium was changed every three days until the stem cells reached 70% fusion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Analysis of DPSC surface markers by flow cytometry\u003c/h2\u003e \u003cp\u003eThird-passage DPSCs were harvested at 70\u0026ndash;80% confluence using 0.25% trypsin-EDTA solution (Gibco, USA), followed by centrifugation at 1000 \u0026times;g for 5 min. Cells were washed twice and resuspended in PBS supplemented with 2% FBS for subsequent staining. For immunophenotypic characterization, cell suspensions were incubated with fluorochrome-conjugated antibodies targeting human surface markers: CD44-FITC, CD73-PE, CD90-APC, CD105-PerCP (positive markers), CD31-FITC, and HLA-DR-PE (negative markers), along with isotype-matched controls (BD Biosciences, USA). All incubations were performed in the dark at 4\u0026deg;C for 30 min. After antibody staining, cells were washed and resuspended in 200 \u0026micro;L PBS for analysis. A minimum of 10,000 events per sample were acquired on a CytoFLEX LX flow cytometer (Beckman Coulter, USA), with fluorescence compensation adjusted using single-stained controls. Data analysis was performed using FlowJo v10.8 software (TreeStar Inc., USA), with gating strategies based on isotype control thresholds (\u0026lt;\u0026thinsp;1% background staining).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.3 Multilineage differentiation of DPSCs\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTo assess the ability of DPSCs to differentiate in multiple directions, third-generation DPSCs were differentiated into adipocytes, osteoblasts, or chondrocytes using adipogenic, osteogenic, or chondrogenic differentiation-inducing medium (OriCell, China) according to the manufacturer's instructions after reaching 80% fusion. For osteogenic differentiation, DPSCs were cultured in an osteogenic induction medium (OriCell, China), which was changed every 3 days for 21 days. To observe mineralization deposition, cells were fixed with 4% paraformaldehyde for staining with alizarin red S (ARS) solution. For adipogenic differentiation, DPSCs were cultured in an adipogenic induction medium (OriCell, China). After 21 days, they were stained with fresh oil red O solution. For chondrogenic differentiation, DPSCs were cultured in a complete chondrogenic differentiation induction medium (OriCell, China) for 21 days and then stained with alcian blue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Conditioned medium (CM) preparation from DPSCs (DPSC-CM)\u003c/h2\u003e \u003cp\u003eFor DPSC-CM preparation, cells were plated at a density of 5 \u0026times; 10⁵ cells per 10 cm culture dish. Upon reaching 80\u0026ndash;90% confluence, complete culture medium was replaced with serum-free medium to initiate CM production. Following 48-hour incubation, the conditioned supernatant was collected and subjected to sequential processing: initial centrifugation at 1,000 rpm for 5 min to remove cellular debris, followed by sterile filtration through a 0.22 \u0026micro;m membrane. The clarified CM was subsequently concentrated using centrifugal filtration devices (Millipore UFC900324, Germany) and cryopreserved at -80\u0026deg;C for downstream experimental applications in both in vitro and in vivo systems.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Characterization of DPSC-CM\u003c/h2\u003e \u003cp\u003eFor transmission electron microscopy (TEM) analysis of DPSC-CM microstructure, 20 \u0026micro;L of DPSC-CM sample was deposited onto carbon-coated copper grids and allowed to adsorb for 3\u0026ndash;5 minutes. Excess liquid was carefully removed by filter paper blotting, followed by air-drying at room temperature for 10 minutes. The grids were then washed with DPBS and negatively stained with 2% (w/v) uranyl oxalate for 1\u0026ndash;2 minutes, after which excess stain was blotted and the grids were air-dried again. Samples were imaged with TEM (HITACHI, HT7700, Japan). Particle size distribution was determined using a Nanoparticle tracking analysis (NTA) instrument (Zetasizer Nano ZS, Malvern Panalytical, UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Cell culture and osteogenic differentiation of mouse embryonic osteoblasts cells (MC3T3-E1 cells)\u003c/h2\u003e \u003cp\u003eFor osteogenic differentiation, cells were seeded in 6-well plates at a density of 1 \u0026times; 10⁵ cells/well in complete growth medium. Upon reaching 70% confluence, the culture medium was replaced with osteogenic induction medium consisting of: α-MEM base supplemented with 10% FBS; 50 \u0026micro;g/mL ascorbic acid (Merck, USA); 4 mM β-glycerophosphate (Merck, USA).Cells were maintained in this differentiation cocktail for 14 days with medium changes every third day to ensure consistent nutrient availability and metabolic waste removal.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Cell freezing and resuscitation\u003c/h2\u003e \u003cp\u003eCell freezing and resuscitation experiments were performed using MC3T3-E1 cells and human umbilical vein endothelial cells (HUVECs), both of which were purchased from Pricella Biotechnology Company(Procell, China). Three cryopreservation groups were established: 1. DMSO-only group: 100%DMSO; 2. DMSO\u0026thinsp;+\u0026thinsp;FBS group: 10% DMSO\u0026thinsp;+\u0026thinsp;90% FBS; 3. DMSO\u0026thinsp;+\u0026thinsp;DPSC-CM group: 10% DMSO\u0026thinsp;+\u0026thinsp;90% DPSC-CM. An equal number of cells (1\u0026times;10⁵ cells/cryopreservation tube) were cryopreserved using a gradient freezing protocol (-1\u0026deg;C/min) at -80\u0026deg;C, and cell recovery was initiated 24 hours later. To allow cells to fully adhere and spread, cell digestion and counting were performed 24 hours post-thaw.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Ki67 fluorescent staining assay\u003c/h2\u003e \u003cp\u003eResuscitated cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature and then rinsed with PBS (3 \u0026times; 5 min). 0.1% Triton X-100 was osmotically stabilized in PBS for 10 min (room temperature). Treat with blocking buffer (5% normal goat serum\u0026thinsp;+\u0026thinsp;1% BSA, PBS) for 1 hour at room temperature to reduce nonspecific binding. Primary antibody incubation: Incubate with anti-Ki-67 antibody (1:200, ABclone, China) at 4\u0026deg;C overnight. Rinse with PBS (3 \u0026times; 5 min). Add ABflo\u0026reg; 488-conjugated Goat anti-Rabbit IgG (1:200, ABclone, China) and incubate for 1 hour at room temperature away from light. The nuclei were stained with DAPI (1:1000, Beyotime, China). The stained sections were observed using a microscope (IX71, Olympus, Japan) and photographed at 100\u0026times; magnification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Animal and moral statement\u003c/h2\u003e \u003cp\u003eMale C57BL/6 mice (8-week-old, 30\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g body weight) were used in this study, and random allocation into 5 experimental groups (n\u0026thinsp;=\u0026thinsp;6/group). The experiments were conducted in the animal house of Wuhan University People's Hospital, and the mice were housed in separate cages at a temperature of 25\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, relative humidity of 50% \u0026plusmn; 15%, and a light/dark cycle of 12 h. All mice were allowed to drink and eat freely. All animal studies complied with the Wuhan University Guidelines for the Care and Use of Laboratory Animals (Institutional Animal Care and Use Committee No. 20231102B) and internationally recognized principles. The work has been reported in line with the ARRIVE guidelines 2.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Surgery and treatment\u003c/h2\u003e \u003cp\u003eAll surgeries were performed under isoflurane (4%) anesthesia. In the cranial defect model, mice were anesthetized using isoflurane (4%) by inhalation, and a median cranial defect of 5 mm in diameter was constructed using an electric dental drill at a low drilling speed under continuous saline perfusion. The harvested cranial flaps were preserved in cryoprotective solutions (1 mL per tube) containing respective components: 0.9 percent NaCl solution (NS), dimethyl sulfoxide (DMSO), α-MEM, or DPSC-CM, and the cryopreservation tubes were stored in liquid nitrogen at -196\u0026deg;C in a gradient freezing mode. In the subcutaneous preservation (SP) group, a 1.5 cm longitudinal incision was surgically created in the femoral region of C57BL/6 mice under aseptic conditions, followed by meticulous dissection to establish a lateral subcutaneous pocket for cranial flaps implantation. Postoperative protocols included intramuscular penicillin prophylaxis and topical povidone-iodine (10%) wound disinfection for 3 days.\u003c/p\u003e \u003cp\u003eAfter 4 weeks of cranial flaps preservation, the cryopreservation tubes containing the cranial flaps were removed from liquid nitrogen, warmed to room temperature in a gradient, and the flap was implanted in situ in the defect site. The wound was closed in layers (periosteum, skin) by absorbable sutures. Postoperative care included analgesia with buprenorphine and antibiotic treatment with penicillin. After surgery, the animals were kept individually under constant conditions. No animal deaths were noted during or after surgery. According to the American Veterinary Medical Association (AVMA) 2020 edition of Guidelines for Euthanasia of Animals, animals were euthanized at weeks 4 and 8 after implantation of the cranial flaps, and all animals were euthanized using carbon dioxide gas. Regenerated tissue was harvested from the defect area for further evaluation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Micro-CT analysis of mouse cranial flaps\u003c/h2\u003e \u003cp\u003eFollowing in situ cranial flaps implantation, samples were scanned using a Skyscan 1176 micro-computed tomography scanner (Bruker microCT, Germany) at a resolution of 6.5 \u0026micro;m at weeks 0, 2, 4, and 8. Raw data were reconstructed using NRecon software (Bruker, Germany), followed by 3D model generation with CTVox (Bruker, Germany). For quantitative analysis, a cylindrical volume of interest (VOI; 5 mm diameter \u0026times; 1 mm height) was aligned with the original defect boundary using CTAn software (Bruker, Germany). Key parameters calculated included: Bone volume (BV), bone surface area (BS), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and bone mineral density (BMD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Western blot\u003c/h2\u003e \u003cp\u003eThe co-cultured MC3T3-E1 cells and HUVECs were lysed in lysis buffer (Beyotime, China) with a mixture of protease and phosphatase inhibitors (MCE, USA). Following in situ cranial flaps implantation, animals were euthanized at 4 and 8 weeks, and fresh bone tissue samples were homogenized in lysis buffer (Beyotime, China) containing a protease/phosphatase inhibitor cocktail (HY-K0013, MCE, USA) using a 3D cryo-mill (Servicebio, China) for 45 s at high speed to extract total proteins, whose concentrations were subsequently quantified with the Enhanced BCA Protein Detection Kit (Thermo, USA). A total of 10 \u0026micro;g of protein from each sample was electrophoresed in SDS-PAGE gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Germany), which were blocked with 5% nonfat milk and incubated with primary antibodies at 4\u0026deg;C overnight. The primary antibodies used to detect protein expression were runt-related transcription factor 2 (RUNX2) (1:500, Proteintech, China), osteocalcin (OCN) (1:500, ABclone, China), platelet endothelial cell adhesion molecule-1 (CD31) (1:500, Proteintech, China), tumor necrosis factor-α (TNF-α) (1:500, Proteintech, China), interleukin 6 (IL-6) (1:500, Proteintech) and interleukin 10 (IL-10) (1:500, Proteintech, China). After washing, membranes were incubated with HRP-conjugated goat anti-rabbit or mouse IgG secondary antibodies (1:1000, Proteintech, China) for 1 h at 37\u0026deg;C, followed by protein visualization using an Enhanced Chemiluminescence Kit (Biology, China). Relative protein expression levels were quantified via densitometric analysis of immunoreactive bands using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Histological analysis for bone regeneration\u003c/h2\u003e \u003cp\u003eHistological evaluation of bone regeneration was performed through hematoxylin and eosin (H\u0026amp;E) and Masson trichrome staining, wherein cranial specimens harvested at 4 and 8 weeks post-cranioplasty were fixed in 4% paraformaldehyde, decalcified in 0.5 M EDTA for 8 weeks at room temperature, and paraffin-embedded before being sectioned into 5 \u0026micro;m slices using a tissue microtome. Deparaffinized sections underwent sequential xylene immersion (Shanghai Huawei Pharmaceutical Co., Ltd., China) for 15 min and ethanol gradient dehydration (anhydrous, 90%, and 75% ethanol, 10 min each), followed by H\u0026amp;E staining (Beyotime, China) to evaluate bone architecture and pathological alterations, and Masson trichrome staining (Reckitt Benckiser This Biotech, China) to visualize collagen fiber distribution in regenerated bone. Stained sections were imaged under a light microscope (Olympus, Japan) for qualitative assessment of osteogenic activity and matrix remodeling.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Immunofluorescent staining\u003c/h2\u003e \u003cp\u003eAfter being dewaxed and dewatered, the prepared cranial sections (5 \u0026micro;m) were softened by incubation with 0.1% Triton X-100 for 15 min and blocked with 3% bovine serum albumin for 30 min. Sections were then incubated with primary antibodies RUNX2 (1:500, Proteintech, China), OCN (1:500, Proteintech, China), and CD31 (1:500, Proteintech, China) overnight at 4\u0026deg;C, followed by incubation with secondary antibodies for 1 h at room temperature. The nuclei were stained with DAPI (1:1000, Beyotime, China). The stained sections were observed using a microscope (Olympus, Japan) and photographed at 100\u0026times; magnification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Cell co-culture\u003c/h2\u003e \u003cp\u003eA Transwell co-culture system (0.4 \u0026micro;m pore membrane; BD Biosciences, Franklin Lakes, NJ, USA) was employed to investigate cell-cell interactions. The experimental setup included: Lower chamber (6-well plate): MC3T3-E1 cells seeded at 1 \u0026times; 10⁶ cells/well in α-MEM medium. Upper chamber (3 experimental groups): Control (CON) group: 2 mL α-MEM basal medium; DPSCs group: 1 \u0026times; 10⁵ DPSCs in 2 mL α-MEM; DPSC-CM group: 2 mL DPSC-CM. The co-culture system was maintained for 14 days under standard conditions (37\u0026deg;C, 5% CO₂), with medium replacement every 3 days to ensure metabolic homeostasis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16 Cell viability assay\u003c/h2\u003e \u003cp\u003eMC3T3-E1 cell viability was quantified using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). The MC3T3-E1 cells treated with α-MEM or DPSCs or DPSC-CM were seeded into 96-well plates at 1 \u0026times; 10⁴ cells/well and cultured under standard conditions (37\u0026deg;C, 5% CO₂). Cellular metabolic activity was evaluated at five timepoints (0, 12, 24, 36, and 48 hours post-seeding) by adding 10 \u0026micro;L CCK-8 reagent to each well, followed by 1-hour incubation. Absorbance was measured at 450 nm using a microplate reader (Molecular Devices, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17 Wound-healing and transwell assay\u003c/h2\u003e \u003cp\u003eIn order to detect the effects of DPSCs and DPSC-CM on the migration ability of MC3T3-E1 cells, we performed wound-healing and transwell experiments. In the wound-healing experiment, MC3T3-E1 cells were seeded into six-well plates and cultured to a density of 90%; the original medium was discarded, and the cells were starved in serum-free medium for 12 h. A scratch wound was created in the middle of each well with a sterile 200 \u0026micro;L pipette tip, and the cellular debris was washed away with PBS, and then the cells were co-culture with α-MEM or DPSCs or DPSC-CM, respectively, for 24 h. Using an inverted microscope (Olympus, Japan) to take pictures at 0/12/24h, respectively, to monitor the migration rate of cells to the cell-free area. The area of cells migrating into the scratched area was calculated using ImageJ analysis software for quantitative analysis. The specific formula was: cell migration rate = (0 h scratch area \u0026minus;\u0026thinsp;12 h scratch area)/0 h scratch area. This procedure was repeated three times for each set of samples.\u003c/p\u003e \u003cp\u003eCell migration capacity was assessed using 24-well Transwell chambers with 6.5 \u0026micro;m pore membranes (Corning, NY, USA). MC3T3-E1 cells (1 \u0026times; 10⁵ cells/mL in 100 \u0026micro;L serum-free α-MEM) were seeded in the upper chamber, while the lower chamber contained 600 \u0026micro;L of either α-MEM complete medium or DPSC-cultured α-MEM or DPSC-CM. After 24 h incubation under standard conditions (37\u0026deg;C, 5% CO₂), the transwell chamber were fixed with 4% paraformaldehyde (Merck, USA) for 15 min and stained with 0.1% crystal violet solution for 30 min. The stained cells were photographed using an inverted microscope (Olympus, Japan). Subsequently, the number of migrated cells was calculated using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.18 Tube formation, wound-healing and transwell assay\u003c/h2\u003e \u003cp\u003eAngiogenesis is essential for bone regeneration, so in order to detect the effects of DPSCs and DPSC-CM on the migration ability of HUVECs, We performed wound-healing and transwell assay experiments. The steps were consistent with the wound-healing and transwell assay experiments described above. To compare the in vitro angiogenic ability of each group, a tubule formation assay was performed. Matrigel (BD Biosciences, USA) was dissolved in a refrigerator at 4\u0026deg;C overnight, and Matrigel was rapidly added to 48-well plates in a volume of 100 \u0026micro;L per well and left at 37\u0026deg;C for 30 min to form a gel. HUVECs were seeded on Matrigel at a density of 2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well, and HUVECs were co-cultured with α-MEM or DPSCs or DPSC-CM for 6 hours. The cells were imaged using phase contrast microscopy (Leica, German) to assess endothelial cell tubule formation. The total length of tubules, number of junctions, and reticulation were calculated using ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e2.19 Alkaline phosphatase (ALP) staining and activity assay\u003c/h2\u003e \u003cp\u003eTo evaluate DPSCs/DPSC-CM effects on osteogenic differentiation, MC3T3-E1 cells were analyzed through ALP histochemical staining and enzymatic activity quantification. MC3T3-E1 cells were cultured in 6-well plates at a density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well, and MC3T3-E1 cells were incubated in the differentiation process with α-MEM or DPSCs or DPSC-CM and incubated at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e, and the differentiation medium was changed every three days. After 14 days of culture, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature, rinsed with PBS, and stained for ALP using the BCIP/NBT Alkaline Phosphatase Chromogenic Kit (Beyotime, China). To determine ALP activity, proteins were extracted after treatment of MC3T3-E1 cells as described above. ALP activity was measured using an Alkaline Phosphatase Assay Kit (Beyotime, China) according to the manufacturer's instructions. The absorbance of alkaline phosphatase activity was measured at 405 nm using a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e2.20 Alizarin Red S (ARS) staining and quantification for mineralization measurement\u003c/h2\u003e \u003cp\u003eTo evaluate extracellular matrix mineralization, MC3T3-E1 cells (2 \u0026times; 10⁵ cells/well) were co-cultured with α-MEM or DPSCs or DPSC-CM under osteogenic conditions for 14 days. MC3T3-E1 cells were rinsed with PBS and fixed with 4% paraformaldehyde for 30 min. ARS (1 mL) (Beyotime, China) was added to the culture wells and incubated for 30 min at room temperature, then rinsed 5 times with distilled water to remove excess dye. Images of the stained cells were taken under an inverted fluorescence microscope (Olympus, Japan). For quantitative analysis, the stained cells were lysed with 10% cetylpyridinium chloride (Solarbio, China) for 1 h at room temperature, then transferred to a 96-well plate and quantified by measuring the absorbance at 570 nm with a microplate reader (Biotek Instruments Inc., USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e2.21 Statistical analysis\u003c/h2\u003e \u003cp\u003eData from three independent experiments are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The significance between the two groups was analyzed using two-tailed Student's t-test or one-way analysis of variance (ANOVA). For multiple comparisons, Tukey post-hoc tests were used. All experimental data were statistically analyzed using GraphPad Prism (9.5.1) software. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant; *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Characterization of dental pulp stem cells (DPSCs) and dental pulp stem cell-conditioned medium (DPSC-CM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSystematic characterization confirmed that dental pulp stem cells (DPSCs) retained typical morphological features (spindle-shaped fibroblast-like morphology) (Figure S1A), MSCs-specific surface markers (CD44/CD73/CD90/CD105) (Figure S1C), and trilineage differentiation potential (osteogenic/chondrogenic/adipogenic) (Figure S1B), ensuring homogenization of cell extraction.\u003c/p\u003e\n\u003cp\u003eNumerous studies have demonstrated the significant advantages of mesenchymal stem cells-conditioned medium (MSC-CM) in bone repair and regeneration\u003csup\u003e26-28\u003c/sup\u003e. To further explore the therapeutic potential of MSC-CM, we selected dental pulp stem cells (DPSCs)\u0026nbsp;\u0026ndash;\u0026nbsp;a widely accessible cell source with minimal ethical concerns\u0026nbsp;\u0026ndash;\u0026nbsp;to prepare dental pulp stem cell-conditioned medium (DPSC-CM). Chouaib et al. previously characterized DPSC-CM composition using human growth factor antibody arrays, revealing that DPSC-CM contains a complex mixture of bioactive factors responsible for its biological effects\u003csup\u003e29\u003c/sup\u003e. In our study, transmission electron microscopy (TEM) analysis identified abundant extracellular vesicles within DPSC-CM (Figure 1A). Nanoparticle tracking analysis (NTA) further demonstrated that these vesicles exhibited a size distribution ranging from 58 nm to 290 nm, with an average diameter of approximately 79 nm (Figure 1B) \u0026ndash; characteristics consistent with exosome. These findings suggest that extracellular vesicles and growth factors in DPSC-CM may serve as critical mediators of bone repair and regeneration. Based on this evidence, we subsequently investigated the potential preserving capacity of DPSC-CM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 DPSC-CM can reduce mechanical damage caused by ice crystals during cell cryopreservation and provide nutritional support\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDimethyl sulfoxide (DMSO) is widely recognized as a potent cryoprotectant in cell preservation, functioning by permeating cell membranes to reduce intracellular water freezing points and inhibit ice crystal formation during cryopreservation. However, its cytotoxicity at concentrations exceeding 10% necessitates cautious formulation design\u003csup\u003e30,31\u003c/sup\u003e. Standard cryopreservation protocols typically combine 10% DMSO with either a mixture of basal medium (40-70%) and fetal bovine serum (FBS, 20-50%) or 90% FBS alone. The basal medium supplies essential nutrients and pH buffering, while FBS acts as an extracellular protective matrix and metabolic support through its rich repertoire of growth factors and proteins\u003csup\u003e32\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo validate the preservation and nutritional support effects of DPSC-CM on cells, we conducted cell experiments by replacing serum with DPSC-CM for cell preservation and observed the post-thaw cell state and proliferation efficiency. In the context of bone repair and regeneration, bone regeneration and vascular network repair are particularly critical. Therefore, we selected two cell types for cryopreservation experiments: mouse embryonic osteoblast cells (MC3T3-E1 cells) and human umbilical vein endothelial cells (HUVECs). It was found that the DMSO + DPSC-CM group exhibited a higher cell count compared to the DMSO-only and DMSO + FBS groups, with statistical significance. CCK-8 assay results demonstrated that the proliferation rates of both MC3T3-E1 cells and HUVECs in the DMSO + DPSC-CM group were faster than those in the other two groups, with significant differences (P＜0.05) (Figure 1C-D). Additionally, Ki67 fluorescence staining was performed, and the fluorescence statistical data showed similar results(Figure 1E-H). These experimental findings indicate that DPSC-CM can replace serum by providing extracellular molecular barriers and nutritional support, reducing mechanical damage and metabolic stress on cells, making it an excellent alternative cryopreservation solution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Cranial flaps preserved by DPSC-CM have greater bone repair capacity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the in vitro findings, we applied DPSC-CM to the cryopreservation of calvarial cranial flaps to validate its efficacy as a cryoprotectant solution. In this section, we applied DPSC-CM as a bone preservation fluid in the deep cryopreservation of mouse cranial flaps. We established a mouse cranial defect model in which surgically excised cranial flaps were preserved in designated solutions. The experimental setup included clinically standard solutions: 0.9% sodium chloride (normal saline, NS), dimethyl sulfoxide (DMSO), and subcutaneous pouch preservation. DPSC-CM served as the experimental group. Additionally, an \u0026alpha;-MEM control group was established to account for potential confounding effects of culture medium nutrients on bone preservation. Subsequently reimplanted via cranioplasty after 4 weeks (Figure 2A-B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLongitudinal micro-CT imaging and analysis at 0, 2, 4, and 8 weeks post-cranioplasty revealed distinct outcomes across groups. Figure 2C displays 3D reconstructed models of cranial flaps at 4 and 8 weeks, demonstrating significant bone resorption in the NS and SP groups, while the remaining three groups exhibited favorable healing outcomes. However, due to incomplete fixation of the bone flaps during cranioplasty, minor displacement was observed in all groups. Longitudinal micro-CT quantitative analysis demonstrated that, with the accumulation of time, bone surface (BS), bone volume (BV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) gradually increased compared to baseline measurements on postoperative day 1, while trabecular separation (Tb.Sp) decreased (Figure 2E).\u003c/p\u003e\n\u003cp\u003eNotably, the DPSC-CM group demonstrated superior osteogenic outcomes, with significantly elevated BS, BV, Tb.N, and Tb.Th values (p \u0026lt; 0.05) and reduced Tb.Sp compared to control groups at 2-, 4-, and 8-week intervals. These morphological improvements were paralleled by enhanced bone mineral density (BMD) in the DPSC-CM treatment group (Figure 2D). Collectively, these findings indicate that calvarial cranial flaps cryopreserved with DPSC-CM exhibit accelerated and sustained maturation of cranial sutures following cranioplasty.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo systematically evaluate the osteogenesis of cranial sutures, histological analyses were conducted at 4 and 8 weeks postoperatively. H\u0026amp;E staining revealed enhanced osteogenic activity in the DPSC-CM group, characterized by dense lamellar bone formation within connective tissue bridging the defect margins, with no evidence of bone resorption. In contrast, the DMSO and \u0026alpha;-MEM groups exhibited localized resorption at cranial flap centers, while severe resorption was observed in NS and SP groups, including cranial flap degradation in some specimens (Figure 3A). Masson trichrome staining showed that the cranial flaps preserved with DPSC-CM formed a large amount of bone matrix and collagen with strong continuity at suture of the cranial flaps, whereas the other groups showed only a small amount of collagen fibers (Figure 3B). Notably, DPSC-CM-treated specimens displayed progressive osteogenic enhancement from week 4 to 8, evidenced by increased osteoblast density on H\u0026amp;E staining and expanded bone matrix and collagen production on trichrome staining (Figure 3A-B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 DPSC-CM promotes bone and vascular regeneration-related proteins expression and modulates the inflammatory microenvironment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo substantiate the osteopreservative effects of DPSC-CM at the molecular level, we performed protein-level validation of its biological activity. Total protein extracts from fresh bone tissues harvested at 4 and 8 weeks post-cranioplasty underwent immunoblotting analysis. DPSC-CM-preserved cranial flaps demonstrated significantly elevated expression of osteogenic regulators RUNX2 and OCN, with quantitative densitometry revealing 2.3- and 3.1-fold increases respectively compared to other preservation groups at 4-week (Figure 4A-B). Concurrently, enhanced neovascularization was evidenced by upregulated CD31 expression (1.8-fold increase vs\u0026nbsp;\u0026alpha;-MEM groups, p＜0.05), indicating superior angiogenic potential relative to comparator groups. Protein expression profiles in the DPSC-CM group at week 8 exhibited comparable results, maintaining consistent molecular signatures with previous observations (Figure 4E-F).\u003c/p\u003e\n\u003cp\u003eNotably, DPSC-CM treatment both exerted dual regenerative-immunomodulatory effects at week 4 and week 8, as reflected by: (1) reductions in pro-inflammatory mediators TNF-\u0026alpha; and IL-6; (2) elevation of anti-inflammatory IL-10 (Figure 4C-D,G-H). This coordinated modulation of osteogenesis, vascularization, and inflammatory microenvironment underscores DPSC-CM\u0026apos;s multifaceted therapeutic profile as a preservation solution for cranial flaps in cranial reconstruction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition, we also performed immunofluorescence staining on bone tissue sections from different groups at weeks 8 to evaluate the expression levels of important proteins in the bone formation process. In both NS and SP groups, substantial bone resorption following reimplantation resulted in incomplete visualization of the grafted cranial flaps, accompanied by markedly reduced expression of osteogenic markers (RUNX2 and OCN) and angiogenic marker CD31 (Figure 5). Multiplex immunofluorescence analysis revealed that DPSC-CM-preserved cranial flaps significantly enhanced the expression of osteogenic regulators (OCN and RUNX2) at cranial sutures, concomitant with upregulated CD31 expression, indicating robust neovascularization (Figure 5). These findings were fully consistent with complementary western blot data from both in vivo and in vitro experiments, thereby validating the reliability of our observations across multiple analytical platforms.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 DPSC-CM promotes the proliferation and osteogenic differentiation of MC3T3-E1 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the notable \u0026nbsp; preservation effects of DPSC-CM observed in vivo, we sought to investigate its intrinsic regenerative potential in bone repair and angiogenesis. This premise prompted subsequent cellular-level investigations to systematically characterize DPSC-CM\u0026apos;s mechanistic contributions to these biological processes. Research indicates that promoting bone repair and regeneration in vivo primarily involves enhancing the homing and proliferation of osteoblasts\u003csup\u003e33\u003c/sup\u003e. Therefore, we selected mouse embryonic osteoblast cells (MC3T3-E1 cells) to investigate the role of DPSC-CM in promoting cell proliferation and osteogenesis.To evaluate the effects of DPSC-CM on MC3T3-E1 cell proliferation and migration, we conducted CCK-8, wound-healing, and transwell assays. And we used untreated cells as a negative control and cells co-cultured with DPSCs as a positive control.CCK-8 analysis demonstrated that DPSC-CM co-culture induced a significant enhancement in MC3T3-E1 cell viability relative to both control and DPSCs-treated groups \u0026nbsp;(Figure 6A). Migration assessments revealed that while DPSCs and DPSC-CM both stimulated directional movement of MC3T3-E1 cells, DPSC-CM exhibited superior migratory induction capacity. Quantitative analysis of wound closure rates \u0026nbsp;(Figure 6C, F) and transwell membrane penetration \u0026nbsp;(Figure 6D, G) confirmed statistically greater migration potential in DPSC-CM-treated cells compared to DPSCs-co-cultured counterparts (p\u0026lt;0.01).\u003c/p\u003e\n\u003cp\u003eTo investigate the osteogenic regulatory effects of DPSC-CM on MC3T3-E1 cells, we performed systematic biochemical characterization. Western blot analysis revealed significantly elevated expression levels of key osteogenic transcription factors RUNX2 and matrix protein OCN in DPSC-CM-treated cells compared to controls (Figure 6B, E). Subsequent evaluation of early osteogenic differentiation through ALP activity assays demonstrated that DPSC-CM exposure induced pronounced alkaline phosphatase activation. Quantitative analysis of ALP staining intensity showed statistically significant differences (p\u0026lt;0.01) between DPSC-CM-treated cells and both CON and DPSCs groups (Figure 6H, J), with enzymatic activity measurements corroborating these findings.\u003c/p\u003e\n\u003cp\u003eTo assess terminal differentiation capacity, mineralization potential was quantified using ARS staining. DPSC-CM-treated specimens exhibited markedly enhanced calcium deposition compared to CON and DPSCs groups, with quantitative spectrophotometric analysis confirming 4.2-fold and 1.3-fold increases in mineralization respectively (Figure 6I, K). This multi-parametric evaluation consistently demonstrated that DPSC-CM not only potentiates osteogenic differentiation of MC3T3-E1 cells but surprisingly exhibits mechanistically superior osteoinductive properties compared to direct DPSCs co-culture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.\u003c/strong\u003e \u003cstrong\u003e6 DPSC-CM exhibits potent pro-angiogenic activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the angiogenic regulatory potential of DPSC-CM, we established a co-culture system with HUVECs to evaluate migratory and angiogenic capacities. Transwell assays demonstrated that both DPSCs and DPSC-CM stimulated HUVECs migration (Figure 7A, E), with pronounced cellular infiltration into lower chambers (Figure 7B, F). Notably, DPSC-CM-treated HUVECs exhibited statistically significant enhancement in migratory capacity compared to both CON and DPSCs groups (p＜\u0026nbsp;0.001).\u003c/p\u003e\n\u003cp\u003eSubsequently, angiogenic potential was assessed through tubulogenesis assays. DPSC-CM-treated HUVECs developed more complex tubular networks compared to controls (Figure 7C), with quantitative analysis revealing significant increases in total vascular structures, evidenced by elevated numbers of tubular segments and branching points \u0026nbsp;relative to CON and DPSCs groups (Figure 7H, I). Furthermore, western blot analysis confirmed upregulated expression of CD31, a key endothelial junction marker, in DPSC-CM-exposed HUVECs (Figure 7D, G). These findings collectively demonstrate that DPSC-CM enhances both migratory competence and angiogenic functionality in endothelial cells.\u0026nbsp;\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCranioplasty involving autologous cranial flaps transplantation or synthetic substitutes is critical for reestablishing physiological cerebrospinal fluid dynamics and restoring cerebral perfusion following decompressive craniectomy (DC)\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. While autologous cranial flaps transplantation remains the clinical gold standard for cranial reconstruction\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, the absence of standardized preservation protocols presents a significant clinical challenge. The standards for cryopreservation of cranial flaps may be summarized as follows: They must ensure non-toxicity (e.g., DMSO\u0026thinsp;\u0026le;\u0026thinsp;10%), sterility, low endotoxin levels, programmed freezing (1\u0026deg;C/min to -150\u0026deg;C), and verification of cell viability\u0026thinsp;\u0026ge;\u0026thinsp;70% and bone structural integrity. The current preservation strategies\u0026mdash;including cryopreservation using saline, DMSO, glycerol, or ethanol solutions, as well as subcutaneous storage\u0026mdash;all have their respective drawbacks. For instance, DMSO exhibits cytotoxic effects on bone cells, ethanol solution storage leads to loss of bone cell viability, and subcutaneous implantation of bone flaps requires additional surgical procedures along with associated pain, often accompanied by bone resorption during preservation. Moreover, these methods frequently result in postoperative complications following cranioplasty, such as microbial colonization, vascular injury, peri-implant fibrosis, chronic pain, impaired osseointegration, and progressive bone resorption\u003csup\u003e\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. These limitations underscore the urgent need for optimized bone preservation methodologies in neurosurgical practice.\u003c/p\u003e \u003cp\u003eContemporary developments in regenerative medicine have validated the bone regenerative capacity of MSCs secretomes across multiple tissue origins. DPSCs emerge as particularly advantageous MSC sources given their abundant availability, minimally invasive harvest procedures, and immunologically favorable characteristics\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The DPSC-CM comprises various bioactive components - including signaling proteins, extracellular vesicles, and genetic regulators - that synergistically mediate tissue repair through immunomodulation and regeneration\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Our transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) of DPSC-CM further confirmed the presence of abundant vesicles with particle sizes consistent with exosomes. These distinctive properties position DPSC-CM as a promising candidate for cranial flaps preservation. Given these results, we employed a combination of DMSO and DPSC-CM for cryopreservation and subsequent revival of osteoblasts (MC3T3-E1) and HUVECs, preliminarily validating its protective effects at the cellular level. Post-thaw observations revealed that the DMSO\u0026thinsp;+\u0026thinsp;DPSC-CM group exhibited significantly accelerated cell proliferation rates. This suggests that DPSC-CM can functionally replace serum by providing essential nutrients (e.g., proteins and growth factors) to cryopreserved cells, thereby supporting viability and mitigating freeze-thaw stress.It has been reported that DPSC-CM contains several growth factors, and the biological effects of DPSC-CM in promoting bone regeneration have been investigated\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Chouaib et al. identified 16 distinct growth factors in DPSC-CM through human growth factor antibody array analysis, all of which exhibited expression levels above the detection limit. Surprisingly, the highly expressed bone morphogenetic proteins (BMP-4, BMP-5, and BMP-7), which belong to the transforming growth factor beta family, were found to play crucial roles in odontogenesis, osteogenesis, and chondrogenesis, as well as in fracture repair and osteoblast differentiation\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.And in vivo, Ogata et al. observed the migration of rat MSCs and endothelial cells to the site of cranial defects using in vivo imaging methods and showed that MSC-CM induced early bone regeneration by accelerating the migration of endogenous MSCs\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Building upon these findings, we propose that these growth factors may critically contribute to the deep cryopreservation of cranial bone flaps. To test this hypothesis, we employed DPSC-CM as a preservation medium for cranial flaps, and to compare it with the existing 0.9 percent NaCl solution (NS), dimethyl sulfoxide (DMSO) and subcutaneous preservation (SP) in clinical practice, investigating its capacity to maintain the viability and structural integrity of autologous cranial flaps during deep cryopreservation (The α-MEM group served as an experimental control to account for potential confounding effects of culture medium nutrients on bone preservation).\u003c/p\u003e \u003cp\u003eAfter 4 weeks of deep cryopreservation in designated preservation solutions, the calvarial bone flaps underwent cranioplasty. Micro-CT 3D reconstruction analysis of postoperative outcomes demonstrated successful osseointegration in the majority of cryopreserved autologous cranial flaps. These autologous cranial flaps showed extensive healing tissue formation at week 4, followed by moderate bone healing at week 8. DPSC-CM-preserved cranial flaps induced extensive bone healing, as evidenced by elevated CT indices such as BS, BV, and BMD. In contrast, some degree of bone resorption, deformation, and fracture occurred in the subcutaneously preserved and 0.9 percent NaCl solution-preserved groups. In fact, significant bone formation was observed in vivo at an early stage in the DPSC-CM group (about a 1.2-fold higher osteogenic ratio at week 2 compared to the other groups). Interestingly, in histological analysis, the DPSC-CM group had significantly more newly generated osteoblasts (blue) and mature osteocytes (red) at the bone defect site than the other groups, and a certain number of bone unit structures appeared. Using immunofluorescence staining of histological analysis, RUNX2 and OCN showed elevated expression at the bone defects, which was significantly different from other groups.\u003c/p\u003e \u003cp\u003eExtensive research has established that DPSC-CM enhances cellular proliferation and migration, modulates endothelial behavior to stimulate vascular-like structure formation, and upregulates angiogenic gene expression\u003csup\u003e\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Consistent with these findings, our immunohistochemical analysis of DPSC-CM-treated bone sections revealed CD31-positive vascular endothelial cells and neo-vasculature surrounding regenerated bone. Notably, DPSC-CM demonstrates multifaceted therapeutic potential, exhibiting anti-inflammatory properties evidenced by its capacity to mitigate diabetic polyneuropathy and aneurysmal subarachnoid hemorrhage-induced neuroinflammation\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Our study further revealed that DPSC-CM-preserved cranial flaps not only upregulated osteogenic markers (RUNX2/OCN) and angiogenic protein CD31 at surgical sites, but also significantly modulated inflammatory responses by suppressing pro-inflammatory cytokines (TNF-α, IL-6) while enhancing anti-inflammatory IL-10 expression, creating a favorable microenvironment for accelerated healing. These findings suggest DPSC-CM orchestrates bone regeneration through dual mechanisms: activating recipient cells to secrete osteogenic paracrine factors and vascular growth mediators, while simultaneously reshaping the cellular niche through localized immunomodulation. The coordinated regulation of angiogenic, osteogenic, and anti-inflammatory pathways collectively contributes to its enhanced regenerative efficacy in cranial repair.Given the remarkable advantages of DPSC-CM in cryopreserving cranial bone flaps, we sought to further investigate the intrinsic effects of DPSC-CM itself while conducting comparative analyses with DPSCs. We found that DPSC-CM enhanced cell proliferation, migration, osteogenesis, and the expression of osteogenic and angiogenic proteins, including RUNX2, OCN, and CD31. It also promoted capillary sprouting and tube formation in HUVECs. Interestingly, the addition of DPSC-CM had a more dramatic effect on bone regeneration compared to the transplantation of DPSCs themselves.\u003c/p\u003e \u003cp\u003eOur findings demonstrate the multifaceted therapeutic potential of DPSC-CM in cranial flaps preservation, as DPSC-CM-treated cranial bones exhibit concurrent osteogenic, angiogenic, and anti-inflammatory activities following cranioplasty. In summary, DPSC-CM can be used as a new preservation fluid for autologous cranial flaps preservation with deep cryopreservation, which has the potential to promote angiogenesis and bone regeneration to a certain extent, helps to regulate the local microenvironment, and has certain advantages in comparison with relatively common preservation fluids and SP preservation.\u003c/p\u003e \u003cp\u003eThe underlying mechanisms by which DPSC-CM preserves calvarial flaps remain to be fully elucidated. However, the enhanced efficacy of DPSC-CM in flap preservation may be attributed to its unique bioactive composition and multimodal regulatory effects\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. As a cell-free system, DPSC-CM contains a cocktail of trophic factors (e.g., VEGF, FGF-2, and IGF-1), anti-inflammatory cytokines (e.g., IL-10, TGF-β), and extracellular vesicles (EVs) carrying functional miRNAs (e.g., miR-21-5p, miR-146a). These components may collectively orchestrate a regenerative microenvironment through the following mechanisms: mitigation of ischemic and hypoxic damage, suppression of osteoclastic resorption, ECM remodeling and osteogenic activation, as well as immunomodulation and microbial defense\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCritically, DPSC-CM-based strategies eliminate the need for cytotoxic solvents (e.g., DMSO) and surgical trauma, thereby addressing the dual challenges of cellular toxicity and iatrogenic damage inherent in conventional preservation protocols. Future studies should focus on clarifying the specific mechanisms of DPSC-CM in calvarial flap preservation and explore lyophilization techniques to enhance clinical feasibility.\u003c/p\u003e \u003cp\u003eHowever, there are several limitations that remain in this study. Firstly, in cranioplasty, we did not use a medium (e.g., bone cement) for fixation of the cranial flap, which would have led to the displacement of the autologous cranial flap after cranioplasty. Andrade et al. have shown that freezing leads to denaturation of the collagenous components in the bone cortex and that this qualitative change is exacerbated by decreasing temperatures and prolongation of freezing time\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. We did not investigate the degree of degeneration associated with cranial flap freezing preservation and whether it affects further bone regeneration and angiogenesis. No biomechanical tests were also performed to determine the functional load-bearing capacity (e.g., strength and stiffness) of the bone after cranioplasty with autologous cranial flaps\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eTo our knowledge, this study pioneers the application of dental pulp stem cell-conditioned medium (DPSC-CM) as a biologically active cryopreservation solution for autologous cranial bone flaps. Our findings demonstrate that DPSC-CM-preserved cranial flaps exhibit enhanced osteogenic potential and angiogenic capacity post-cranioplasty, while significantly modulating local inflammatory responses at the implantation site. It provides a new vision and a mechanistic basis for determining its clinical application.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all the members at the Center of Regenerative Medicine for providing technical support and valuable suggestions for this project. We declare that they have not use AI-generated work in this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: QY, YL, YH, WX\u003c/p\u003e\n\u003cp\u003eMethodology: YL, LYH, BW, XW, JL, PM\u003c/p\u003e\n\u003cp\u003eInvestigation: QY, WX, JL, LD, ZY\u003c/p\u003e\n\u003cp\u003eVisualization: YL, LYH, BW, CY, XW, ZY\u003c/p\u003e\n\u003cp\u003eSupervision: QY, YH\u003c/p\u003e\n\u003cp\u003eWriting\u0026mdash;original draft: YL, LYH, BW\u003c/p\u003e\n\u003cp\u003eWriting\u0026mdash;review \u0026amp; editing: YL, QY, YH, WX\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the key Project of Ministry of Science and Technology China (YFXM2022000264 from QY), Chutian Researcher Project (X22020024 from YH) and the National Natural Science Foundation of China (U22A20314 from YH), Jiangxi\u0026quot;Ganpo Talents Program\u0026quot;(gpyc20240204 from WX).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data needed to evaluate the conclusions in the paper are present in the paper and/or the supplementary information. The raw data supporting the findings of this study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the Declaration of Helsinki.The experimental method of dental pulp stem cell extraction was approved by the Clinical Research Ethics Committee of Wuhan University People\u0026apos;s Hospital (protocol code: WDRY-2022-K025, approval date: December 1, 2021) Approved Project Name: Extraction of Human Dental Pulp Stem Cells and Basic Research. About animal testing, study entitled \u0026quot; Application of dental pulp stem cell conditioned medium combined with cryogenic preservation of autologous skull flap\u0026quot; has been approved by the Experimental Animal Ethics Committee of Wuhan University People\u0026apos;s Hospital (date: November 4, 2023, No. 20231102B) and was conducted in accordance with AVMA guidelines. We had institutional approval for performing experiments using human cells. HUVECs were obtained from Pricella Biotechnology(Wuhan, China). Pricella Biotechnology Company has confirmed that there was initial ethical approval for collection of human cells, and that the donors had signed informed consent.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe animal experiment protocol was approved by the Ethics Committee of Animal Experimentation of Wuhan University (protocol code: 20231102B, approval date: November 4, 2023). Title of the approved project: Application of dental pulp stem cell conditioned medium combined with cryogenic preservation of autologous skull flap. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consent for publication.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFootnotes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher\u003c/strong\u003e\u003cstrong\u003e\u0026rsquo;\u003c/strong\u003e\u003cstrong\u003es Note\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYe Liu, Yonghao Liu and Zhifei Ye contributed equally to this work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributor Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQingsong Ye, Email: [email protected].\u003c/p\u003e\n\u003cp\u003eYan He, Email: [email protected].\u003c/p\u003e\n\u003cp\u003eWei Xiong, Email: [email protected].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHofmeijer, J.\u003cem\u003e et al.\u003c/em\u003e Surgical decompression for space-occupying cerebral infarction (the Hemicraniectomy After Middle Cerebral Artery infarction with Life-threatening Edema Trial [HAMLET]): a multicentre, open, randomised trial. \u003cem\u003eThe Lancet. 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D.\u003cem\u003e et al.\u003c/em\u003e Neural crest-derived mesenchymal progenitor cells enhance cranial allograft integration. \u003cem\u003eStem cells translational medicine\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 797-809, doi:10.1002/sctm.20-0364 (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Decompressive craniectomy, Autologous cranial flaps, Deep cryopreservation, Dental pulp stem cells, Dental pulp stem cell-conditioned medium, Bone regeneration, Vascular regeneration","lastPublishedDoi":"10.21203/rs.3.rs-5909455/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5909455/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAutologous cranial flaps preservation after decompressive craniectomy (DC) is crucial for cranioplasty, yet standard cryopreservation carries high complication rates (15-35%), primarily infections and bone resorption. These complications frequently necessitate surgical revisions and increase morbidity risks. Current methods lack standardized preservation solutions that simultaneously ensure osteocyte survival and prevent microbial growth. Developing integrated bacteriostatic and osteoprotective storage media remains an urgent unmet need to enhance patient outcomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjectives\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study investigates optimized preservation protocols for autologous cranial flaps to mitigate post-cranioplasty complications, while evaluating the preservative efficacy and clinical translation potential of dental pulp stem cell-conditioned medium (DPSC-CM) as a novel osteogenic storage solution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDental pulp stem cells (DPSCs) were cultured in serum-free medium to generate DPSC-CM. To evaluate preservation efficacy of DPSC-CM, first, DPSC-CM was preliminarily evaluated by examining the cell viability after freezing and resuscitation. Second, a murine critical-size calvarial defect model was surgically established. Autologous cranial flaps underwent 4-week storage in experimental preservation solutions (DPSC-CM versus conventional cryoprotectants) were reimplanted. Postoperative bone regeneration was systematically quantified through high-resolution micro-CT analysis and histomorphometric evaluation of bone regeneration capacity. Given DPSC-CM's osteopreservative potential, in vitro analyses confirmed DPSC-CM's osteogenic/angiogenic capacity through proliferation/migration assays, osteogenic differentiation, and biomarker quantification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDPSC-CM demonstrated superior efficacy in cell preservation. Studies in a mouse model of cranial defects showed that the cranial flaps preserved with DPSC-CM in combination with deep cryopreservation (-196°C) showed significantly better bone healing after cranioplasty than the other groups, and their neoangiogenic and anti-inflammatory abilities were also significantly better than those of the other groups. DPSC-CM was found to be superior to DPSCs in the osteogenesis of mouse embryonic osteoblast cells (MC3T3-E1 cells) and the angiogenesis of human umbilical vein endothelial cells (HUVECs).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsidering the superiority of osteogenesis and vascularization in vivo and in vitro, as well as the modulating of the local inflammatory microenvironment, DPSC-CM synergistic combination deep cryopreservation emerges as a novel strategy of preserving cranial flaps after DC. This multidisciplinary approach establishes a transformative framework for advancing autologous cranial flaps storage technologies, demonstrating translational promise through biological optimization of traditional cryopreservation protocols.\u003c/p\u003e","manuscriptTitle":"Application of dental pulp stem cell-conditioned medium combined with deep crypreservation of autologous cranial flaps","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-17 08:28:39","doi":"10.21203/rs.3.rs-5909455/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-04-15T14:36:21+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-15T08:48:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-14T23:57:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Stem Cell Research \u0026 Therapy","date":"2025-04-10T06:09:17+00:00","index":"","fulltext":""},{"type":"decision","content":"Minor Revision","date":"2025-02-17T04:24:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8b5975b2-f1b1-4ff3-872b-45af949b1de6","owner":[],"postedDate":"April 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-09T15:58:41+00:00","versionOfRecord":{"articleIdentity":"rs-5909455","link":"https://doi.org/10.1186/s13287-025-04407-1","journal":{"identity":"stem-cell-research-and-therapy","isVorOnly":false,"title":"Stem Cell Research \u0026 Therapy"},"publishedOn":"2025-06-02 15:56:58","publishedOnDateReadable":"June 2nd, 2025"},"versionCreatedAt":"2025-04-17 08:28:39","video":"","vorDoi":"10.1186/s13287-025-04407-1","vorDoiUrl":"https://doi.org/10.1186/s13287-025-04407-1","workflowStages":[]},"version":"v1","identity":"rs-5909455","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5909455","identity":"rs-5909455","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}