Journal Pre-proof

Optimizing Methods for Bovine Dental Pulp Decellularization

Hengameh Bakhtiar, DDS, MSc, Sarah Rajabi, PhD, Mohammad Pezeshki-

Modaress, PhD, Mohammad Reza Ellini, BSc, DDS Candidate, Mahsa Panahinia,
DDS Candidate, Solmaz Alijani, DDS Candidate, Amir Mazidi, DDS, Amir Kamali,
DVM, PhD, Amir Azarpazhooh, DDS, MSc, PhD, FRCD, Anil Kishen, BDS, MDS, PhD

PII: S0099-2399(20)30751-2
DOI: https://doi.org/10.1016/j.joen.2020.08.027
Reference: JOEN 4686

To appear in: Journal of Endodontics

Received Date: 18 June 2020

Revised Date: 23 August 2020
Accepted Date: 26 August 2020

Please cite this article as: Bakhtiar H, Rajabi S, Pezeshki-Modaress M, Ellini MR, Panahinia M,
Alijani S, Mazidi A, Kamali A, Azarpazhooh A, Kishen A, Optimizing Methods for Bovine Dental Pulp
Decellularization, Journal of Endodontics (2020), doi: https://doi.org/10.1016/j.joen.2020.08.027.

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Copyright © 2020 Published by Elsevier Inc. on behalf of American Association of Endodontists.

Title Page (Only file with author names)

Optimizing Methods for Bovine Dental Pulp Decellularization

Hengameh Bakhtiar* DDS, MSc 1, 2, 3, Sarah Rajabi PhD* 4, Mohammad Pezeshki-Modaress

PhD 5, Mohammad Reza Ellini BSc, DDS Candidate 1,3, Mahsa Panahinia DDS Candidate 1, 3,

Solmaz Alijani DDS Candidate 1, 3, Amir Mazidi DDS 3, Amir Kamali DVM, PhD 6, Amir

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Azarpazhooh DDS, MSc, PhD, FRCD 2, 7, 8, Anil Kishen BDS, MDS, PhD 2, 8

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1 Department of Endodontics, Faculty of Dentistry, Tehran Medical Sciences, Islamic Azad

University, Tehran, Iran

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2 Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada
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3 Stem Cell Research Center, Tissue Engineering and Regenerative Medicine Institute, Tehran
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Central Branch, Islamic Azad University, Tehran, Iran

4 Department of Cell Engineering, Cell Science Research Center, Royan Institute for Stem
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Cell Biology and Technology, ACECR, Tehran, Iran

5 Burn Research Center, Iran University of Medical Sciences, Tehran, Iran

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6 AO Research Institute Davos, Davos, Switzerland

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7 Clinical Epidemiology and Health Care Research, Institute of Health Policy, Management

and Evaluation, University of Toronto, Toronto, Ontario, Canada

8Department of Dentistry, Mount Sinai Hospital, Toronto, Ontario, Canada

Corresponding author:

Professor Anil Kishen; Faculty of Dentistry, University of Toronto, 124 Edward St, Toronto,

ON M5G 1G6, Anil.Kishen@dentistry.utoronto.ca. Phone; 416-864-8224.

* = Co-first authors, Hengameh Bakhtiar and Sarah Rajabi, contributed equally to this

manuscript

Acknowledgment:

This research was supported by a grant from the National Institute for Medical Research

Development, Iran.
All authors declare no conflict of interest.

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ABSTRACT

Aim: This study aimed to characterize the decellularization effects of different treatment protocols

on the bovine dental pulp extracellular matrix (ECM) for tissue regeneration.

Methods and Materials: Seven different decellularization protocols consisting of

Trypsin/Ethylenediaminetetraacetic acid (Trypsin/EDTA, for 1-hour, 24-hours, or 48-hours), Sodium

dodecyl sulfate (SDS, for 24-hours or 48-hours), TritonX-100 (for 1-hour) and DNase treatments

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were tested on bovine dental pulp tissue. The post-treatment samples were evaluated for remaining

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DNA and cellular contents, structural durability, immunofluorescence analysis and in vivo immune

responses.
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Results: A complete decellularization process in all the experimental groups was observed. The
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protocol that included 1-hour TritonX-100 and 12 hours Trypsin/EDTA treatment with no SDS
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treatment (P7 (12E-0S-1T)) showed the highest retention of glycosaminoglycan (GAG) and absence

of nuclei in DAPI. All groups showed significantly lower DNA content compared to native pulp
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tissue (P<0.05), while compared to other protocols the P1 (1E-24S-1T) and P4 (1E-48S-0T) resulted
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in the highest DNA contents (P<0.05). Based on these results P7 was further evaluated by

immunofluorescence and in vivo immunogenicity. P7 specimens preserved collagen type-I, while

mononuclear cells infiltration along with neovascularization was observed in vivo.

Conclusion: All tested treatments displayed potential ability to decellularize pulp tissue and are

viable options for xenogeneic dental pulp ECM scaffold. P7 (12E-0S-1T) protocol resulted in

decellularized ECM with minimal organic matrix/ultrastructural detriments, with acceptable host

immune response.

Keywords: Pulp tissue, extracellular matrix, decellularization

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INTRODUCTION

With the current momentum in regenerative dentistry, the concepts of vital pulp therapy and

regenerative endodontics procedures (REPs) have emerged at the forefront (1). Regenerative

endodontic procedures comprise of therapeutic interventions that promote regeneration of healthy

tissue within root canals that is structurally and functionally comparable to the native pulp (2). These

include approaches that use blood clots or products (platelet-rich plasma and platelet-rich fibrin), tri-

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calcium phosphate materials and etc. (3, 4).

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The extracellular matrix (ECM) of native pulp tissue offer specific advantages over other

biomaterials (5). Pulp ECM incorporates necessary collagenous microstructure along with bioactive
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molecules that are crucial for dental pulp regeneration (2, 6). On the other hand, it has been
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demonstrated previously that scaffold resulting from decellularized pulp promotes proliferation and
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differentiation of stem cells from apical papilla (7). Donor site morbidity and limited sources
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seriously limits the feasibility of human dental pulp ECM for therapeutic applications (8, 9). In this
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respect, xenogenous sources (e.g. bovine) are a practical solution for such applications (2),

Nevertheless, they have the potential to generate antigenic response in host tissue.

Decellularization is a process that removes the cellular contents of a tissue, resulting in an ECM that

is ideally deprived of antigenicity and risk of disease transmission while its biostructure is

maintained (10). The process of decellularization is carried out mechanically, chemically /

enzymatically or in combination (5). The copious irrigations of mechanical decellularization

protocols (e.g., freeze-thaw) in many cases are incapable of eliminating antigenicity but does not

pose toxicity risk (10, 11). Chemical decellularization [e.g., with sodium dodecyl sulfate (SDS)] is

destructive in particular concentrations resulting in altered ultrastructural properties (12, 13). Further,
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residual SDS in the xenogenic tissue can compromise the outcome of the regenerative procedures

(14). TritonX-100, as a non-ionic detergent, is commonly used for washing out SDS remnants and as

an independent decellularization agent. Compared to SDS, TritonX-100 is a milder agent which

better preserves the tissue structure (15), while offering improved cytocompatibility (16).

The current study aims to develop and optimize, treatment methods (mechanical / chemical) for

bovine pulp tissue decellularization to remove antigenic and cellular contents, while preserving the

microstructure / ultrastructural characteristics.

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MATERIALS AND METHODS

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Decellularized pulp preparation
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Harvesting and decellularization of pulp tissue: Pulp tissues were excised from fresh bovine
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molar teeth and maintained on dry ice while transferred to −80 °C until subjected to seven different

decellularization protocols (Figure 1). The pulp tissues were thawed at room temperature, cut into
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0.5*0.5 cm2 pieces and washed with deionized water for one hour (changed water every 15 minutes).
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The samples were subjected to Ethylenediaminetetraacetic acid (EDTA)/Trypsin (on shaker) and 1%
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SDS treatment for different durations. After being washed with PBS for three hours, the tissues were

subjected to TritonX-100 for 1-hour. The duration of each treatment is as follows:

- P1: 1-hour EDTA/Trypsin, 24 hours SDS and 1-hour TritonX-100 (1E-24S-1T)

- P2: 1-hour EDTA/Trypsin, 24 hours SDS and no TritonX-100 (1E-24S-0T)

- P3: no EDTA/Trypsin, 48 hours SDS and 1-hour TritonX-100 (0E-48S-1T)

- P4: 1-hour EDTA/Trypsin, 48 hours SDS and no TritonX-100 (1E-48S-0T)

- P5: 1-hour EDTA/Trypsin, 48 hours SDS and 1-hour TritonX-100 (1E-48S-1T)

- P6: 24-hour EDTA/Trypsin, no SDS and 1-hour TritonX-100 (24E-0S-1T)

- P7: 12-hour EDTA/Trypsin, no SDS and 1-hour TritonX-100 (12E-0S-1T)

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All samples were washed with phosphate buffered saline (PBS) for 24 hours and then treated with

deoxyribonuclease (DNase, 0.5 mL DNase per each milligram of tissue) at 37 °C for 1 hour.

Evaluation of the decellularization process

Samples were evaluated using histological analysis, DNA content analysis, immunofluorescence and

animal immunogenicity testing.

Histological evaluation: Samples were fixed for 24 hours in 10% neutral buffered formalin solution

(pH 7.4) at 25 °C for 24 hours followed by double deionized water wash. They were then serially

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dehydrated with ethanol and embedded in paraffin. Samples were serially sectioned at 5 µm

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thickness, stained with hematoxylin and eosin (H&E, Sigma-Aldrich, St. Louis, MO, USA),
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Masson’s Trichrome (MT, Sigma-Aldrich, St. Louis, MO, USA) and Safranin O (SO, Sigma-
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Aldrich, St. Louis, MO, USA) to evaluate the cellular, collagen and glycosaminoglycan (GAG)
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content, respectively (17, 18). The slides were examined under a light microscope (BX51, Olympus,
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Japan) to quantify the collagen and GAG contents. The original images of five random microscopic

fields of view (X400) from each sample were analyzed to determine the integrated optical density
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(IOD) of MT and SO satin intensity, using Image-Pro Plus software (version 6, Media cybernetics,
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MD, USA).

DNA quantification: DNA was extracted based on previously described protocol (2). Briefly, 50mg

of native and decellularized samples were dissolved in 1 mL of lysis buffer (50 mM Tris-HCl, 50

mM EDTA, 1% SDS, 10 mM NaCl, pH 8.0) after being homogenized. Samples were digested in

proteinase K (Sigma-Aldrich, St. Louis, MO, USA) overnight in a water bath at 65 °C. Then, DNA

was precipitated using 100% ethanol and then washed with ethanol 70%. After drying, 50 µl Tris-

EDTA buffer (Sigma-Aldrich, St. Louis, MO, USA) was added to each plate. The samples were
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stored at 4 °C. DNA was quantified using spectrophotometric nanodrop read mechanism (2000c,

Thermo Fisher Scientific Inc, Waltham, MA, USA) at 230, 260, and 280 nm wavelengths.

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Immunofluorescence and DAPI: P7 (12E-0S-1T) protocol samples were chosen based on DNA

content and histological results. For immunostaining analysis, tissue slides were permeabilized and

incubated with primary antibodies, including rabbit polyclonal anti-collagen II (Abcam, ab90395)

overnight at 4 °C. Secondary antibodies were added for 2 hours at RT. Images were taken with an

Olympus DP72 digital camera that was mounted on the microscope. To confirm the DNA content

results, the samples were stained with 4,6-diamidino-2- phenylindole (DAPI, Sigma-Aldrich,

Canada) to assess the absence of nucleic acids in the decellularized pulp using fluorescent

microscopy (IX70, Olympus, Shinjuku, Tokyo, Japan).

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In vivo immunogenicity: To observe immune compatibility of the pulp ECM derived from the
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decellularization P7 (12E-0S-1T) samples, Sprague Dawley rats (Female, 250–300 gram, 6-8 weeks
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old) were anesthetized with ketamine hydrochloride (Bremer, Bremerhaven, Germany) and sedative
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(KELA, Hoogstraten, Belgium). After decellularization procedures, the specimens were freeze-dried
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and were subcutaneously implanted on the dorsum of the rats. After two weeks, the rats were

euthanized, and the harvested tissue (implanted site) was fixed in the 10% neutral buffered formalin
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(NBF, PH. 7.26) for 48 hours, then processed and embedded in paraffin. The 5µm thick sections
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were prepared and stained with H&E and MT to observe host immune cell infiltration. Five sections

from the sample were randomly selected and evaluated using computer software Image-Pro Plus by

an independent pathologist who was blinded to the experimental group.

Statistical analysis: All data were expressed as mean ± standard error. Statistical analysis was

performed using student t-test for two groups or one-way analysis of variance (ANOVA) for more

than two groups followed by proper post hoc test in prism software (Prism 6.01, Graphpad Prism). P

value less than 0.05 was considered as significant.

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RESULTS

Evaluation of the decellularization process

Histological evaluation: H&E, MT, and SO-stained micrographs of native and decellularized dental

pulp tissue sections are shown in Figure 2A. Histological staining shows a complete decellularization

process in all groups. Semi-quantitative analysis of SO stained sections revealed a significant

difference between GAG density (IOD) of different protocols and native pulp GAG content, while

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P7 (12E-0S-1T) demonstrated the highest GAG density (Figure 2B). However, the

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histomorphometric analysis showed no significant difference between the tested protocols and the

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native pulp tissues in terms of collagen content (P>0.05).
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DNA content analysis: As shown in figure 2C, compared to DNA concentration of native pulp
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(336.6 ± 7.07 ng/mg), all protocols showed significantly less DNA content, indicative of successful
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decellularization. The highest DNA contents were recorded for P1 (1E-24S-1T) and P4 (1E-48S-0T),
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while the other protocols showed less than 10 ng/mg (P<0.05) (Figure 2C). The minimum DNA
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content was observed as 0.079 ± 0.006 ng/mg for the protocol P6 (24E-0S-1T), which was

significantly less than other protocols (P<0.05).

Immunofluorescence and DAPI: Collagen type-I (Col-I) was measured in P7 (12E-0S-1T) protocol

and native pulp. Immunofluorescent staining showed that ECM Col-I was retained in P7 (12E-0S-

1T) protocol compared to native pulp tissue. While DAPI staining of P7 (12E-0S-1T) shows the

complete removal of cells and nuclear debris (Figure 3).

In-Vivo immunogenicity: Fibroblasts infiltrated the implanted materials (P7 (12E-0S-1T)) and

collagen deposition was observed after 2 weeks (figure 4). Numerous macrophages, lymphocytes,
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and other chronic inflammatory cells were also evident. The implanted materials gradually degraded

over 14 days and were replaced by newly formed, highly vascularized connective tissues and fibrous

encapsulation (figure 4).

DISCUSSION

Xenograft pulp ECM has promising potential for dentin-pulp tissue engineering if prepared using

optimized and efficient decellularization protocols (2, 19, 20). Decellularization, as a critical process

is anticipated to eliminate xenograft antigenicity without impacting the matrix microstructure. An

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ideal procedure is anticipated to provide a treated tissue with clinically acceptable antigenicity and

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preserved microstructural integrity (2, 19, 20). The current decellularization procedures of xenograft
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ECMs involve different steps. Therefore, methods that are economical and requires a shorter
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duration and considered desirable. In this study, seven different chemical/enzymatical
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decellularization methods were qualitatively compared for bovine dental pulp ECM
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decellularization.
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EDTA - a chelating agent, is commonly employed in conjunction with trypsin to disrupt cell
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adhesion to ECM structure during decellularization (21). Schenke-Layland and colleagues suggested

that to avoid any immune response from xenogenic soft tissue grafts, a minimum of 24 hours solo

EDTA/trypsin treatment is required (21). However, a considerable impairment of tissue GAG

content, as well as its mechanical strength, is anticipated with this approach (10, 21). The studies on

the influence of detergents, particularly SDS, are inconsistent. Jank et al. showed that 50 hours of

1% SDS treatment results in a well-preserved ECM microstructure (22). Other studies suggested that

SDS treatment would degrade the tissue collagen structure (10, 23, 24). In addition, the SDS

treatment presents the risk of residual toxicity (10).

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Tissue collagen and GAG content plays a crucial role in regulating the cell-ECM interaction and

subsequent intercellular events (18, 25). It also plays an essential role in the process of

regeneration/repair by presenting matrix-bound adhesion / growth factors (18, 25). In this study all

decellularization protocols reduced tissue GAG content except P7 (12E-0S-1T), which displayed the

minimal effect on the tissue GAG content. Furthermore, a comparison between groups P1 (1E-24S-

1T) vs. P2 (1E-24S-0T) and P4 (1E-48S-0T) vs. P5 (1E-48S-1T), which only differed in the

application of TritonX-100, highlighted that TritonX-100 had no effect on the tissue GAG content.

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The DNA content in the ECM is considered the most potent xenogeneic antigen in terms of

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generating host immune response (26). It is also reported that the immune reaction to ECM with 50
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ng/mg DNA contents or less is acceptable with no significantly differences (8). In the current study,
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all decellularization methods reduced DNA content below 50 ng/mg level, which indicates the
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efficacy of all decellularization protocols tested in the study (27-29). The P7 (12E-0S-1T) DNA
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content was less than that of P1 (1E-24S-1T) and P4 (1E-48S-0T), which proves the efficacy of

EDTA/trypsin in removing DNA contents compared to SDS treatment. Compared to P7 (12E-0S-

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1T), the DNA content for P6 (24E-0S-1T) protocol was lower; nevertheless, P6 protocol
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demonstrated less GAG retention. Since residual DNA contents for P7 (12E-0S-1T) and P6 (24E-0S-

1T) were within the acceptable values as indicated previously with no statistically significant

difference (28, 29), P7 (12E-0S-1T) protocol was chosen as the optimal protocol for further testing.

The in vivo evaluation of the specimens treated with P7 (12E-0S-1T) protocol showed physiologic

infiltration of mononuclear cells and biodegradability, indicating acceptable biocompatibility.

Immunofluorescent staining showed that P7 (12E-0S-1T) protocol retained the majority of Col-I

while DAPI staining demonstrated nearly complete removal of cell nuclei; further confirming P7 as

the optimal protocol. P7 protocol required the shortest treatment time, also making it an
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economically more favorable process besides causing less micro- / ultra-structural changes to the

pulp tissue. Since SDS contributes to residual toxicity, the absence of SDS in P7 (12E-0S-1T) is

considered advantageous. Despite MT and Safranin O staining of the ECM, suggesting

microstructural integrity of pulp tissue, the bioactive and the regenerative potential of P7 (12E-0S-

1T) protocol requires further investigations. In summary, the series of experiments conducted in the

current study highlights the benefits of P7 (12E-0S-1T) protocol, which constitutes 12 hours of

EDTA/Trypsin and 1-hour TritonX-100 treatment with no SDS application for the decellularization

of bovine dental pulp xenograft.

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CONCLUSION

This study compared different protocols of chemical/enzymatic decellularization of bovine dental

pulp ECM and suggested the optimal protocol that decellularized the pulp tissue to a clinically

acceptable level. Microscopic evaluations and DNA quantifications highlight the benefits of protocol

P7 (12E-0S-1T) containing 12 hours of EDTA/Trypsin and 1-hour TritonX-100 treatment with no

SDS treatment for the decellularization process of bovine dental pulp xenograft for regenerative

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applications.

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References

1. Hameed MH, Gul M, Ghafoor R, Badar SB. Management of immature necrotic permanent teeth with
regenerative endodontic procedures - a review of literature. JPMA The Journal of the Pakistan Medical
Association. 2019;69(10):1514-20.
2. Bakhtiar H, Pezeshki-Modaress M, Kiaipour Z, Shafiee M, Ellini MR, Mazidi A, et al. Pulp ECM-derived
macroporous scaffolds for stimulation of dental-pulp regeneration process. Dental materials : official
publication of the Academy of Dental Materials. 2019.
3. Bakhtiar H, Mazidi SA, Mohammadi Asl S, Ellini MR, Moshiri A, Nekoofar MH, et al. The role of stem
cell therapy in regeneration of dentine-pulp complex: a systematic review. Progress in biomaterials.
2018;7(4):249-68.
4. Murray PE. Platelet-Rich Plasma and Platelet-Rich Fibrin Can Induce Apical Closure More Frequently
Than Blood-Clot Revascularization for the Regeneration of Immature Permanent Teeth: A Meta-Analysis of
Clinical Efficacy. Frontiers in bioengineering and biotechnology. 2018;6:139.

of
5. Yao Q, Zheng YW, Lan QH, Kou L, Xu HL, Zhao YZ. Recent development and biomedical applications
of decellularized extracellular matrix biomaterials. Mater Sci Eng C Mater Biol Appl. 2019;104:109942.

ro
6. Bakhtiar H, Mazidi A, Mohammadi-Asl S, Hasannia S, Ellini MR, Pezeshki-Modaress M, et al. Potential
of Treated Dentin Matrix Xenograft for Dentin-Pulp Tissue Engineering. J Endod. 2019.
7. -p
Song JS, Takimoto K, Jeon M, Vadakekalam J, Ruparel NB, Diogenes A. Decellularized Human Dental
Pulp as a Scaffold for Regenerative Endodontics. Journal of dental research. 2017;96(6):640-6.
re
8. Ramm R, Goecke T, Theodoridis K, Hoeffler K, Sarikouch S, Findeisen K, et al. Decellularization
combined with enzymatic removal of N-linked glycans and residual DNA reduces inflammatory response and
improves performance of porcine xenogeneic pulmonary heart valves in an ovine in vivo model.
lP

Xenotransplantation. 2019:e12571.
9. Isidan A, Liu S, Li P, Lashmet M, Smith LJ, Hara H, et al. Decellularization methods for developing
na

porcine corneal xenografts and future perspectives. Xenotransplantation. 2019;26(6):e12564.

10. Gilpin A, Yang Y. Decellularization Strategies for Regenerative Medicine: From Processing Techniques
to Applications. Biomed Res Int. 2017;2017:9831534.
ur

11. Xing Q, Yates K, Tahtinen M, Shearier E, Qian Z, Zhao F. Decellularization of fibroblast cell sheets for
natural extracellular matrix scaffold preparation. Tissue engineering Part C, Methods. 2015;21(1):77-87.
Jo

12. Elder BD, Eleswarapu SV, Athanasiou KA. Extraction techniques for the decellularization of tissue
engineered articular cartilage constructs. Biomaterials. 2009;30(22):3749-56.
13. Zhou J, Fritze O, Schleicher M, Wendel HP, Schenke-Layland K, Harasztosi C, et al. Impact of heart
valve decellularization on 3-D ultrastructure, immunogenicity and thrombogenicity. Biomaterials.
2010;31(9):2549-54.
14. Friedrich EE, Lanier ST, Niknam-Bienia S, Arenas GA, Rajendran D, Wertheim JA, et al. Residual
sodium dodecyl sulfate in decellularized muscle matrices leads to fibroblast activation in vitro and foreign
body response in vivo. J Tissue Eng Regen Med. 2018;12(3):e1704-e15.
15. Luo Z, Bian Y, Su W, Shi L, Li S, Song Y, et al. Comparison of various reagents for preparing a
decellularized porcine cartilage scaffold. American journal of translational research. 2019;11(3):1417-27.
16. Fernandez-Perez J, Ahearne M. The impact of decellularization methods on extracellular matrix
derived hydrogels. Sci Rep. 2019;9(1):14933.
17. Agarwal T, Maiti TK, Ghosh SK. Decellularized caprine liver-derived biomimetic and pro-angiogenic
scaffolds for liver tissue engineering. Mater Sci Eng C Mater Biol Appl. 2019;98:939-48.
18. Uhl FE, Zhang F, Pouliot RA, Uriarte JJ, Rolandsson Enes S, Han X, et al. Functional role of
glycosaminoglycans in decellularized lung extracellular matrix. Acta Biomater. 2019.
19. Pall O, Cuisinier F, Panayotov I, Orti V, Collart-Dutilleul P-Y. Pulp Regeneration Concepts for Nonvital
Teeth: From Tissue Engineering to Clinical Approaches. 2018.
 13

20. Staffoli S, Plotino G, Nunez Torrijos BG, Grande NM, Bossù M, Gambarini G, et al. Regenerative
Endodontic Procedures Using Contemporary Endodontic Materials. Materials (Basel, Switzerland).
2019;12(6).
21. Schenke-Layland K, Vasilevski O, Opitz F, Konig K, Riemann I, Halbhuber KJ, et al. Impact of
decellularization of xenogeneic tissue on extracellular matrix integrity for tissue engineering of heart valves.
J Struct Biol. 2003;143(3):201-8.
22. Jank BJ, Xiong L, Moser PT, Guyette JP, Ren X, Cetrulo CL, et al. Engineered composite tissue as a
bioartificial limb graft. Biomaterials. 2015;61:246-56.
23. Mangold S, Schrammel S, Huber G, Niemeyer M, Schmid C, Stangassinger M, et al. Evaluation of
decellularized human umbilical vein (HUV) for vascular tissue engineering - comparison with endothelium-
denuded HUV. J Tissue Eng Regen Med. 2015;9(1):13-23.
24. O'Neill JD, Anfang R, Anandappa A, Costa J, Javidfar J, Wobma HM, et al. Decellularization of human
and porcine lung tissues for pulmonary tissue engineering. The Annals of thoracic surgery. 2013;96(3):1046-
55; discussion 55-6.
25. Linhardt RJ, Toida T. Role of glycosaminoglycans in cellular communication. Accounts of chemical

of
research. 2004;37(7):431-8.
26. Wang Y, Bao J, Wu Q, Zhou Y, Li Y, Wu X, et al. Method for perfusion decellularization of porcine

ro
whole liver and kidney for use as a scaffold for clinical-scale bioengineering engrafts. Xenotransplantation.
2015;22(1):48-61.
27. Lee JS, Shin J, Park HM, Kim YG, Kim BG, Oh JW, et al. Liver extracellular matrix providing dual
-p
functions of two-dimensional substrate coating and three-dimensional injectable hydrogel platform for liver
tissue engineering. Biomacromolecules. 2014;15(1):206-18.
re
28. Uygun BE, Soto-Gutierrez A, Yagi H, Izamis ML, Guzzardi MA, Shulman C, et al. Organ reengineering
through development of a transplantable recellularized liver graft using decellularized liver matrix. Nature
lP

medicine. 2010;16(7):814-20.
29. Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization
processes. Biomaterials. 2011;32(12):3233-43.
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Figure legends

Figure 1. Schematic presentation of the different decellularization protocols and treatment.

Figure 2. (A) Histological evaluation of the different groups compared to the native pulp tissue, (B)

GAG and collagen content of different groups, (C) DNA content of each group. P1: 1E-24S-1T, P2:

1E-24S-0T, P3: 0E-48S-1T, P4: 1E-48S-0T, P5: 1E-48S-1T, P6: 24E-0S-1T, P7: 12E-0S-1T.

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Figure 3. DAPI and Col-1 immunofluorescence evaluation of the P7 (12E-0S-1T) group.

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Figure 4. Histopathological findings of implanted materials P7 (12E-0S-1T) treated bovine pulp
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tissue. Black thick arrow: implanted material, black thin arrows: inflammatory cells, red arrows:
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angiogenesis, FL: fibrous layer.

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