Conditioned medium derived from 3D tooth germs: A novel cocktail for stem cell priming and early in vivo pulp regeneration

Abstract Objectives Conditioned medium (CM) from 2D cell culture can mitigate the weakened regenerative capacity of the implanted stem cells. However, the capacity of 3D CM to prime dental pulp stem cells (DPSCs) for pulp regeneration and its protein profile are still elusive. We aim to investigate the protein profile of CM derived from 3D tooth germs, and to unveil its potential for DPSCs‐based pulp regeneration. Materials and Methods We prepared CM of 3D ex vivo cultured tooth germ organs (3D TGO‐CM) and CM of 2D cultured tooth germ cells (2D TGC‐CM) and applied them to prime DPSCs. Influences on cell behaviours and protein profiles of CMs were compared. In vivo pulp regeneration of CMs‐primed DPSCs was explored using a tooth root fragment model on nude mice. Results TGO‐CM enhanced DPSCs proliferation, migration, in vitro mineralization, odontogenic differentiation, and angiogenesis performances. The TGO‐CM group generated superior pulp structures, more odontogenic cells attachment, and enhanced vasculature at 4 weeks post‐surgery, compared with the TGC‐CM group. Secretome analysis revealed that TGO‐CM contained more odontogenic and angiogenic growth factors and fewer pro‐inflammatory cytokines. Mechanisms leading to the differential CM profiles may be attributed to the cytokine–cytokine receptor interaction and PI3K‐Akt signalling pathway. Conclusions The unique secretome profile of 3D TGO‐CM made it a successful priming cocktail to enhance DPSCs‐based early pulp regeneration.

and compromised regenerative functions. 5 Donor systemic diseases and senescence can also impair the MSC reparative capacity, 6,7 with unsatisfactory clinical trial outcomes. [8][9][10] In vitro MSC priming is a promising technique in circumventing these shortcomings by modulating the secretome, 11 with hypoxia, cytokines, and 3D cell culture proposed as in vitro priming stimuli. [12][13][14][15] Conditioned medium (CM), the supernatant collected from the cell culture medium, contains the MSC secretome, which includes extracellular matrix components, growth factors, and cytokines. 16 Already being broadly used, 17 CM priming can promote tissue regeneration, angiogenesis, immunomodulation, and anti-fibrosis. 18 DPSC-derived CMs can recapitulate parent MSC functions, suggesting potential in dental and extra-oral tissue engineering applications. 19 Most current CM types are harvested from 2D cell culture, 17 including tooth germ cell-derived CM (TGC-CM). [20][21][22] Nevertheless, living systems like tooth germ organs (TGOs) exist in well-organized 3D arrangements with intricate cellular and extracellular interactions; these in vivo bioprocesses can barely be replicated by 2D monolayer cell cultures. In contrast, 3D cell cultures create more accurate physiological simulations of in vivo microenvironments, with gene and protein expression closely resembling those within original living systems. 23,24 As pulpogenesis is orchestrated by bio-factors secreted by mesenchymal and epithelial cells within the tooth germ during embryonic development, 25 we reasonably deduced that tooth germ-derived CM would contain a semblable tooth germ protein profile which could orchestrate pulp repair under physiological and pathological scenarios.
While the literature on 2D CM-based DPSC-priming abounds, few studies have addressed 3D CM-based DPSC-priming for pulp regeneration. 19 Only a few studies have analysed the protein profile of 2D CM derived from DPSCs. 26,27 It remains unclear whether and how CM of 3D tooth germ organ (TGO-CM) will impact on pulp regeneration.
Due to its secretome biomimicry, we hypothesized that 3D TGO-CM would triumph over its 2D counterpart in promoting early pulp regeneration. We compared the effects of 3D TGO-CM and 2D TGC-CM on in vitro DPSC proliferation, migration, differentiation, and mineralization, and on in vivo pulp regeneration after 4 weeks. Additionally, we analysed the compositional differences between the two CMs and explored the resulting biomechanisms. To the best of our knowledge, this study is the first to report the protein profile of 3D cultured tooth germs and the first to unveil its potential in priming DPSCs for early in vivo pulp regeneration. Our results may shed light on stem cell priming for pulp regeneration using a trace-back-to-organ approach. Mandibular first molar tooth germs from newborn CD-1 mice were surgically dissected following a previously reported protocol. 28 All animal experiments conformed to related ethical principles.
Human DPSCs and mouse TGCs were isolated as previously reported 28 and cultured in L-DMEM supplemented with 15% foetal bovine serum (FBS), 100 U/ml penicillin-G, and 100 mg/ml streptomycin, under 5% CO 2 at 37°C. The culture medium was refreshed every 2-3 days. Cells were passaged when they reached 80% confluency and expanded for all experiments at passages 3-5.

| Immunofluorescent staining of mouse TGCs
Immunofluorescent staining was performed as per the manufacturer's instructions, using anti-CK14 and anti-vimentin antibodies (Abcam, Cambridge, UK) and treated with related secondary antibodies. Samples were blocked using Vectashield mounting medium containing DAPI. Images were captured using a confocal immunofluorescence microscope. 29

| Preparation of 2D TGC-CM
At 80% confluency, the TGC medium was replaced with FBS-free L-DMEM. After 16 h, the resulting medium was collected, filtered, and centrifuged at 1,000 rpm for 5 min at 4°C. The supernatant was harvested as 2D TGC-CM, stored at −80°C, and used without concentration.

| Preparation of 3D TGO-CM
Fifteen TGOs of newborn mice were 3D ex vivo cultured in 1-ml FBS-free L-DMEM under 5% CO2 at 37°C. The medium was refreshed at the same time intervals as the TGC-CM culture. At 8, 16, and 24 h, the resulting medium was collected, filtered, and centrifuged at 1,000 rpm for 5 min at 4°C. The supernatant was harvested as 3D TGO-CM, stored at −80°C, and used without concentration.

| DPSCs priming with CMs
Conditioned mediums harvested at 16 h were used to prime DPSCs.
L-DMEM supplemented with 15% FBS was used in the control medium group in all assays. In the TGC-CM and TGO-CM group, L-DMEM was supplemented with 15% FBS for cell proliferation and migration assays or supplemented with 15% FBS containing related osteoinductive ingredients (1% penicillin/streptomycin, 50 μg/ ml ascorbic acid, 10 nmol/L dexamethasones, and 5 mmol/L βglycerophosphate) for in vitro odontogenic/osteogenic differentiation assays. All groups were incubated under 5% CO 2 at 37℃.

| Cell proliferation assay
Dental pulp stem cells were seeded at 10,000/well in 96-well plates and cultured for 5 days. CCK-8 assay was conducted as per the manufacturer's protocol on days 1-5, and cell viability was measured via UV spectrophotometry using absorbance at 450 nm.   Table S1. 30

| Western Blot assay
Seven days after osteoinduction, Western blotting was conducted as described in our previous study, 28

| Preparation of root fragments of human teeth
Mature single-root premolars without previous root canal treatment were collected from adult patients. Tooth root fragments were prepared as previously reported. 32 Briefly, 5-mm fragments were sectioned with pulp tissue, pre-dentin, and partial dentin removed.
Canals were enlarged to 3 mm in diameter, treated with 5% EDTA for 5 min, and subjected to ultrasonic treatment for 10 min. The larger end was then sealed with mineral trioxide aggregate. Fragments were stored in PBS containing 50 mg/ml streptomycin and 50 U/ ml penicillin at 4℃ and disinfected by UV sterilization. The fluid hydrogel containing 4 × 10 5 DPSCs from each group was injected into the canal cavity of root fragments. The hydrogel-DPSCs compounds jelled quickly to prevent leakage. were performed as previously reported. 32 Briefly, surgery was performed on 6-week-old immunocompromised nude mice (n = 12) under general anaesthesia. A single hydrogel-DPSCs filled fragment was transplanted subcutaneously into the dorsal side of each mouse.

| Subcutaneous implantation into nude mice
All mice were euthanized at 4 weeks post-surgery, and all fragments were collected.

Histomorphology was observed using Scanscope CS and Image
Scope software (Aperio, Sausalito, CA). Quantitative analysis was conducted using ImageJ software to measure the percentage of regenerated pulp tissue area inside the root canal, the percentage of pulpal blood vessel area, and the number of cells attached to the dentin wall.

| Statistical analysis
Data analysis and graphical preparation were conducted using GraphPad Prism 8.3. Data are described as mean ±standard deviation and replicated for three independent experiments. One-way ANOVA with Tukey's multiple comparison test was performed to detect statistical differences. Statistical significance was set at p < 0.05.

| RE SULTS
The study design is depicted in Figure 1.

| Cell types within tooth germs
Tooth germ cells from digested tooth germ explants ( Figure 2

| TGO-CM enhanced in vitro DPSCs behaviour
Transwell migration assay revealed that the TGO-CM group pre- Quantitatively, DPSCs primed by TGO-CM proliferated more quickly and had increased by 0.56 ± 0.28 and 0.22 ± 0.14 folds, respectively, compared to the control group and TGC-CM group at day 5 (p < 0.05, Figure 4J). The number of migrated DPSCs in the TGO-CM group was 1.09 ± 0.19 and 0.55 ± 0.12 folds greater than those of the control group and TGC-CM group, respectively (p < 0.0001, Figure 4K). The ARS unit count in the TGO-CM group was significantly larger, by 13.81 ± 2.96 and 8.81 ± 2.48 folds, compared to those of the control group and TGC-CM group, respectively (p < 0.01, Figure 4L). folds greater (p < 0.05, Figure 5F).

| Possible mechanisms leading to the protein profile differences
GO cellular component analysis ( Figure 8A) revealed that the differential proteins existed in forms of the receptor complex, secretory granule, growth factor complex, and extracellular matrix, with 5, 5, 6, and 23 detected protein types, respectively (p < 0.05). GO biological process analysis confirmed that these differential proteins were linked to cytokine production, positive cell adhesion regulation, and inflammatory processes ( Figure 8B). These results aligned with the downregulated inflammation-related cytokine activity in TGO-CM ( Figure 7C, p < 0.05). GO molecular function analysis ( Figure 8C) revealed that these proteins were functionally related to the activity and receptor bindings of growth factors, chemokines, and cytokines (p < 0.05). KEGG analysis identified possible signalling mechanisms that led to such differential protein profile, including cytokinecytokine receptor interaction, TNF signalling pathway, and PI3K-Akt signalling pathway ( Figure 8D).

| DISCUSS ION
Tooth germ comprises mesenchymal and epithelial components, and its organogenesis is a temporal interplay involving diverse biofactors between these two elements. 25,33 Successful CM production requires careful collection timing; 2D CM can be harvested between 24-72 h and every 2-4 days after seeding. 19 3D ex vivo culture of tooth germs mimics the physiology of the original microenvironment, with hierarchical tissue structures and graded chemical distribution. 23,24 In our study of the 3D culture, hypoxia, undernutrition, and accumulation of metabolic waste resulted in greater damage at the organ core. The elevated apoptosis rate in tissue culture compared to that in cell line culture can distort the protein secretion profile 34 ; excessive tissue debris and by-products may interfere with the subsequent CM content analysis. 35 Thus, to counter the tissue damage presented at 24 h and the low protein secretion at 8 h, 16 h was adopted as the harvest timing.

F I G U R E 8
Possible mechanisms that led to the protein profile differences. (A) GO cellular component analysis: differential proteins mainly existed in forms of the receptor complex, secretory granule, growth factor complex, and extracellular matrix. (B) GO biological process analysis: differential proteins were mainly linked to cytokine production, positive cell adhesion regulation, and inflammatory processes. (C) GO molecular function analysis: differential proteins were functionally related to the activity and receptor bindings of growth factors, chemokines, and cytokines (D) KEGG analysis: cytokine-cytokine receptor interaction, TNF signalling pathway, and PI3K-Akt signalling pathway may be involved Vectors-mediated MSCs engineering has also been reported to successfully prime stem cells. 36 47 Here, lacking labelling analysis, we speculated that the neovascularization was angiogenesis which was derived from host vessel ingrowth through the end opening. 48 Summarily, in vitro and in vivo evidence confirmed the potential of 3D TGO-CM as a priming cocktail to enhance DPSCsmediated pulp regeneration.
Proteome cytokine array, GOEA, and KEGG analysis revealed significant differences between the two CM secretome profiles.
TGO-CM contains more growth factors such as FGF and EGF, which are related to cell proliferation and odontogenic differentiation. 2 Higher levels of angiogenesis-related factors such as angiopoietin-1, PDGF, VEGF, and FGF, in TGO-CM, contributed to vasculature formation. 49 Higher level of CX3CL-1, a type of chemokine with an angiogenic role, 50 was also detected in TGO-CM. In a clinical scenario, pulp blood perfusion is often limited by a narrow apical foramen or progressive inflammation, 51 potentially causing hypoxia in implanted DPSCs. 3D ex vivo culture of tooth germ mimics the in vivo graded oxygen distribution during germ development, with its inner core exhibiting increased hypoxia. As a self-protective response, the tooth germ elevates related gene expression, secreting more biofactors associated with cell survival, proliferation, and angiogenesis. Our results correspond with these observations, demonstrating that hypoxia can guide MSCs towards a pro-angiogenic phenotype and promote angiogenesis. [52][53][54] Inflammation plays an important role in pulp regeneration, 55 as tissue repair can only occur at a lower inflammation resolution level. 56 An inflammatory biomolecules influx would impede regen- hypoxia-inducible factor-1α (HIF-1α) and the P13K/AKT signalling pathway. 67 In this study, the biomimic-graded hypoxia inside 3D cultured tooth germs may induce the production of pro-angiogenic biomolecules as an adaptive response.
Due to budget and time constraints, we were unable to address certain limitations. Future efforts may focus on the following aspects: influence of factors such as different embryonic stages of tooth germs, priming duration, and pathological status on protein profiles; more thorough filtering of effective CM components; in-depth mechanisms leading to different secretome profiles; use of orthotopic models of larger animals and longer-term follow-up; optimizing and standardizing the fabrication protocols of 3D CM before use.

| CON CLUS ION
Dental pulp stem cells-based pulp regeneration exhibit shortcomings, and its therapeutic outcomes rely heavily on the unreliable post-implantation functional status of stem cells. In this study, we prepared CM of 3D tooth germ organ and investigated its preconditioning capabilities on DPSCs for early dental pulp regenera- To the best of our knowledge, this is the first study to investigate the protein profile of 3D tooth germs and the first to apply it for early in vivo pulp regeneration. Taken together, our results confirmed that 3D TGO-CM can be applied as a priming cocktail to enhance DPSCs-mediated early pulp regeneration. This study may shed light on personalized stem cell priming for early pulp regeneration using a trace-back-to-organ approach.

ACK N OWLED G EM ENTS
We give special thanks to Ziyi Liu for valuable discussion.

CO N FLI C T O F I NTE R E S T
There are no conflicts of interest in this study.