Construction of dental pulp decellularized matrix by cyclic lavation combined with mechanical stirring and its proteomic analysis

The decellularized matrix has a great potential for tissue remodeling and regeneration; however, decellularization could induce host immune rejection due to incomplete cell removal or detergent residues, thereby posing significant challenges for its clinical application. Therefore, the selection of an appropriate detergent concentration, further optimization of tissue decellularization technique, increased of biosafety in decellularized tissues, and reduction of tissue damage during the decellularization procedures are pivotal issues that need to be investigated. In this study, we tested several conditions and determined that 0.1% Sodium dodecyl sulfate and three decellularization cycles were the optimal conditions for decellularization of pulp tissue. Decellularization efficiency was calculated and the preparation protocol for dental pulp decellularization matrix (DPDM) was further optimized. To characterize the optimized DPDM, the microstructure, odontogenesis-related protein and fiber content were evaluated. Our results showed that the properties of optimized DPDM were superior to those of the non-optimized matrix. We also performed the 4D-Label-free quantitative proteomic analysis of DPDM and demonstrated the preservation of proteins from the natural pulp. This study provides a optimized protocol for the potential application of DPDM in pulp regeneration.


Introduction
The incidence of pulpitis, the inflammation of the pulp, and periapical periodontitis is on the rise due to increasingly refined diets and aging population [1,2].Traditionally, during pulpitis treatment, the inflamed dental pulp is extirpatedand replaced with a specialized dental material to fill the root canal.After root canal therapy (RCT), small number of number of patients still continues to experience persistent pain possibly due to a combination of odontogenic and non-odontogenic causes [3].The pulp tissue contains blood vessels, that are responsible for tooth sensation and play an important role in tooth development and physiological function.Given the importance of pulp function, dental pulp regeneration using tissue engineering is currently actively investigated [4].To date, complete dental pulp regeneration in large nonprimate animals has been performed using dental pulp stem cells (DPSCs) [5].Furthermore, a pilot clinical study demonstrated the biosafety of isolated human mobilized DPSCs transplanted into the pulp cavity [6].
The proliferation and differentiation of DPSCs in the host require an appropriate microenvironment provided by the scaffold material that contains the structural elements necessary for tissue regeneration [7].Decellularized matrix has a great potential as a scaffold for the bioengineered organoid microenvironment [8,9].An ideal organoid matrix should possess high biocompatibility, good mechanical properties, degradability, and the ability to simulate the microenvironment to support and enhance cellular activities, such as proliferation, migration and differentiation [10].To date, several natural and synthetic materials, including collagen [11], gelatin [12], polyethylene glycol [13], chitosan [14], and hyaluronic acid [15], have been used to generate organoids; these materials do not meet all of the requirements of the ideal organoid matrix [10].A decellularized matrix retains the native tissue structure and provides an optimal microenvironment for cell survival, as well as a porous nanofiber scaffold that facilitates stem cell attachment, proliferation, and infiltration, while preserving the structural integrity of nanofibers [16].Furthermore, this type of matrix has already been evaluated as a scaffold in several organs, such as the heart [17], liver [18], esophagus [19], blood vessels [20], nerves [21] and teeth [22].
The objective of decellularization is to eliminate cellular components (CC) and genetic material from tissues, while preserving structural integrity, including the thickness, density, and three-dimensional (3D) structure of the tissues [23].Currently, decellularization strategies primarily include physical, chemical, and enzymatic methods, as well as their combinations [24].Although decellularized matrices have better biocompatibility than other biological materials, each treatment method causes different types of damage to the natural structures of tissues and organs [25].As a result, the 3D structure and active components of the decellularized matrix obtained from different tissues or organs using different decellularization methods would not be the same [26].At present, the investigation of decellularized dental pulp matrices using bovine [27], porcine [28] and human [29] tissues has been reported.
The dental pulp is a loose connective tissue, suitable for biochemical decellularization; however, there are no uniform standard protocols for the detergent type selection and concentration, as well as the washing procedures, which significantly affect the biological properties of the decellularized matrix.The release of bioactive factors is the key advantage of these biomaterials, and proteins retained after preparation play an important role in the subsequent induction of cell proliferation, differentiation, and tissue regeneration [30].Therefore, to fabricate a biomaterial using dental pulp decellularization matrix (DPDM), it is imperative to retain the natural properties of acellular tissues,which would allow the generation of an optimized scaffold for tissue engineering.
In this study, we comprehensively evaluated various decellularization techniques (table 1) and optimized the preparation process for decellularized dental pulp to create an ideal environment for pulp tissue regeneration, ensuring maximum preservation of structural integrity while minimizing damage caused by the decellularization procedure itself.

Determination of pulp tissue volume
To evaluate the initial volume and the volume after decellularization for sample.(n = 5), the drainage method was used as previously described [32].The samples were completely submerged in water, the volume of the increasing water was calculated as their volume.

Hematoxylin and eosin(H&E) staining
Samples (n = 3) were fixed in 4% paraformaldehyde for 24 h at 37 • C. Next, the samples were embedded in paraffin and cut into 5 µm sections.The sections were stained with H&E (G1120-3, Solarbio, China) using the following protocol:samples were rehydrated with gradient alcohols, stained with hematoxylin for 2 min, washed with water, incubated in 1% hydrochloric alcohol for 10 s, stained with eosin for 2 min, dehydrated using gradient alcohols, cleared with xylene, mounted, and seal (564 422, Jiangyuan, China).C for 30 min, and then washed again 3 × 5 min in PBS using the ultrasonic water bath.This process was defined as one decellularization cycle.DPDM samples that completed one decellularization cycle were defined as the C1 group, and the total of six cycles was performed (groups C1-C6, respectively; n = 3) (figure 2(A)).

Decellularization rate calculation
Samples (n = 3/group) from C1-C6 groups were fixed with 4% paraformaldehyde for 24 h.Next, the samples were embedded in paraffin wax, cut into 5 µm sections, and.The samples were stained with H&E.For each sample, the distance between the outer edge of the tissue to the tissue containing the remaining nuclei was determined.Based on our analysis, the C3 group was selected, treated with SDS for 36 h without cycling, and then designated as control (DPDM-36H).

DNA content measurement of DPDM-36H and DPDM-C3 samples
To determine the extent of decellularization, residual DNA content was measured (n = 5).Refer to the methods in the literature for DNA content detection [33].

Detection of matrix protein in DPDM-36H and DPDM-C3 samples
Immunofluorescence staining was performed on sections of natural (untreated) porcine dental pulp and decellularized dental pulp (DPDM-36H and DPDM-C3) tissue sections using anti-laminin, antifibronectin, anti-integrinβ1, and anti-vimentin.DPDM-36H and DPDM-C3 samples (n = 3/group) were prepared as described above, freeze-dried for 4_6 h, and imaged using SEM.Next, DPDM-36H and DPDM-C3 samples were sterilized for 8 h using ethylene oxide, and 1 g DPDM was incubated in 5 ml αMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin for 48 h under aseptic conditions at 37 • C. The extract was obtained after centrifugation at 24 • C, 1000 r, 5 min.The release of Use Elisa kit to detect the release of dentine salivary phosphoric protein (DSPP), dentin matrix acidic phosphoprotein 1 (DMP-1), collagen type I (COL-1), and collagen type III (COL-3) was detected using an ELISA kit according to the manufacturer's instructions (n = 3).

hDPSCs culture
Pulp tissues were collected from healthy teeth extracted for orthodontics of 14-24 years old patients.
(Ethics committee ethical approval number: WCHS-IRB-CT-2021-151).Only teeth without the evidence of caries, pulpitis, or periapical periodontitis were used.hDPSCs were cultured using the tissue block method as previously described [34].When cells reached 80% confluence, they were incubated with trypsin and passaged.The cell passaged 3-5 times were used in this study.

Cell proliferation and viability assessment
The cells were cultured in a medium containing DPDM extract (1 g DPDM in 5 ml αMEM).At the specified time point, the Cell Counting Kit-8 (CCK8) (KeyGEN, China) working solution was added, cells were inoculated for 1 h, and then absorbance at 450 nm was measured using a microplate spectrophotometer (Molecular Devices, USA).To evaluate cell viability, hDPSCs (5000 cells/well) were plated on the glass bottom culture dishes and 500 µl/well of DPDM extract was added.After three days, the live & dead viability/cytotoxicity assay kit (KGA9501-1000, KeyGEN, China) was used to determine cell viability.Images were captured using confocal microscopy (FV3000RS; Olympus Corporation, Tokyo, Japan).

Cell migration
hDPSCs(1 × 10 5 cells/well) were plated in Transwell chambers inside a 24-well plate and 500 µl of DPDM extract or α-MEM was added to each well.After 48 h, the cells were stained with 0.1% crystal violet (Biosharp, China).

4 D-Label-free quantitative proteomic detection 2.5.1. Sample collection
For the experimental group, the pulp (3 mm × 3 mm × 3 mm) from a porcine tooth was decellularized for three cycles.For the control group, the natural (untreated) porcine pulp of was washed using an ultrasonic water bath to remove blood and impurities (n = 3/group).

Protein extraction and digestion
To extract proteins, the samples were lysed using the SDT buffer (4% SDS, 100 mM DTT, 150 mM Tris/HCl, pH 8.0) and proteins concentrations were quantified using the bicinchoninic acid method.Proteins from each sample were collected using the Filter aided proteome preparation method for trypsin enzymatic hydrolysis, followed by peptide desalting using C18 Cartridge (Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 ml, Sigma).Next, the sample were lyophilized and redissolved in 40 µl 0.1% formic acid solution.Based on the frequency of tryptophan and tyrosine in vertebrate proteins, the peptide content was estimated using the ultraviolet spectral density of 0.1% (g l −1 ) solution with an extinction coefficient of 1.1 at 280 nm.

Liquid chromatography-mass spectrometry (LC-MS)/MS analysis
LC-MS/MS analysis was performed using a the tim-sTOF Pro Mass Spectrometer (Bruker) coupled to a NanoElute (Bruker Daltonics) for 60/120/240 min.The peptides were loaded onto a reverse phase trap column (Thermo Scientific Acclaim PepMap100, 100 µm * 2 cm, nanoViper C18).The trap column was connected to the C18-reversed phase analytical column (Thermo Scientific Easy column, 10 cm long, 75 µm inner diameter, 3 µm resin) for separation in buffer A(0.1% formic acid) and linear gradient separation with buffer B(84% acetonitrile and 0.1% formic acid).The mass spectrometer was operated in the positive ion mode.The mass spectrometer collected an ion mobility mass spectrum with a mass range of m/z 100-1700 and a mass range of 1/k0 0.6-1.6 with a target intensity of 1.5 k and a threshold of 2500 for 10 PASEF MS/MS cycles.Active exclusion was enabled at a release time of 0.4 min.

Identification and quantitation of proteins
For identification and quantitative analysis, MaxQuant 1.5.3.17software was used to combine and retrieve the raw MS data for each sample.The protein database used was uni-prot_Sus_scrofa_329834_20220802.fasta (www.uniprot.org).The related parameters and instructions were as follows: Related parameters and instructions for identification and quantitative analysis of proteins.

Expression difference analysis
For the screening of significantly differentially expressed proteins, the expression ratio (Fold Change, FC) (up-regulated > 2 times or downregulated < 0.50) and P-value < 0.05 (t-test) were used as the criteria to obtain the number of upregulated and down-regulated proteins between the comparison groups.

Culturing of SD rat dental papilla cells (sdDPCs)
SD rats (7 d old) were sacrificed, their mandibles were removed and placed into PBS containing a 1% Penicillin-Streptomycin Solution.Using a stereomicroscope, the mucosal tissue of the alveolar crest was peeled off with micro tweezers, and the first molar tooth germ was removed from the alveolar socket.The dental follicles were then carefully removed.The dental papilla tissues were collected from the hard tissues inside of the calcified crown, cut into 1 mm pieces, and incubated with 200 U ml −1 type I collagenase at 5% CO 2 37 • C for 20 min.The tissues were shaken every 5 min.Next, the samples were centrifuged at 1000 r min −1 for 5 min, the supernatant was removed, and the samples were incubated inα-MEM containing 20% FBS in a 37 • C, 5% CO2 incubator.The medium was replaced after 48 h of incubation.Upon reaching 85% confluence, cells were trypsinized for 3-5 min, the digestion was stopped using 10% FBS in α-MEM, and cells were centrifuged at 1200 r min −1 for 5 min.The supernatant was discarded and the cells were resuspended and cultured in 10% FBS α-MEM.The third passage cells were used for experiments.

Preparation of porcine treated dental matrix (pTDM)
The anterior teeth of 6-month-old miniature pigs were extracted.The periodontal ligament, tooth enamel, cementum, dental pulp, predentin, and dentin were removed using a high-speed handpiece.The length of pTDM was about 3-4 mm.Samples were placed in double-distilled water (ddH2O), washed foe 3 × 15 min using an ultrasonic water bath.Next the samples were demineralized at room temperature using 17%, 10%, 5% ethylene diamine tetraacetic acid for 20 min, freeze-dried for 8 h, sterilized with ethylene oxide, and stored at −20 • C.

Implantation of DPDM in simulated mineralized environment
The prepared DPDM-C3 was placed into the medullary cavity of the pTDM.DPDM-C3 combined with sdDPCS was used as the experimental group (n = 3), while DPDM-C3 alone was used as the control group (n = 3).Samples were implanted under the skin of 8-week-old SD rats and then collected at 1 month.Samples were fixed, dehydrated, sectioned, and stained using H&E.

Statistical analysis
Statistical significance was performed using a T-test or one-way or two-way ANOVA, with the results are presented as the mean ± standard deviation of at least three independent experiments.P < 0.05 was considered as statistically significant (0.01 < P < 0.05 is depicted as * , 0.001 < P < 0.01 is depicted as * * , and P < 0.001 is depicted as * * * ).

Evaluation of optimal SDS concentration
The pulp tissue of 6-month-old pig canine teeth was obtained (figure 1(B)), treated with SDS for 12 h, and the tissue volume was measured (figure 1(C)).
The results of the volume change determined by the drainage method were 73.8 ± 0.80% for the 10% SDS group, 90.4 ± 0.10% for the 1% SDS group, and 93.9 ± 0.09% for the 0.1% SDS group (figure 1(D)).The results showed that the higher concentration of SDS corresponded to the greater tissue deformation.The higher concentrations of SDS increased the disordered degree of fiber arrangement and the porosity in the pulp tissue after decellularization (figure 1(E)).The fibers in the 1% SDS group were disordered with obvious fracture holes (figure 1(E), Yellow Arrow).The 0.5% SDS group maintained the parallel structure of the natural pulp; however, someobvious fracture holes could still be observed.The fiber orientation and tissue structure of the 0.1% SDS group were more similar to those of the natural; pulp While the cells in the 0.01% SDS group were not effectively cleared, and obvious nuclei were still visible (figure 1(E)).Therefore, subsequent experiments were performed using 0.1% SDS concentration to decellularize the pulp tissue.

Evaluation of matrix proteins in DPDM-C3 and DPDM-36H samples
Our results demonstrated that laminin, fibronectin, integrin β1, and vimentin were were expressed in DPDM-C3 and DPDM-36H samples (figure 2(D)); however, the expression levels in the DPDM-36H group were significantly lower than those in the native group, and the expression levels in the DPDM-36H group were significantly lower than those in the DPDM-C3 group (figure 2(E)).The concentration of DSPP and DMP-1 in the DPDM-C3 group was 5.308 ± 0.189 ng ml −1 and 7.405 ± 0.266 ng ml −1 , respectively, and it was higher than that in the DPDM-36H group; however, there was no significant difference in COL-1 and COL-3 concentrations between the groups (figure 2(F)).

Evaluation of micromorphology in DPDM-C3 and DPDM-36H
The surface of DPDM-C3 group is more flat than that of DPDM-36H group, and the porosity of truncated surface is more uniform (figure 2(G)).The crosssectional image reveals that the pores of the DPDM-C3 group exhibit a diminutive and uniform morphology.The pore structure can influence curvature of the pore, which can influence cell behavior.

Effect of DPDM on proliferation and migration of hDPSCs
To evaluate the effect of DPDM on cell proliferation, hDPSCs (S1(B)) were cultured in the presence of decellularized pulp freeze-dried powder (S1(C)).On days 1, 3, 5, and 7 of culture, cell proliferation in the DPDM-C3 group was higher than that in the DPDM-36H group, while cell proliferation in the DPDM-36H group was lower than that in the control group on days 1, 5, and 7 (figure 2(H)).On days 1 and 3, the cell viability/cytotoxicity results were consistent with those of the proliferation assay (figure 2(I)).Transwell assay showed that cell migration was inhibited in the DPDM-36H group compared to the control group (figure 2(J)).

Expression difference analysis
In total, 141 up-regulated and 725 down-regulated protein groups were identified (S1(F)).The significantly down-regulated proteins were labeled in blue (FC < 0.50 and P < 0.05), the significantly up-regulated proteins were labeled in red (FC > 2 and P < 0.05), while the non-differentially affected proteins were labeled in gray (figure 3(A)).Significantly up-regulated proteins included holocytochrome c-type synthase, collagen alpha-1 (XI) chain, trinucleotide repeat-containing gene 18 protein, decorin, fibrillin-1.The cluster analysis of the differentially expressed is presented as a heatmap (figure 3(B)).

Domain analysis
The domain prediction software InterProscan was used to predict the domains of differentially expressed proteins.The number of proteins for each domains, including WD domain [35], G-beta repeat, Ras Family [36], collagen triple helix repeat (20 copies) [37], Protein kinase domain [38], Fibronectin type III domain [39], von Willebrand factor type A domain [40] and others, are presented as a bar chart (top 20, figure 3(D)).The Fisher's exact test was used for domain enrichment analysis of differentially expressed proteins (red color represents the lowest P-value, and the highest significance under the corresponding domain classification) (figure 3(E)).
The most significant domains were the TCP-1/cpn60 chaperonin family, acyl-CoA dehydrogenase, linker histone H1, and the H5 family.Pyridine nucleotidedisulfide oxidoreductase is involved in the formation of cytoskeletal motor proteins [41], cell metabolism [42], chromatin composition [43,44], and regulation of mitochondrial function [45].Blast2Go (www.blast2go.com/)software was used for GO functional annotation of all differentially expressed proteins in GO Level 2 to statistical differences in protein numbers (figure 4(A)).GO Level 2, which includes biological processes, CC, and molecular functions, is depicted in purple, green, and orange, repectively.The top ten bits with the highest degree of enrichment were considered as the primary nodes of the directed acyclic graph and were represented by boxes.The associated GO entries were displayed together through an inclusion relationship, represented by a circle.The color indicates the degree of enrichment: the closer it is to red, the higher the degree of enrichment (S2).

KEGG annotation
KEGG pathway annotation and quantitative statistics were performed for all differentially expressed proteins, and each pathway was classified according to the seven branches of KEGG (figures 4(B)-(D), table 2).The most significant difference was found in ribosomal proteins (figure 4(F)).

Protein-protein interaction analysis
Circular nodes represent differentially expressed proteins, and lines represent protein-protein interactions.The color of the circle indicates the difference in protein expression (blue for down-regulation and red for up-regulation), while the size of the circle indicates protein connectivity (i.e. the number of proteins that directly interact with a protein) Mitogen-activated protein kinase (S3).Based on topological structure recognition, proteins with a high degree of aggregation in the interaction network diagram were divided into clusters (figure 4(E)).The ten significantly different proteins with the highest interaction scores were Elongation factor 1-gamma, T-complex protein 1 subunit delta, Ribonucloprotein, 60 S ribosomal protein L9, Large ribosomal subunit protein uL5, 40 S ribosomal protein S2, Elongation factor 1-beta, Large ribosomal subunit protein eL6, Large ribosomal subunit protein uL14, 60S ribosomal protein L17.The significantly different proteins were divided into 5 different clusters, among which the protein with the highest score in each group was Decorin, Large ribosomal subunit protein eL6, Glycine cleavage system T-protein, ATP synthase subunit e, Hemebinding protein 1 (table 3).The significance of differences between the two was obtained through Fisher's exact test.Thus, all the differentially expressed protein enrichment pathway categories were found (P < 0.05) (figure 4(F)).Six of the top ten proteins were ribosomal core proteins, including SNU13, RPL9, RPL11, RPL6, RPL23, and RPL17.

In vivo transplantation of DPDM
H&E staining of the subcutaneous transplantation transplants showed increased cell aggregation in the native pulp group; however, there was no apparent cell aggregation in the experimental group; furthermore, the presence of blood vessels could be observed at the junction of the experimental group material and host tissue.Blood cells was observed in the lumen.Immunofluorescence staining showed that the expression of IL-1 in the DPDM group appeared to be lower than thst in the control group, indicating a decreased immune response in the experimental group.Expression of the vascular markers CD34 and CD31 also appeared to be higher in the experimental group compared to that in the control group (figure 5(A)).

Discussion
The decellularized matrix has low immunogenicity, retains the original structure and matrix components of the organ, maintains the cellular microenvironment, promotes cell survival, proliferation, and differentiation, and guides cell ular infiltration of the scaffold [46].RCT requires removal the entire pulp tissue; however, without the support of the vital blood-rich pulp tissue' , the hard tissue becomes fragile.The tooth and neighboring tissues become susceptible to infection, resulting in widespread chronic inflammation [47].To restore the physiological function of teeth after RCT, it is necessary to regenerate the replacement tissue of the natural pulp to provide nutrition and support to the remaining hard tissue.In this study, we optimized the protocol for porcine pulp decellularization and validated its application using in vitro and in vivo models.Tissue-engineered pulp regeneration requires the use of suitable stem cells and scaffolds.DPSCs, a type of mesenchymal stem cells isolated from the dental papillary tissue [48], are the preferred seed cells for dental pulp regeneration.Ideal tissue-engineered scaffolds need to provide support structures for cell growth and regulate the biological properties of cells.As a natural tissue source, the decellularized matrix retains the structure and composition of natural tissues, and is rich in proteins, polysaccharides, cell regulatory factors, and other components.Cells should be able to grow directly on a scaffold without affecting the cell activity, which is important for tissue regeneration [49].Decellularized matriceshave a great potential for regenerating damaged or missing tissues and organs, for example, using DPDM for dental pulp regeneration [50].In this study, we used the porcine dental pulp is widely available in relatively large volumes, and has better plasticity and less ethical restrictions than human pulp tissues.
Different decellularization procedures have several advantages and disadvantages.Freeze-thaw decellularization can effectively remove cells in the tissue without significant changes to the mechanical properties of the tissue [51,52].; however, it might damage the tissue ultrastructure, even though this damage could be reduced by freezing protective agents [31].Mechanical stirring combined with perfusion is a technique commonly used to produce decellularized tissues such as the bladder [53], esophagus [54], trachea [55], skeletal muscle [56], heart valve [51], spinal cord [57],and cartilage [58].
'Washing' method refers to the use of an ionic or nonionic detergent combined with mechanical agitation to decellularize tissues [59].Nonionic detergents are mild detergents that can dissolve proteins while maintaining their natural structure and enzymatic activity.Ionic detergents are stronger than nonionic detergents, and can completely remove cell membranes and denatured proteins.SDS and TritonX-100 are the two most commonly used ionic detergents that can effectively dissolve cell membranes, lipids and DNA [60].SDS has been reported to remove cells while retaining collagen, glycoproteins, and other natural tissue components [61].In this study, we obtained similar results as we adjusted the SDS concentration: a higher SDS concentration resulted in increased tissue damage, disordered fiber orientation, and less favorable tissue surface for cell adhesion.However, it is difficult to remove SDS from the tissues, which could negatively affect biocompatibility during in vivo experiments.Therefore, in this project,we adopted a short-term, multi-cycle approach to reduce the contact time between SDS and pulp tissue.
The time necessary for decellularization primarily depends on the type and concentration of the detergent, as well as the thickness and density of the tissue.Thicker and denser tissues would decrease solvent perfusion.For example, the mucosa of the bladder or small intestine is decellularized using a milder acetic acid, while relatively dense tissues, such as the dermis and trachea, need longer contact times with the detergent or higher detergent concentration [59].Decellularization using SDS and Triton-100X will change the morphology of the tissue and reduce the extracellular matrix content.Therefore, in this study we selected the appropriate detergent concentration (0.1% SDS) and processing time (three cycles) to reduce the impact of the decellularization procedure on tissue morphology.Our results showed that the organization of laminin, fibronectin and integrin β1 was more disordered in DPDM than in the native pulp.Laminin is expressed around the vascular basement membrane, suggesting that it may be involved in blood vessels regeneration [62].The high expression levels of fibronectin and vimentin indicate that these two proteins may be involved in the maintenance of pulp structure.The specific expression of proteins in both decellularized and natural pulp suggests that decellularized pulp can be used as a scaffold for pulp regeneration.
During preparation of the acellular matrix, the retention of bioactive factors significantly affects tissue regeneration.Therefore, we performed domain detection analysis using the DPDM proteome, and identified several domains, including the WD domain [35], Ras Family [36], and collagen triple helix repeat (20 copies) [37].Collagen triple helix repeat (20 copies) was ranked third and Fibronectin type III domain [39] was ranked fifth, and both domains are related to fiber formation.Collagen triple helix repeat containing 1 is an essential molecule involved in the synthesis and deposition of fibrotic scar [63].Fibronectin type III is a myokine that leads to increased energy expenditure by stimulating the 'browning' of white adipose tissue [64].Ras Family [36], protein kinase domain [38], and RNA recognition motif (also known as RRM, RBD, or RNP domain) [65], SH3 [66], and calponin homology (CH) domains are associated with cellular structure or cellular activity.The different proteins and their enriched domains facilitate the processes of cell signaling [38], cell proliferation [67], and collagen fiber generation [63] during pulp regeneration.
Protein interaction network analysis of differentially expressed proteins identified elongation factor 1-gamma (eEF1G) as the most interacting protein.eEF1G is a subunit of the eEF1 complex that plays a role in the transport of AmAcyl-tRNAs to the ribosomes for protein synthesis [68].The N-terminal region of the eEF1G protein contains a glutathione transferase domain, which may be involved in the regulation of the assembly of multi-subunit complexes containing eEF1G and AmAcyl-tRNA synthetase.The eEF1G protein is involved in regulating the vimentin gene expression by interacting with RNA polymerase II, binding to the vimentin promoter region, and shuttling/nursing vimentin mRNA [69].Science vimentin is the main component of the cytoskeleton, this result suggested that cell components were effectively removed during decellularization.Beside eEF1G, the highest correlation was found with in complex protein 1 subunit delta (CCT4).Chaperones are a family of proteins that encapsulate their substrates, assist with protein folding, and function in an ATP-dependent manner [70].CCT4 silencing induces oxidative stress, inhibits glycolysis in esophageal squamous cell carcinoma (ESCC) cells, significantly inhibits cell proliferation and migration, promotes apoptosis, and causes ESCC cell cycle arrest [71].Since CCT4 is closely related to cell metabolism and protein activity, the reduction of CCT4 content after decellularization suggests a new approach for improving DPDM application in the later stages and pulp regeneration.The genes encoding ribosomal proteins are the most highly expressed genes in most cell types.These proteins are often necessary for the synthesis of ribosomes, the building blocks for cell growth and proliferation [72].The function of ribosomes is to translate the genetic code into functional units, leading to cell growth and proliferation.The initial identification of ribosomal protein binding MDM2 (a negative regulator of p53) has evolved into an identification of ribosomal protein-MDM2-p53 signaling that involves a number of processes, from energy metabolism to proliferation [73].Our findings indicated that there could be a loss of fiber components and effective removal of cell components during DPDM preparation.
Decellularized matrix has been reported to promote vascular regeneration [50,74], a key step in the restoration of pulp viability.In this study, the porcine decellularized dental pulp matrix reduced the immune response after subcutaneous transplantation in rats, and cell aggregation was significantly reduced compared to natural dental pulp.TDM has been reported to promote tooth sac cell differentiation and biological root regeneration through the release bioactive factors [75][76][77].In the presence of mineralized microenvironment, the optimized DPDM demonstrated the generation of pulpdentin complex-like tissue, indicating that xenodecellular pulp could be used as a potential scaffold for pulp regeneration.However, there are still some limitations in this study, this hypothesis obviously needs to be tested in orthotopic dental pulp regeneration models.

Conclusions
In this study, the effect of different detergent concentrations on the pulp tissue structure was investigated, and the detergent concentration and decellularity rate suitable for porcine pulp tissue were selected.DPDM was prepared by incubating with 0.1% SDS and three washing cycles.Our results indicated that the optimized preparation of DPDM could release more odontogenesis-related factors and maintain protein content similar that of natural pulp.To validate the application of DPDM in tissue regeneration, we performed subcutaneous transplantation experiments in rats and demonstrated that the optimized DPDM reduced inflammation and promoted tissue vascularization in vivo.Our findings indicate that in in a mineralized microenvironment, a pulp-like tissue can be regenerated and degraded, which is necessary for tissue remodeling, in order to maintain a long-term stable structure.

Figure 3 .
Figure 3. Proteomic detection of DPDM.(A) Significantly differentially expressed protein volcano map.(The horizontal coordinate represents the logarithm of the difference multiplied by base two.the ordinate represents the logarithm, with base ten, of the significance P-value difference.Red denotes significantly up-regulated differentially expressed proteins, blue indicates significantly down-regulated differentially expressed proteins, and gray represents proteins without any differential expression changes.)(B) Cluster analysis of significantly differentially expressed proteins.(B), (C) Subcellular pie map of differentially expressed proteins.(D) Enrichment analysis of significantly differentially expressed protein domains.(E) Domain enrichment analysis diagram.(The horizontal coordinate represents the significance of domain classification enrichment, while the color indicates the magnitude of the enrichment factor, with larger values depicted in red.Additionally, bubble size corresponds to the number of distinct proteins within each domain classification.).

Figure 4 .
Figure 4. Bioinformatic analysis of DPDM.(A) Goannotated statistical map of differentially expressed proteins (level 2).(B) Up-regulated differential protein pathway enrichment butterfly map.(C) KEGG pathway annotation and attribution histogram of differentially expressed proteins.(D) Annotated Statistical map of KEGG Pathway of Differentially expressed Proteins (Top20).(E) Interaction network diagram for function classification diagram.(Theproteins with high aggregation degree in the interaction network diagram were divided into different clusters.)(F) KEGG channel enrichment bubble diagram.

Figure 5 .
Figure 5.In vivo subcutaneous transplantation of DPDM.(A) Subcutaneous transplantation of natural pulp and DPDM with HE staining and immunofluorescence staining (n = 3).(B) Experimental design of subcutaneous simulated mineralized environment transplantation.(C) Images of HE staining for 1 month (n = 3).(D) Biochemical detection of blood.