# MSCs-Derived Decellularised Matrix: Cellular Responses and Regenerative Dentistry

Suphalak Phothichailert, Shirel Samoun, Benjamin P Fournier, Juliane Isaac, Sindy Cornelia Nelwan, Thanaphum Osathanon, Nunthawan Nowwarote

► To cite this version:

Suphalak Phothichailert, Shirel Samoun, Benjamin P Fournier, Juliane Isaac, Sindy Cornelia Nelwan, et al.. MSCs-Derived Decellularised Matrix: Cellular Responses and Regenerative Dentistry. International Dental Journal, 2024, 74, pp.403 - 417. 10.1016/j.identj.2024.02.011. hal-04587078

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Distributed under a Creative Commons Attribution 4.0 International License
MSCs-Derived Decellularised Matrix: Cellular
Responses and Regenerative Dentistry

Suphalak Phothichailert, Shirel Samoun, Benjamin P Fournier, Juliane Isaac, Sindy Cornelia Nelwan, Thanaphum Osathanon, Nunthawan Nowwarote

► To cite this version:

Suphalak Phothichailert, Shirel Samoun, Benjamin P Fournier, Juliane Isaac, Sindy Cornelia Nelwan, et al.. MSCs-Derived Decellularised Matrix: Cellular Responses and Regenerative Dentistry. International Dental Journal, 2024, 74, pp.403 - 417. 10.1016/j.identj.2024.02.011. hal-04587078

HAL Id: hal-04587078

https://hal.sorbonne-universite.fr/hal-04587078v1

Submitted on 24 May 2024

**HAL** is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire **HAL**, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

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Distributed under a Creative Commons Attribution 4.0 International License
Concise Review

MSCs-Derived Decellularised Matrix: Cellular
Responses and Regenerative Dentistry

Suphalak Phothichailert<sup>a</sup>, Shirel Samoun<sup>b</sup>, Benjamin P. Fournier<sup>b,c</sup>, Juliane Isaac<sup>b,c</sup>, Sindy Cornelia Nelwan<sup>d</sup>, Thanaphum Osathanon<sup>a,e*</sup>, Nunthawan Nowwarote<sup>b,c**</sup>

ARTICLE INFO

Article history:

Received 14 August 2023

Received in revised form

12 February 2024

Accepted 16 February 2024

Available online xxx

Key words:

Regenerative dentistry

Extracellular matrix

Decellularisation

Mesenchymal stem cell

Cell proliferation

Cell differentiation

A B S T R A C T

The decellularised extracellular matrix (dECM) of *in vitro* cell culture is a naturally derived biomaterial formed by the removal of cellular components. The compositions of molecules in the extracellular matrix (ECM) differ depending on various factors, including the culture conditions. Cell-derived ECM provides a 3-dimensional structure that has a complex influence on cell signalling, which in turn affects cell survival and differentiation. This review describes the effects of dECM derived from mesenchymal stem cells (MSCs) on cell responses, including cell migration, cell proliferation, and cell differentiation *in vitro*. Published articles were searched in the PubMed databases in 2005 to 2022, with assigned keywords (MSCs and decellularisation and cell culture). The 41 articles were reviewed, with the following criteria. (1) ECM was produced exclusively from MSCs; (2) decellularisation processes were performed; and (3) the dECM production was discussed in terms of culture systems and specific supplementations that are suitable for creating the dECM biomaterials. The dECM derived from MSCs supports cell adhesion, enhances cell proliferation, and promotes cell differentiation. Importantly, dECM derived from dental MSCs shows promise in regenerative dentistry applications. Therefore, the literature strongly supports cell-based dECMs as a promising option for innovative tissue engineering approaches for regenerative medicine.

© 2024 The Authors. Published by Elsevier Inc. on behalf of FDI World Dental Federation.

This is an open access article under the CC BY license

(http://creativecommons.org/licenses/by/4.0/)

Introduction

The extracellular matrix (ECM) serves as a scaffold, offer-
ing support to cells and tissues. It affects cell responses
through interactions with receptors and acts as a reservoir
for signalling molecules, indirectly governing cell behav-
iour. Its properties are vital for development, growth, and
regeneration.¹ The ECM substrate is widely used as a bio-
material in regenerative medicine and tissue engineering.
To create an ECM scaffold, genetic materials (eg, DNA) are
removed using techniques such as chemical, mechanical,
or enzyme digestion methods. This process leads to the
decellularised extracellular matrix (dECM). Studies involv-
ing animal models have shown successful use of dECM to

Introduction

<footnote>ª Center of Excellence for Dental Stem Cell Biology, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand</footnote>

<footnote>ᵇ Centre de Recherche des Cordeliers, Université Paris Cité, Sorbonne Université, INSERM UMR51138, Molecular Oral Pathophysiology, Paris, France</footnote>

<footnote>Department of Oral Biology, Faculty of Dentistry, Université Paris Cité, Paris, France</footnote>

<footnote>d Department of Pediatric Dentistry, Faculty of Dental Medicine, Universitas Airlangga, Airlangga, Indonesia</footnote>

<footnote>e Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand</footnote>

<footnote>* Corresponding author. Faculty of Dentistry, Center of Excellence for Dental Stem Cell Biology, Chulalongkorn University, Bangkok 10330, Thailand. ** Corresponding author. Centre de Recherche des Cordeliers, Université Paris Cité, Sorbonne Université, INSERM UMR1138, Molecular Oral Pathophysiology, Paris, France; Dental Faculty, Department of Oral Biology, Université Paris Cité, Paris, France.</footnote>

<footnote>E-mail addresses: thanaphum.o@chula.ac.th (T. Osathanon),
p_now@hotmail.com, nunthawan.nowwarote@u-paris.fr
(N. Nowwarote).</footnote>

<footnote>Juliane Isaac: http://orcid.org/0000-0001-7898-022X</footnote>

<footnote>Thanaphum Osathanon: http://orcid.org/0000-0003-1649-6357</footnote>

<footnote>Nunthawan Nowwarote: http://orcid.org/0000-0001-5315-5565</footnote>

<footnote>https://doi.org/10.1016/j.identj.2024.02.011</footnote>

<footnote>0020-6539/© 2024 The Authors. Published by Elsevier Inc. on behalf of FDI World Dental Federation. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)</footnote>
Concise Review

MSCs-Derived Decellularised Matrix: Cellular
Responses and Regenerative Dentistry

Suphalak Phothichailert<sup>a</sup>, Shirel Samoun<sup>b</sup>, Benjamin P. Fournier<sup>b,c</sup>, Juliane Isaac<sup>b,c</sup>, Sindy Cornelia Nelwan<sup>d</sup>, Thanaphum Osathanon<sup>a,e*</sup>, Nunthawan Nowwarote<sup>b,c**</sup>

ARTICLE INFO

Article history:

Received 14 August 2023

Received in revised form

12 February 2024

Accepted 16 February 2024

Available online xxx

Key words:

Regenerative dentistry

Extracellular matrix

Decellularisation

Mesenchymal stem cell

Cell proliferation

Cell differentiation

A B S T R A C T

The decellularised extracellular matrix (dECM) of *in vitro* cell culture is a naturally derived biomaterial formed by the removal of cellular components. The compositions of molecules in the extracellular matrix (ECM) differ depending on various factors, including the culture conditions. Cell-derived ECM provides a 3-dimensional structure that has a complex influence on cell signalling, which in turn affects cell survival and differentiation. This review describes the effects of dECM derived from mesenchymal stem cells (MSCs) on cell responses, including cell migration, cell proliferation, and cell differentiation *in vitro*. Published articles were searched in the PubMed databases in 2005 to 2022, with assigned keywords (MSCs and decellularisation and cell culture). The 41 articles were reviewed, with the following criteria. (1) ECM was produced exclusively from MSCs; (2) decellularisation processes were performed; and (3) the dECM production was discussed in terms of culture systems and specific supplementations that are suitable for creating the dECM biomaterials. The dECM derived from MSCs supports cell adhesion, enhances cell proliferation, and promotes cell differentiation. Importantly, dECM derived from dental MSCs shows promise in regenerative dentistry applications. Therefore, the literature strongly supports cell-based dECMs as a promising option for innovative tissue engineering approaches for regenerative medicine.

© 2024 The Authors. Published by Elsevier Inc. on behalf of FDI World Dental Federation.

This is an open access article under the CC BY license

(http://creativecommons.org/licenses/by/4.0/)

Introduction

The extracellular matrix (ECM) serves as a scaffold, offer-
ing support to cells and tissues. It affects cell responses
through interactions with receptors and acts as a reservoir
for signalling molecules, indirectly governing cell behav-
iour. Its properties are vital for development, growth, and
regeneration.¹ The ECM substrate is widely used as a bio-
material in regenerative medicine and tissue engineering.
To create an ECM scaffold, genetic materials (eg, DNA) are
removed using techniques such as chemical, mechanical,
or enzyme digestion methods. This process leads to the
decellularised extracellular matrix (dECM). Studies involv-
ing animal models have shown successful use of dECM to

Introduction

<footnote>ª Center of Excellence for Dental Stem Cell Biology, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand</footnote>

<footnote>ᵇ Centre de Recherche des Cordeliers, Université Paris Cité, Sorbonne Université, INSERM UMR51138, Molecular Oral Pathophysiology, Paris, France</footnote>

<footnote>Department of Oral Biology, Faculty of Dentistry, Université Paris Cité, Paris, France</footnote>

<footnote>d Department of Pediatric Dentistry, Faculty of Dental Medicine, Universitas Airlangga, Airlangga, Indonesia</footnote>

<footnote>e Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand</footnote>

<footnote>* Corresponding author. Faculty of Dentistry, Center of Excellence for Dental Stem Cell Biology, Chulalongkorn University, Bangkok 10330, Thailand. ** Corresponding author. Centre de Recherche des Cordeliers, Université Paris Cité, Sorbonne Université, INSERM UMR1138, Molecular Oral Pathophysiology, Paris, France; Dental Faculty, Department of Oral Biology, Université Paris Cité, Paris, France.</footnote>

<footnote>E-mail addresses: thanaphum.o@chula.ac.th (T. Osathanon),
p_now@hotmail.com, nunthawan.nowwarote@u-paris.fr
(N. Nowwarote).</footnote>

<footnote>Juliane Isaac: http://orcid.org/0000-0001-7898-022X</footnote>

<footnote>Thanaphum Osathanon: http://orcid.org/0000-0003-1649-6357</footnote>

<footnote>Nunthawan Nowwarote: http://orcid.org/0000-0001-5315-5565</footnote>

<footnote>https://doi.org/10.1016/j.identj.2024.02.011</footnote>

<footnote>0020-6539/© 2024 The Authors. Published by Elsevier Inc. on behalf of FDI World Dental Federation. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)</footnote>
regenerate damaged kidneys, liver, heart, lungs, and other organs.²⁻⁴

The purpose of dECM utilisation is to create an appropriate microenvironment for cells to proliferate and differentiate, subsequently restoring damaged or diseased tissues. Critical steps in the production of dECM are considered, including the source of ECM, the decellularisation process, and postmodification. Therefore, investigations of the biological properties of the dECM process using various techniques are required. This review describes a brief overview of cellular responses to dECM derived from cultured mesenchymal stem cells (MSCs) and strategies for application in regenerative medicine.

## Material and methods

A PubMed database search (Jan 2000-Dec 2022) used keywords: MSCs [All Fields] AND decellularization [All Fields] AND cell culture techniques [All Fields]. Article titles and abstracts are reviewed. Inclusion: (1) MSC-derived ECM, (2) decellularization, and (3) dECM production with culture systems and supplements.

## Results and discussions

From PubMed, 124 articles were found with assigned keywords. Among the 41 meeting criteria, 4 articles related to regenerative dentistry were identified. These selected articles formed the basis of this narrative review.

### Classification of the ECM

The ECM proteins can be categorised into several main categories: glycoproteins, proteoglycans, and fibrous proteins, based on their molecular structure. The concept of the ECM matrisome has been introduced, consisting of 2 groups: the core matrisome proteins (collagens, glycoproteins, and proteoglycans) and matrisome-associated proteins (ECM

regulators and secreted factors) (Table 1). Mass spectrometry is used to quantify peptides and proteins, allowing for protein identification.⁵ Matrisome is a proteomic database available for humans, mice, and nematodes (Caenorhabditis elegans) (http://matrisomeproject.mit.edu).⁶,⁷ Comprehending composition and function aids targeted interventions, modulating the ECM for tissue repair and regeneration.⁸ Essentially, the matrisome represents the complete set of ECM proteins and associated factors synthesised and secreted by cells, playing crucial roles in cell adhesion, signalling, and other processes.

### Decellularisation processes

Cell culture-derived dECM is a naturally occurring biomaterial obtained by removing cellular components from isolated culture conditions. The decellularisation process aims to retain the ECM's structure and bioactive molecules while eliminating cellular elements. The surface chemistry of the substrate appears to have no influence on the microarchitecture or composition of the cell culture-derived dECM. Decellularisation techniques involve the use of chemical or detergent solutions, sometimes coupled with mechanical methods, to completely remove cellular components, preserving the ECM structures.⁹⁻¹² During the process, several limitations occur, including: (1) the ECM protein loss, (2) the decellularisation is incomplete due to high cellular confluence, (3) the chemicals' concentration and incubation time varies, (4) ECM detaches from the substrate before recellularisation, and (5) the technique should not cause cytotoxicity or immunological response.¹¹⁻¹³ Several decellularisation methods have been developed for the production of MSC-derived dECM, including chemical, enzymatic, and physical methods. Various decellularised formulas have been developed (Table 2). For example, decellularisation with 2M potassium chloride (KCL) and 0.2% Triton X-100 yields ECM rich in laminin.¹⁴ The use of 1% Triton X-100 with 20 mM NH₄OH in PBS preserves the ECM structure.¹⁵ A combination of Triton X-100 and freeze-thaw cycles

<table><caption>**Table 1 – Classification of extracellular matrix proteins by protein structural and proteomic matrisome database.**</caption><thead><tr><th colspan="2">Classical ECM</th></tr></thead><tbody><tr><td>Glycoproteins</td><td>Proteins that have attached oligosaccharide chains to the amino acid</td></tr><tr><td>Proteoglycans</td><td>The protein comprises attached sulphated GAGs linked to the core proteins.</td></tr><tr><td>Fibrous proteins</td><td>Proteins composed of polypeptide chains that exhibit elon- gated and fibrous structures or sheet-like structures.</td></tr></tbody><thead><tr><th colspan="2">ECM matrisome database</th></tr></thead><tbody><tr><td colspan="2"><strong>Core matrisome proteins</strong></td></tr><tr><td>Glycoprotein</td><td>eg, fibrins, laminins, tenascins, thrombospondins, fibrins, fibulins, and others.</td></tr><tr><td>Proteoglycans</td><td>-</td></tr><tr><td>Collagens</td><td>Including transmembrane collagens</td></tr><tr><td colspan="2"><strong>Matrisome-associated proteins</strong></td></tr><tr><td>ECM-affiliated proteins</td><td>- Proteins that could be considered ECM proteins (eg, mucins, c-type lectins, syndecans, glypicans)<br/>- Proteins that appear repeatedly in ECM-enriched preparations (eg, annexins, galectins)<br/>- Secreted factors and associated with solid-phase complexes (eg, semaphorins and their homologous, collagen-related proteins and their homologous)</td></tr><tr><td>ECM regulators</td><td>- ECM crosslinking (eg, lysin oxidases, transglutaminases)<br/>- ECM-modifying enzymes (eg, sulphatase, extracellular kinases)</td></tr><tr><td>Secreted factors</td><td>Protein and other cytokines, eg, transforming growth factor β, bone morphogenetic</td></tr></tbody></table>

ECM, extracellular matrix; GAGs, glycosaminoglycans.
regenerate damaged kidneys, liver, heart, lungs, and other organs.²⁻⁴

The purpose of dECM utilisation is to create an appropriate microenvironment for cells to proliferate and differentiate, subsequently restoring damaged or diseased tissues. Critical steps in the production of dECM are considered, including the source of ECM, the decellularisation process, and postmodification. Therefore, investigations of the biological properties of the dECM process using various techniques are required. This review describes a brief overview of cellular responses to dECM derived from cultured mesenchymal stem cells (MSCs) and strategies for application in regenerative medicine.

## Material and methods

A PubMed database search (Jan 2000-Dec 2022) used keywords: MSCs [All Fields] AND decellularization [All Fields] AND cell culture techniques [All Fields]. Article titles and abstracts are reviewed. Inclusion: (1) MSC-derived ECM, (2) decellularization, and (3) dECM production with culture systems and supplements.

## Results and discussions

From PubMed, 124 articles were found with assigned keywords. Among the 41 meeting criteria, 4 articles related to regenerative dentistry were identified. These selected articles formed the basis of this narrative review.

### Classification of the ECM

The ECM proteins can be categorised into several main categories: glycoproteins, proteoglycans, and fibrous proteins, based on their molecular structure. The concept of the ECM matrisome has been introduced, consisting of 2 groups: the core matrisome proteins (collagens, glycoproteins, and proteoglycans) and matrisome-associated proteins (ECM

regulators and secreted factors) (Table 1). Mass spectrometry is used to quantify peptides and proteins, allowing for protein identification.⁵ Matrisome is a proteomic database available for humans, mice, and nematodes (Caenorhabditis elegans) (http://matrisomeproject.mit.edu).⁶,⁷ Comprehending composition and function aids targeted interventions, modulating the ECM for tissue repair and regeneration.⁸ Essentially, the matrisome represents the complete set of ECM proteins and associated factors synthesised and secreted by cells, playing crucial roles in cell adhesion, signalling, and other processes.

### Decellularisation processes

Cell culture-derived dECM is a naturally occurring biomaterial obtained by removing cellular components from isolated culture conditions. The decellularisation process aims to retain the ECM's structure and bioactive molecules while eliminating cellular elements. The surface chemistry of the substrate appears to have no influence on the microarchitecture or composition of the cell culture-derived dECM. Decellularisation techniques involve the use of chemical or detergent solutions, sometimes coupled with mechanical methods, to completely remove cellular components, preserving the ECM structures.⁹⁻¹² During the process, several limitations occur, including: (1) the ECM protein loss, (2) the decellularisation is incomplete due to high cellular confluence, (3) the chemicals' concentration and incubation time varies, (4) ECM detaches from the substrate before recellularisation, and (5) the technique should not cause cytotoxicity or immunological response.¹¹⁻¹³ Several decellularisation methods have been developed for the production of MSC-derived dECM, including chemical, enzymatic, and physical methods. Various decellularised formulas have been developed (Table 2). For example, decellularisation with 2M potassium chloride (KCL) and 0.2% Triton X-100 yields ECM rich in laminin.¹⁴ The use of 1% Triton X-100 with 20 mM NH₄OH in PBS preserves the ECM structure.¹⁵ A combination of Triton X-100 and freeze-thaw cycles

<table><caption>Table 1 – Classification of extracellular matrix proteins by protein structural and proteomic matrisome database.</caption><thead><tr><th colspan="2">Classical ECM</th></tr></thead><tbody><tr><td>Glycoproteins</td><td>Proteins that have attached oligosaccharide chains to the amino acid</td></tr><tr><td>Proteoglycans</td><td>The protein comprises attached sulphated GAGs linked to the core proteins.</td></tr><tr><td>Fibrous proteins</td><td>Proteins composed of polypeptide chains that exhibit elon- gated and fibrous structures or sheet-like structures.</td></tr></tbody><thead><tr><th colspan="2">ECM matrisome database</th></tr></thead><tbody><tr><td colspan="2"><strong>Core matrisome proteins</strong></td></tr><tr><td>Glycoprotein</td><td>eg, fibrins, laminins, tenascins, thrombospondins, fibrins, fibulins, and others.</td></tr><tr><td>Proteoglycans</td><td>-</td></tr><tr><td>Collagens</td><td>Including transmembrane collagens</td></tr><tr><td colspan="2"><strong>Matrisome-associated proteins</strong></td></tr><tr><td>ECM-affiliated proteins</td><td>- Proteins that could be considered ECM proteins (eg, mucins, c-type lectins, syndecans, glypicans)<br/>- Proteins that appear repeatedly in ECM-enriched preparations (eg, annexins, galectins)<br/>- Secreted factors and associated with solid-phase complexes (eg, semaphorins and their homologous, collagen-related proteins and their homologous)</td></tr><tr><td>ECM regulators</td><td>- ECM crosslinking (eg, lysin oxidases, transglutaminases)<br/>- ECM-modifying enzymes (eg, sulphatase, extracellular kinases)</td></tr><tr><td>Secreted factors</td><td>Protein and other cytokines, eg, transforming growth factor β, bone morphogenetic</td></tr></tbody></table>

ECM, extracellular matrix; GAGs, glycosaminoglycans.
<table><caption>**Table 2 – (A) Decellularisation solutions.**</caption><thead><tr><th>Reagents</th><th>Removal DNA content</th><th>Number of studies</th></tr></thead><tbody><tr><td>0.1% Triton X-100 containing 10 mM NH<sub>4</sub>OH and 5 mM EDTA in PBS</td><td>DNase and RNase A</td><td>1 (84)</td></tr><tr><td>0.25% Triton X-100 containing 10 mM NH<sub>4</sub>OH in PBS</td><td>DNase and RNase A</td><td>1 (39)</td></tr><tr><td>0.5% Triton X-100 solution containing 20 mM NH<sub>4</sub>OH in PBS</td><td>-</td><td>15 (10, 16, 30, 32, 38, 47, 48, 50, 51, 56, 59, 67, 85-87)</td></tr><tr><td>0.5% Triton X-100 solution containing 20 mM NH<sub>4</sub>OH in PBS</td><td>DNase</td><td>14 (13, 19, 27-29, 35, 40, 43, 45, 46, 49, 53, 58, 88)</td></tr><tr><td>0.5% Triton X-100 solution containing 20 mM NH<sub>4</sub>OH in PBS</td><td>DNase and RNase A</td><td>1 (18)</td></tr><tr><td>0.5% Triton X-100 solution containing 20 mM NH<sub>4</sub>OH in PBS</td><td>DNase, RNase A, and lipase or urea</td><td>2 (34, 54)</td></tr><tr><td>0.5% Triton X-100 solution containing 25 mM NH<sub>4</sub>OH in PBS</td><td>DNase</td><td>1 (36)</td></tr><tr><td>0.5% Triton X-100 in PBS and freeze-thaw cycle</td><td>-</td><td>1 (11)</td></tr><tr><td>1% Triton X-100 solution in dH<sub>2</sub>O or containing 20 mM NH<sub>4</sub>OH in PBS</td><td>DNase</td><td>1 (15)</td></tr><tr><td>1% Triton X-100 solution containing 25 mM NH<sub>4</sub>OH in PBS</td><td>DNase</td><td>1 (17)</td></tr><tr><td>25 mM NH<sub>4</sub>OH in PBS and freeze-thaw cycle</td><td>-</td><td>1 (12)</td></tr><tr><td>Double-distilled water (DDW) containing 20 mM NH<sub>4</sub>OH</td><td>DNase</td><td>2 (41, 42)</td></tr></tbody></table>

PBS, phosphate-buffered saline.

leads to the best-preserved structure and components of the ECM.¹¹ Furthermore, 1% to 5% of sodium dodecyl sulphate (SDS) slightly affects the ECM and increases collagen.¹⁶ DNases are essential to remove the remaining DNA content in dECM.¹⁷⁻¹⁹ Optimised processes can be done by adjusting time, chemical agent, temperature, and physical application methods (ie, agitation). MSC-derived dECM requires optimising numerous parameters, including:

1. ECM structure preservation is crucial for tissue's bio-mechanical properties.

2. The cellular elements (ie, DNA, RNA, and debris) must be efficiently removed, but the ECM proteins must be retained.

3. The dECM should be nontoxic and nonimmunogenic. It should also support cell attachment, proliferation, and differentiation.

4. Uniform dECM scaffold properties must be ensured for consistency and reproducibility.

5. Decellularisation must be scalable for large-scale dECM scaffold production in clinical use.

Therefore, the decellularised process should ideally retain the native structure and composition while minimising immunogenicity and preserving bioactivity. Additionally, the process of obtaining dECM involves a variety of methods. Each method presents specific advantages and disadvantages, as shown in Table 2B.

### The effect of the microenvironment on cellular-derived ECM production

The microenvironment has a significant effect on ECM production by regulating the behaviour of cells responsible for producing and depositing ECM molecules. The microenvironment comprises physical and biological factors that influence ECM production, altering composition, structure, and function.²⁰,²¹ In regard to physical factors, cyclic loading strain raised elastin and collagen levels in smooth muscle cells, resulting in an improvement in tissue organisation.²² Meanwhile, the mechanical loading force enhanced the synthesis of ECM while seeding on scaffold material.²³ However, further investigation is needed to understand the impact of

<table><caption>**Table 2 – (B) The decellularisation methods present specific advantages and disadvantages.**</caption><thead><tr><th>Methods</th><th>Advantages</th><th>Disadvantages</th></tr></thead><tbody><tr><td>Chemicals</td><td><ul><li>Relatively simple and straightforward process applicable to cell culture systems.</li><li>Can be used with a variety of cell types.</li><li>Preserves the structural integrity of the ECM</li></ul></td><td><ul><li>Damage to certain ECM components.</li></ul></td></tr><tr><td>Enzymatic digestion</td><td><ul><li>Essential to remove DNA content.</li></ul></td><td><ul><li>May affect the bioactivity of growth factors.</li></ul></td></tr><tr><td>Chemicals and enzymatic digestion</td><td><ul><li>Completely remove intracellular components.</li></ul></td><td><ul><li>May affect both ECM component and bioactivity of growth factors.</li></ul></td></tr><tr><td>Freeze-thaw cycling</td><td><ul><li>Relatively simple and cost-effective.</li><li>A gentle method, preserving some ECM structure.</li></ul></td><td><ul><li>May not be as effective in fully removing intracellular components.</li><li>Effectiveness might vary based on cell and tissue types.</li></ul></td></tr></tbody></table>

ECM, extracellular matrix.
<table><caption>**Table 2 – (A) Decellularisation solutions.**</caption><thead><tr><th>Reagents</th><th>Removal DNA content</th><th>Number of studies</th></tr></thead><tbody><tr><td>0.1% Triton X-100 containing 10 mM NH<sub>4</sub>OH and 5 mM EDTA in PBS</td><td>DNase and RNase A</td><td>1 (84)</td></tr><tr><td>0.25% Triton X-100 containing 10 mM NH<sub>4</sub>OH in PBS</td><td>DNase and RNase A</td><td>1 (39)</td></tr><tr><td>0.5% Triton X-100 solution containing 20 mM NH<sub>4</sub>OH in PBS</td><td>-</td><td>15 (10, 16, 30, 32, 38, 47, 48, 50, 51, 56, 59, 67, 85-87)</td></tr><tr><td>0.5% Triton X-100 solution containing 20 mM NH<sub>4</sub>OH in PBS</td><td>DNase</td><td>14 (13, 19, 27-29, 35, 40, 43, 45, 46, 49, 53, 58, 88)</td></tr><tr><td>0.5% Triton X-100 solution containing 20 mM NH<sub>4</sub>OH in PBS</td><td>DNase and RNase A</td><td>1 (18)</td></tr><tr><td>0.5% Triton X-100 solution containing 20 mM NH<sub>4</sub>OH in PBS</td><td>DNase, RNase A, and lipase or urea</td><td>2 (34, 54)</td></tr><tr><td>0.5% Triton X-100 solution containing 25 mM NH<sub>4</sub>OH in PBS</td><td>DNase</td><td>1 (36)</td></tr><tr><td>0.5% Triton X-100 in PBS and freeze-thaw cycle</td><td>-</td><td>1 (11)</td></tr><tr><td>1% Triton X-100 solution in dH<sub>2</sub>O or containing 20 mM NH<sub>4</sub>OH in PBS</td><td>DNase</td><td>1 (15)</td></tr><tr><td>1% Triton X-100 solution containing 25 mM NH<sub>4</sub>OH in PBS</td><td>DNase</td><td>1 (17)</td></tr><tr><td>25 mM NH<sub>4</sub>OH in PBS and freeze-thaw cycle</td><td>-</td><td>1 (12)</td></tr><tr><td>Double-distilled water (DDW) containing 20 mM NH<sub>4</sub>OH</td><td>DNase</td><td>2 (41, 42)</td></tr></tbody></table>

PBS, phosphate-buffered saline.

leads to the best-preserved structure and components of the ECM.¹¹ Furthermore, 1% to 5% of sodium dodecyl sulphate (SDS) slightly affects the ECM and increases collagen.¹⁶ DNases are essential to remove the remaining DNA content in dECM.¹⁷⁻¹⁹ Optimised processes can be done by adjusting time, chemical agent, temperature, and physical application methods (ie, agitation). MSC-derived dECM requires optimising numerous parameters, including:

1. ECM structure preservation is crucial for tissue's biochemical properties.

2. The cellular elements (ie, DNA, RNA, and debris) must be efficiently removed, but the ECM proteins must be retained.

3. The dECM should be nontoxic and nonimmunogenic. It should also support cell attachment, proliferation, and differentiation.

4. Uniform dECM scaffold properties must be ensured for consistency and reproducibility.

5. Decellularisation must be scalable for large-scale dECM scaffold production in clinical use.

Therefore, the decellularised process should ideally retain the native structure and composition while minimising immunogenicity and preserving bioactivity. Additionally, the process of obtaining dECM involves a variety of methods. Each method presents specific advantages and disadvantages, as shown in Table 2B.

### The effect of the microenvironment on cellular-derived ECM production

The microenvironment has a significant effect on ECM production by regulating the behaviour of cells responsible for producing and depositing ECM molecules. The microenvironment comprises physical and biological factors that influence ECM production, altering composition, structure, and function.²⁰,²¹ In regard to physical factors, cyclic loading strain raised elastin and collagen levels in smooth muscle cells, resulting in an improvement in tissue organisation.²² Meanwhile, the mechanical loading force enhanced the synthesis of ECM while seeding on scaffold material.²³ However, further investigation is needed to understand the impact of

<table><caption>**Table 2 – (B) The decellularisation methods present specific advantages and disadvantages.**</caption><thead><tr><th>Methods</th><th>Advantages</th><th>Disadvantages</th></tr></thead><tbody><tr><td>Chemicals</td><td><ul><li>Relatively simple and straightforward process applicable to cell culture systems.</li><li>Can be used with a variety of cell types.</li><li>Preserves the structural integrity of the ECM</li></ul></td><td><ul><li>Damage to certain ECM components.</li></ul></td></tr><tr><td>Enzymatic digestion</td><td><ul><li>Essential to remove DNA content.</li></ul></td><td><ul><li>May affect the bioactivity of growth factors.</li></ul></td></tr><tr><td>Chemicals and enzymatic digestion</td><td><ul><li>Completely remove intracellular components.</li></ul></td><td><ul><li>May affect both ECM component and bioactivity of growth factors.</li></ul></td></tr><tr><td>Freeze-thaw cycling</td><td><ul><li>Relatively simple and cost-effective.</li><li>A gentle method, preserving some ECM structure.</li></ul></td><td><ul><li>May not be as effective in fully removing intracellular components.</li><li>Effectiveness might vary based on cell and tissue types.</li></ul></td></tr></tbody></table>

ECM, extracellular matrix.
mechanical loading on MSCs' ECM production and its compo-
nents, as well as to identify the optimal conditions for ECM
production.

Biological factors such as growth factors and cytokines
also play a crucial role in regulating the production of ECM.
Transforming growth factor-β (TGF-β)²⁴ and bone morphoge-
netic protein (BMP)²⁵ stimulate ECM synthesis by promoting
the differentiation of precursor cells into ECM-producing cells
and increasing the production of ECM proteins. Similarly,
cytokines such as interleukin-1 (IL-1) and tumour necrosis
factor-α (TNF-α) enhance the production of ECM-degrading
enzymes, leading to the breakdown of the ECM.²⁶ Culture
conditions play a vital role in producing ECM that influences
desired cell responses. Under a chondrogenic medium, dECM
enhances MSCs viability, spreading, and proliferation, redif-
ferentiation, and anti-inflammatory properties. Conversely,
dECM from an ascorbic acid-supplemented medium or osteo-
genic medium culture promotes the highest calcium accumu-
lation.¹³,²⁷⁻²⁹ Three-dimensional dECM provides a specific
environment for specific cell type to retain their differentia-
tion ability but does not affect cell proliferation.³⁰

Using dECM to mimic the *in vivo* extracellular microenvi-
ronment has been shown to be a useful strategy to stimulate
cell proliferation and survival.³¹ The 3-dimensional struc-
tures of the ECM support cell growth and serve as a reservoir
of growth factors and cytokines that control cell destiny and
function.¹⁹,³¹,³² This complexity contributes to the formation
of optimal niches for cells to reside in. Furthermore, 3-dimen-
sional culture conditions could lead to an ECM that physiolog-
ically resembles native tissue more closely than those derived
from the 2-dimensional culture system.³³ Together, these
results emphasise the significance of microenvironment and
culture conditions in the bioactive characteristics of dECM.

Effect of dECM derived from MSCs on cellular responses

MSCs possess self-renewal and proliferation abilities, contrib-
uting to regeneration through direct differentiation towards
specific cell types and the secretion of regenerative-related
factors. MSCs can be extracted from bone marrow, umbilical
cord, fat, cartilage, urine, and dental tissues. The *in vitro*
expansion of MSCs has limitations, such as cell senescence
and reduced differentiation capacity. Studying dECM from
diverse MSC sources is crucial for creating targeted microen-
vironments, aiding tissue regeneration.

MSC-derived dECM affects cellular responses diversely, for
example, by promoting cell adhesion, migration, and prolifer-
ation, as well as modulating cell differentiation and immune
responses. MSC-derived dECM exhibits lower rejection risk by
using the patient's own cells. Nevertheless, some studies
show that dECM from MSCs triggers an immune response in
animals, leading to an increased production of cytokines and
immune cell infiltration.¹² However, this immune response
does not appear to affect the functional properties of the
dECM.

Adipose-derived stem cells

Adipose-derived stem cells (ADSCs) are easily accessible and
widely used in regenerative medicine due to their self-

renewing and versatile nature. dECM-ADSCs exhibit more
collagen and glycosaminoglycans when cultured in an osteo-
genic differentiation medium.³⁴ Additionally, dECM from
ADSCs plays a crucial role in regulating retinal progenitor cell
proliferation and differentiation, holding promise for enhanc-
ing the effectiveness of retinal progenitor cell treatment in
retinal degenerative diseases.³⁵ The microstructure and
native constituents preserved in the dECM could provide a
useful platform for studying the role of ECM in wound heal-
ing.¹⁷ Furthermore, it also affects pathologic changes by sup-
porting repopulation in cancer cells during chemotherapy
treatment.³⁶

Bone marrow MSCs

Bone marrow MSCs (BMSCs)-derived dECM contains multiple
proteins that dramatically promote the proliferation, migra-
tion, and differentiation of MSCs.37-43 dECM derived from foel-
tal BMSCs promotes MSCs with a higher proliferative and
osteogenic capacity compared to adult BMSCs-dECM.44,45 Fur-
thermore, BMSCs-derived dECM suppressed osteoclastogene-
sis, implying positive anabolic effects on bone regeneration.
dECM derived from BMSCs could benefit in clinical use by
modulating bone remodelling and promoting bone tissue
engineering.46 BMSCs-dECM enhances chondrogenesis of
chondrocytes both in vitro and in vivo47 and modulates human
umbilical vein endothelial cell (HUVECs) responses in the vas-
cularisation process by improving endothelial cell prolifera-
tion and migration.48 Moreover, dECM-derived from BMSCs
enhances dermal fibroblast proliferation.15 Reseeding MSCs
on naïve BMSCs-dECM or transferred BMSCs-dECM signifi-
cantly enhances osteogenic differentiation ability via ERK
and integrin α2β1 pathways.49 Meanwhile, it offers a microen-
vironment that retains the quality of MSCs in terms of repli-
cation, differentiation, and BMP-2 responsiveness.32 The
BMSCs-dECM promotes calvarial bone regeneration in severe
combined immunodeficiency mice.12 Surprisingly, cocultured
HUVECs and BMSCs-dECM boost osteogenesis and
angiogenesis.50

Synovium-derived stem cells

Cartilage tissue engineering has emerged as a potentially
effective treatment option for cartilage repair. The creation of
the cartilage-specific matrix was sustained by dECM derived
from synovium-derived stem cells (SDSCs), which inhibited
the expression of the enzymes that break down the matrix.²⁸
Furthermore, hSDSCs-dECM promoted self-renewal ability,
induced cell proliferation, and enhanced chondrogenic differ-
entiation of SDSCs via the MAPK pathway.⁵¹ Therefore, dECM
derived from SDSCs improves the proliferation and matrix
synthesis of chondrocytes.

Umbilical cord MSCs

Decellularised ECM derived from Wharton's jelly MSCs (WJ-MSCs) reduces immunogenicity and improves proliferation and chondrogenic differentiation through the MAPK pathway of SDSCs and WJ-MSCs.¹⁶ Furthermore, dECM-derived from WJ-MSCs rescues cardiac C-kit positive cells from oxidative stress, offering enhanced *in vivo* transplantation survival and function.⁵² MSCs grown on umbilical cord MSCs (UC-MSCs)-
mechanical loading on MSCs' ECM production and its compo-
nents, as well as to identify the optimal conditions for ECM
production.

Biological factors such as growth factors and cytokines
also play a crucial role in regulating the production of ECM.
Transforming growth factor-β (TGF-β)²⁴ and bone morphoge-
netic protein (BMP)²⁵ stimulate ECM synthesis by promoting
the differentiation of precursor cells into ECM-producing cells
and increasing the production of ECM proteins. Similarly,
cytokines such as interleukin-1 (IL-1) and tumour necrosis
factor-α (TNF-α) enhance the production of ECM-degrading
enzymes, leading to the breakdown of the ECM.²⁶ Culture
conditions play a vital role in producing ECM that influences
desired cell responses. Under a chondrogenic medium, dECM
enhances MSCs viability, spreading, and proliferation, redif-
ferentiation, and anti-inflammatory properties. Conversely,
dECM from an ascorbic acid-supplemented medium or osteo-
genic medium culture promotes the highest calcium accumu-
lation.¹³,²⁷⁻²⁹ Three-dimensional dECM provides a specific
environment for specific cell type to retain their differentia-
tion ability but does not affect cell proliferation.³⁰

Using dECM to mimic the *in vivo* extracellular microenvi-
ronment has been shown to be a useful strategy to stimulate
cell proliferation and survival.³¹ The 3-dimensional struc-
tures of the ECM support cell growth and serve as a reservoir
of growth factors and cytokines that control cell destiny and
function.¹⁹,³¹,³² This complexity contributes to the formation
of optimal niches for cells to reside in. Furthermore, 3-dimen-
sional culture conditions could lead to an ECM that physiolog-
ically resembles native tissue more closely than those derived
from the 2-dimensional culture system.³³ Together, these
results emphasise the significance of microenvironment and
culture conditions in the bioactive characteristics of dECM.

Effect of dECM derived from MSCs on cellular responses

MSCs possess self-renewal and proliferation abilities, contrib-
uting to regeneration through direct differentiation towards
specific cell types and the secretion of regenerative-related
factors. MSCs can be extracted from bone marrow, umbilical
cord, fat, cartilage, urine, and dental tissues. The *in vitro*
expansion of MSCs has limitations, such as cell senescence
and reduced differentiation capacity. Studying dECM from
diverse MSC sources is crucial for creating targeted microen-
vironments, aiding tissue regeneration.

MSC-derived dECM affects cellular responses diversely, for
example, by promoting cell adhesion, migration, and prolifer-
ation, as well as modulating cell differentiation and immune
responses. MSC-derived dECM exhibits lower rejection risk by
using the patient's own cells. Nevertheless, some studies
show that dECM from MSCs triggers an immune response in
animals, leading to an increased production of cytokines and
immune cell infiltration.¹² However, this immune response
does not appear to affect the functional properties of the
dECM.

Adipose-derived stem cells

Adipose-derived stem cells (ADSCs) are easily accessible and
widely used in regenerative medicine due to their self-

renewing and versatile nature. dECM-ADSCs exhibit more
collagen and glycosaminoglycans when cultured in an osteo-
genic differentiation medium.³⁴ Additionally, dECM from
ADSCs plays a crucial role in regulating retinal progenitor cell
proliferation and differentiation, holding promise for enhanc-
ing the effectiveness of retinal progenitor cell treatment in
retinal degenerative diseases.³⁵ The microstructure and
native constituents preserved in the dECM could provide a
useful platform for studying the role of ECM in wound heal-
ing.¹⁷ Furthermore, it also affects pathologic changes by sup-
porting repopulation in cancer cells during chemotherapy
treatment.³⁶

Bone marrow MSCs

Bone marrow MSCs (BMSCs)-derived dECM contains multiple
proteins that dramatically promote the proliferation, migra-
tion, and differentiation of MSCs.37-43 dECM derived from foel-
tal BMSCs promotes MSCs with a higher proliferative and
osteogenic capacity compared to adult BMSCs-dECM.44,45 Fur-
thermore, BMSCs-derived dECM suppressed osteoclastogene-
sis, implying positive anabolic effects on bone regeneration.
dECM derived from BMSCs could benefit in clinical use by
modulating bone remodelling and promoting bone tissue
engineering.46 BMSCs-dECM enhances chondrogenesis of
chondrocytes both in vitro and in vivo47 and modulates human
umbilical vein endothelial cell (HUVECs) responses in the vas-
cularisation process by improving endothelial cell prolifera-
tion and migration.48 Moreover, dECM-derived from BMSCs
enhances dermal fibroblast proliferation.15 Reseeding MSCs
on naïve BMSCs-dECM or transferred BMSCs-dECM signifi-
cantly enhances osteogenic differentiation ability via ERK
and integrin α2β1 pathways.49 Meanwhile, it offers a microen-
vironment that retains the quality of MSCs in terms of repli-
cation, differentiation, and BMP-2 responsiveness.32 The
BMSCs-dECM promotes calvarial bone regeneration in severe
combined immunodeficiency mice.12 Surprisingly, cocultured
HUVECs and BMSCs-dECM boost osteogenesis and
angiogenesis.50

Synovium-derived stem cells

Cartilage tissue engineering has emerged as a potentially
effective treatment option for cartilage repair. The creation of
the cartilage-specific matrix was sustained by dECM derived
from synovium-derived stem cells (SDSCs), which inhibited
the expression of the enzymes that break down the matrix.²⁸
Furthermore, hSDSCs-dECM promoted self-renewal ability,
induced cell proliferation, and enhanced chondrogenic differ-
entiation of SDSCs via the MAPK pathway.⁵¹ Therefore, dECM
derived from SDSCs improves the proliferation and matrix
synthesis of chondrocytes.

Umbilical cord MSCs

Decellularised ECM derived from Wharton's jelly MSCs (WJ-MSCs) reduces immunogenicity and improves proliferation and chondrogenic differentiation through the MAPK pathway of SDSCs and WJ-MSCs.¹⁶ Furthermore, dECM-derived from WJ-MSCs rescues cardiac C-kit positive cells from oxidative stress, offering enhanced in vivo transplantation survival and function.⁵² MSCs grown on umbilical cord MSCs (UC-MSCs)-
dECM show lower level of reactive oxygen species and higher
antioxidative enzyme activity, thus enhancing resistance
oxidative stress.⁵³,⁵⁴ These properties of oxidative stress
improvement reduce cellular senescence and promote cell
proliferation.⁵⁵ Placental MSC-dECM and hTERT-transduced
cell lines share traits, supporting larger dECM production and
aiding primary MSC expansion.⁵⁶

Urine-derived stem cells

Human urine-derived stem cells (hUSCs) are promising due to their low cost and easy collection, making them practical cell sources for ECM production. USCs exhibit immunomodulatory properties and are versatile in differentiation.⁵⁷ dECM derived from USCs is a relatively new area of research. In 2019, USC-dECM was studied in cartilage tissue engineering. USC-dECM supports chondrocyte proliferation and differentiation while promoting the synthesis of ECM proteins.⁵⁸ Moreover, USCs-dECM reduces proinflammation and boosts anti-inflammation in macrophage coculture.⁵⁹ Therefore, hUSCs-dECM has been proposed as a biomaterial for tissue regeneration. Together, this suggests that their ECM would be a better substrate for adult stem cell expansion. However, more research is required for full understanding their dECM characteristics and optimisation process for obtaining dECM.

Effect of dECM derived from dental MSCs on cellular responses

Dental tissue is easily accessed via noninvasive procedures,
allowing MSC isolation from waste tissues from routine den-
tal treatments (eg, tooth extraction and wisdom tooth
removal). Dental MSCs are the promising cell source for
regenerative therapy, especially in craniofacial areas.⁶⁰ Den-
tal tissue-derived MSCs have a faster proliferation rate com-
pared to other MSCs sources like bone marrow or adipose
tissue, indicating that these dental tissue-derived MSCs could
be a candidate MSC source for regenerative therapy.⁶¹,⁶² Sour-
ces of MSCs in the oral region have been reported, including
pulp tissues of permanent teeth (dental pulp stem cells;
DPSCs),⁶³ remaining pulp tissues from primary teeth (stem
cells from human exfoliated deciduous teeth; SHEDs),⁶⁴ peri-
odontal ligament (periodontal ligament stem cells; PDLSCs),⁶⁵
gingival tissues (gingival stem cells),⁶⁶ apical papilla (stem
cells from the apical papilla; SCAPs), and dental follicle (den-
tal follicle stem cells).⁶⁰

The ability of oral cells to adhere, proliferate, and differen-
tiate into osteogenic differentiation can be significantly influ-
enced by the dECM generated from dental MSCs. PDLSCs-
dECM was shown to be more appropriate for osteogenic
induction, while SHEDs-dECM was shown to be more appro-
priate for ex vivo growth of DPSCs.⁶⁷ Furthermore, dECM
derived from hPDLSCs, dental pulp cells, or gingival fibro-
blasts promotes the proliferation and osteogenic differentia-
tion of hPDLSCs.⁶⁸ The dECM derived from hDPSCs enriches
with collagens and elastic fibres, showing the capacity for
osteoinduction by improving the mineralisation of human
gingival fibroblasts (Figure 1).²⁹ hDPSCs-dECM demonstrated
usefulness in modulating dental cell responses in vitro, sug-
gesting the benefit of dental tissue engineering, mineralised
tissue regeneration, or embellishing biomaterials. dECM

derived from dental MSCs promotes angiogenesis, as shown
by the upregulation of proangiogenic growth factors.⁶⁹ These
dECM improve the formation of dental tissues in vivo without
the addition of exogenous factors in subcutaneous implanta-
tion.⁶⁹ dECM derived from hDPSCs on polylactic acid (PLA)
scaffolds promotes calvarial bone healing in rats, as deter-
mined by increased new bone volume and area compared to
PLA alone.⁷⁰ Interestingly, induced pluripotent stem cells
(iPSCs) have been proposed as potential therapeutic cells
because a patient-specific cell line can be generated. iPSCs
can differentiate into numerous cell lineages. Feeder cells are
required for culture during reprogramming and passage to
maintain iPSCs in the stem cell stage.⁷¹ dECM derived from
dental pulp cells is suitable as a feeder-free culture medium
for dental pulp-derived iPSCs by improving iPSC attachment,
cell growth, and proliferation.⁷²

Notch signalling activation in human dental pulp cells upregulated the ECM organisation pathway, as confirmed by RNA sequencing analysis.⁷³ Proteomic and matrisome analysis of Notch ligands, Jagged-1, stimulated hDPSCs demonstrates the protein component related to osteogenic differentiation.⁷⁴ Furthermore, the dECM of the Jagged-1 treated condition exhibited a higher mineralisation and glycosaminoglycan component compared to the dECM of the control condition. Both dECM derived from human IgG Fc fragment (hFc) and Jagged-1 treated hDPSCs support SCAPs attachment, proliferation, and osteogenic differentiation (Figure 2).¹³ However, dECM from Jagged-1-treated hDPSCs promotes higher mineralisation of reseeded SCAPs compared to dECM from the control condition. This strong evidence suggests that the manipulation of dental MSCs can alter the characteristics of the dECM and, subsequently, influence cell responses. Taking all evidence together, ECMs derived from dental cells could potentially be used as a suitable natural biomaterial scaffold for applications in the clinic, such as regenerative treatment. A summary of the effects of dECM-derived from MSCs (B) and dental MSCs (A) on cellular responses is shown in Table 3A, B.

Future study on decellularised dental MSCs-derived ECM for
use in regenerative dentistry

To promote regeneration in dental tissues, dental stem cells need to migrate to the injured site and proliferate. Later, these cells differentiate into mature cells or secrete growth factors, modulating biological processes to enhance regeneration. Furthermore, the biodegradation and biocompatibility of the scaffold are required to provide the appropriate physical and biological interactions. Therefore, dECM is an excellent candidate, as it can be degraded in vivo and is not a cytotoxic substance.²⁹ Cellular responses to the composition, structure, and stiffness properties of the surrounding matrix and signalling pathways to drive the morphogenetic and pathogenic processes should be further investigated.

ECM scaffold combination with dental MSCs

A suitable scaffold material is vital for tissue regeneration,
providing sites for cell adhesion, proliferation, and differenti-
ation. ECM scaffolds offer the advantage of promoting natural
dECM show lower level of reactive oxygen species and higher
antioxidative enzyme activity, thus enhancing resistance
oxidative stress.⁵³,⁵⁴ These properties of oxidative stress
improvement reduce cellular senescence and promote cell
proliferation.⁵⁵ Placental MSC-dECM and hTERT-transduced
cell lines share traits, supporting larger dECM production and
aiding primary MSC expansion.⁵⁶

Urine-derived stem cells

Human urine-derived stem cells (hUSCs) are promising due to their low cost and easy collection, making them practical cell sources for ECM production. USCs exhibit immunomodulatory properties and are versatile in differentiation.⁵⁷ dECM derived from USCs is a relatively new area of research. In 2019, USC-dECM was studied in cartilage tissue engineering. USC-dECM supports chondrocyte proliferation and differentiation while promoting the synthesis of ECM proteins.⁵⁸ Moreover, USCs-dECM reduces proinflammation and boosts anti-inflammation in macrophage coculture.⁵⁹ Therefore, hUSCs-dECM has been proposed as a biomaterial for tissue regeneration. Together, this suggests that their ECM would be a better substrate for adult stem cell expansion. However, more research is required for full understanding their dECM characteristics and optimisation process for obtaining dECM.

Effect of dECM derived from dental MSCs on cellular responses

Dental tissue is easily accessed via noninvasive procedures,
allowing MSC isolation from waste tissues from routine den-
tal treatments (eg, tooth extraction and wisdom tooth
removal). Dental MSCs are the promising cell source for
regenerative therapy, especially in craniofacial areas.⁶⁰ Den-
tal tissue-derived MSCs have a faster proliferation rate com-
pared to other MSCs sources like bone marrow or adipose
tissue, indicating that these dental tissue-derived MSCs could
be a candidate MSC source for regenerative therapy.⁶¹,⁶² Sour-
ces of MSCs in the oral region have been reported, including
pulp tissues of permanent teeth (dental pulp stem cells;
DPSCs),⁶³ remaining pulp tissues from primary teeth (stem
cells from human exfoliated deciduous teeth; SHEDs),⁶⁴ peri-
odontal ligament (periodontal ligament stem cells; PDLSCs),⁶⁵
gingival tissues (gingival stem cells),⁶⁶ apical papilla (stem
cells from the apical papilla; SCAPs), and dental follicle (den-
tal follicle stem cells).⁶⁰

The ability of oral cells to adhere, proliferate, and differen-
tiate into osteogenic differentiation can be significantly influ-
enced by the dECM generated from dental MSCs. PDLSCs-
dECM was shown to be more appropriate for osteogenic
induction, while SHEDs-dECM was shown to be more appro-
priate for ex vivo growth of DPSCs.⁶⁷ Furthermore, dECM
derived from hPDLSCs, dental pulp cells, or gingival fibro-
blasts promotes the proliferation and osteogenic differentia-
tion of hPDLSCs.⁶⁸ The dECM derived from hDPSCs enriches
with collagens and elastic fibres, showing the capacity for
osteoinduction by improving the mineralisation of human
gingival fibroblasts (Figure 1).²⁹ hDPSCs-dECM demonstrated
usefulness in modulating dental cell responses in vitro, sug-
gesting the benefit of dental tissue engineering, mineralised
tissue regeneration, or embellishing biomaterials. dECM

derived from dental MSCs promotes angiogenesis, as shown
by the upregulation of proangiogenic growth factors.⁶⁹ These
dECM improve the formation of dental tissues in vivo without
the addition of exogenous factors in subcutaneous implanta-
tion.⁶⁹ dECM derived from hDPSCs on polylactic acid (PLA)
scaffolds promotes calvarial bone healing in rats, as deter-
mined by increased new bone volume and area compared to
PLA alone.⁷⁰ Interestingly, induced pluripotent stem cells
(iPSCs) have been proposed as potential therapeutic cells
because a patient-specific cell line can be generated. iPSCs
can differentiate into numerous cell lineages. Feeder cells are
required for culture during reprogramming and passage to
maintain iPSCs in the stem cell stage.⁷¹ dECM derived from
dental pulp cells is suitable as a feeder-free culture medium
for dental pulp-derived iPSCs by improving iPSC attachment,
cell growth, and proliferation.⁷²

Notch signalling activation in human dental pulp cells upregulated the ECM organisation pathway, as confirmed by RNA sequencing analysis.⁷³ Proteomic and matrisome analysis of Notch ligands, Jagged-1, stimulated hDPSCs demonstrates the protein component related to osteogenic differentiation.⁷⁴ Furthermore, the dECM of the Jagged-1 treated condition exhibited a higher mineralisation and glycosaminoglycan component compared to the dECM of the control condition. Both dECM derived from human IgG Fc fragment (hFc) and Jagged-1 treated hDPSCs support SCAPs attachment, proliferation, and osteogenic differentiation (Figure 2).¹³ However, dECM from Jagged-1-treated hDPSCs promotes higher mineralisation of reseeded SCAPs compared to dECM from the control condition. This strong evidence suggests that the manipulation of dental MSCs can alter the characteristics of the dECM and, subsequently, influence cell responses. Taking all evidence together, ECMs derived from dental cells could potentially be used as a suitable natural biomaterial scaffold for applications in the clinic, such as regenerative treatment. A summary of the effects of dECM-derived from MSCs (B) and dental MSCs (A) on cellular responses is shown in Table 3A, B.

Future study on decellularised dental MSCs-derived ECM for
use in regenerative dentistry

To promote regeneration in dental tissues, dental stem cells need to migrate to the injured site and proliferate. Later, these cells differentiate into mature cells or secrete growth factors, modulating biological processes to enhance regeneration. Furthermore, the biodegradation and biocompatibility of the scaffold are required to provide the appropriate physical and biological interactions. Therefore, dECM is an excellent candidate, as it can be degraded in vivo and is not a cytotoxic substance.²⁹ Cellular responses to the composition, structure, and stiffness properties of the surrounding matrix and signalling pathways to drive the morphogenetic and pathogenic processes should be further investigated.

ECM scaffold combination with dental MSCs

A suitable scaffold material is vital for tissue regeneration,
providing sites for cell adhesion, proliferation, and differenti-
ation. ECM scaffolds offer the advantage of promoting natural
<figure><img src="image_2.png" /><figcaption>Figure 1 – Decellularised extracellular matrix (dECM) from human dental pulp stem cells (hDPSCs) enhanced the osteogenic differentiation potency of gingival fibroblasts (GFs). GFs were reseeded on N-dECM and OM-dECM cultured with growth medium or osteogenic differentiation medium. A and B, Cell attachment was examined at 24 hours using a scanning electron microscope analysis. C, Cell metabolic activity was examined using MTT assay on days 1, 3, and 7. D, The mRNA expression of the osteogenic marker gene was evaluated using real-time quantitative PCR. GFs were seeded on TCP; after osteogenic differentiation for 14 days, ALP staining and mineral accumulation were determined using BCIP/NBT, Alizarin Red S and Von Kossa staining, respectively (E-H). GFs were reseeded on N-dECM or OM-dECM and subsequently cultured in a growth medium (I-R) or osteogenic induction medium (K-T). Cells seeded on TCP were used as the control. Asterisks indicate a statistically significant difference compared with the control (P-value <.05). Reprinted from Nowwarote N, Petit S, Ferre FC, et al. Extracellular matrix derived from dental pulp stem cells promotes mineralization. Front Bioeng Biotechnol 2022;9:740712, under the terms of the Creative Commons Attribution License (CC BY),²⁹ https://doi.org/10.3389/fbioe.2021.740712.</figcaption></figure>

tissue architecture regeneration in regenerative dentistry. For instance, a porcine-derived ECM scaffold seeded with DPSCs successfully regenerated dentin-pulp-like tissue with morphology, mineralisation, and mechanical properties similar to native dentin-pulp tissue.⁷⁵ Using an ECM scaffold treated with dentin matrix protein 1 and seeded with DPSCs, the results showed the promotion of dental pulp/dentin-like tissue complex formation in both *in vitro* and *in vivo*⁷⁶.

## Decellularisation of dental tissue

Dental tissue decellularisation is a promising approach to the development of tissue-engineered constructs for various dental applications, such as dental pulp and periodontal regeneration. A few studies showed that decellularised bone tissue from porcine mandibles, seeded with MSCs and growth factors, can regenerate bone in the jaw.⁷⁷ Furthermore, the

potential of decellularised PDL tissue for periodontal regeneration in animal models has been demonstrated. These studies showed that decellularised PDL tissue could promote the attachment and proliferation of periodontal cells, induce the formation of new blood vessels, and support the regeneration of periodontal tissues.⁷⁸ However, more research is necessary to evaluate the safety and efficacy of the use of decellularised dental tissues in humans. Furthermore, the development of efficient decellularisation protocols, the optimisation of scaffold properties, and the integration of growth factors and other bioactive molecules could enhance the regenerative potential of decellularised dental tissue.

## dECM derived from dental MSCs

The use of cellular dECM in regenerative dentistry has gained attention in recent years due to its potential to
<figure><img src="image_2.png" /><figcaption>Figure 1 – Decellularised extracellular matrix (dECM) from human dental pulp stem cells (hDPSCs) enhanced the osteogenic differentiation potency of gingival fibroblasts (GFs). GFs were reseeded on N-dECM and OM-dECM cultured with growth medium or osteogenic differentiation medium. A and B, Cell attachment was examined at 24 hours using a scanning electron microscope analysis. C, Cell metabolic activity was examined using MTT assay on days 1, 3, and 7. D, The mRNA expression of the osteogenic marker gene was evaluated using real-time quantitative PCR. GFs were seeded on TCP; after osteogenic differentiation for 14 days, ALP staining and mineral accumulation were determined using BCIP/NBT, Alizarin Red S and Von Kossa staining, respectively (E-H). GFs were reseeded on N-dECM or OM-dECM and subsequently cultured in a growth medium (I-R) or osteogenic induction medium (K-T). Cells seeded on TCP were used as the control. Asterisks indicate a statistically significant difference compared with the control (P-value <.05). Reprinted from Nowwarote N, Petit S, Ferre FC, et al. Extracellular matrix derived from dental pulp stem cells promotes mineralization. Front Bioeng Biotechnol 2022;9:740712, under the terms of the Creative Commons Attribution License (CC BY),²⁹ https://doi.org/10.3389/fbioe.2021.740712.</figcaption></figure>

tissue architecture regeneration in regenerative dentistry. For instance, a porcine-derived ECM scaffold seeded with DPSCs successfully regenerated dentin-pulp-like tissue with morphology, mineralisation, and mechanical properties similar to native dentin-pulp tissue.⁷⁵ Using an ECM scaffold treated with dentin matrix protein 1 and seeded with DPSCs, the results showed the promotion of dental pulp/dentin-like tissue complex formation in both *in vitro* and *in vivo*⁷⁶.

### Decellularisation of dental tissue

Dental tissue decellularisation is a promising approach to the development of tissue-engineered constructs for various dental applications, such as dental pulp and periodontal regeneration. A few studies showed that decellularised bone tissue from porcine mandibles, seeded with MSCs and growth factors, can regenerate bone in the jaw.⁷⁷ Furthermore, the

potential of decellularised PDL tissue for periodontal regeneration in animal models has been demonstrated. These studies showed that decellularised PDL tissue could promote the attachment and proliferation of periodontal cells, induce the formation of new blood vessels, and support the regeneration of periodontal tissues.⁷⁸ However, more research is necessary to evaluate the safety and efficacy of the use of decellularised dental tissues in humans. Furthermore, the development of efficient decellularisation protocols, the optimisation of scaffold properties, and the integration of growth factors and other bioactive molecules could enhance the regenerative potential of decellularised dental tissue.

### dECM derived from dental MSCs

The use of cellular dECM in regenerative dentistry has gained attention in recent years due to its potential to
<figure><img src="image_3.png" /><figcaption>Figure 2 - Biological responses of SCAPs on Jagged1 dECMs. Stem cells isolated from apical papilla (SCAPs) were characterised by flow cytometry to examine surface protein marker expression (A). The mineralisation was examined using Alizarin Red S staining on day 14 after osteogenic induction (B and C). The intracellular lipid accumulation was detected using Oil Red O staining on day 16 after adipogenic induction (D and E). The cell viability of SCAPs on dECM was determined using an MTT assay. The data were presented as mean ± SEM and each dot represented the value from each donor (F). Cell attachment and actin arrangement were examined using phalloidin staining at 30 minutes, 24 hours, and 7 days (G). Cell spreading was observed using scanning electron microscopic analysis (H). dECM-N, decellularised extracellular matrix derived from maintaining cells in normal medium; dECM-OM, decellularised extracellular matrix derived from maintaining cells in osteogenic medium. Reprinted from Phothichailert S, Nowwarote N, Fournier BPJ, et al. Effects of decellularized extracellular matrix derived from Jagged1-treated human dental pulp stem cells on biological responses of stem cells isolated from apical papilla. Front Cell Dev Biol 2022;10:948812, under the terms of the Creative Commons Attribution License (CC BY),¹³ https://doi.org/10.3389/fcell.2022.948812.</figcaption></figure>

promote tissue regeneration in the oral cavity, especially dECM derived from dental MSCs. Dental pulp tissue typically exhibits patterns of expression and distribution of ECM proteins like those of hDPSCs. The characteristics of the donor tooth affect the ECM proteins of DPSCs.⁷⁹ The main issue with dental tissue decellularisation is that it can leave behind residual cellular debris that can trigger an immune response or cause complications during the decellularisation process.⁷⁷,⁷⁸ Therefore, dECM from culture dental MSCs can eliminate those limitations of ECM derived

from tissues, potentially leading to novel and effective therapies for repairing and regenerating damaged or lost dental tissues. dECM derived from dental MSCs has shown promise in tissue engineering and regenerative medicine. It was shown to promote the regeneration of bone, cartilage, and dental tissues.¹³,²⁹,⁵⁸,⁶⁷ Additionally, it exhibits immunomodulatory properties that reduce inflammation and enhance tissue repair.⁵⁸ Despite these positive findings, further research is required to optimise its properties and evaluate its clinical potential.
<figure><img src="image_3.png" /><figcaption>**Figure 2** – Biological responses of SCAPs on Jagged1 dECMs. Stem cells isolated from apical papilla (SCAPs) were characterised by flow cytometry to examine surface protein marker expression (A). The mineralisation was examined using Alizarin Red S staining on day 14 after osteogenic induction (B and C). The intracellular lipid accumulation was detected using Oil Red O staining on day 16 after adipogenic induction (D and E). The cell viability of SCAPs on dECM was determined using an MTT assay. The data were presented as mean ± SEM and each dot represented the value from each donor (F). Cell attachment and actin arrangement were examined using phalloidin staining at 30 minutes, 24 hours, and 7 days (G). Cell spreading was observed using scanning electron microscopic analysis (H). dECM-N, decellularised extracellular matrix derived from maintaining cells in normal medium; dECM-OM, decellularised extracellular matrix derived from maintaining cells in osteogenic medium. Reprinted from Phothichailert S, Nowwarote N, Fournier BPJ, et al. Effects of decellularized extracellular matrix derived from Jagged1-treated human dental pulp stem cells on biological responses of stem cells isolated from apical papilla. Front Cell Dev Biol 2022;10:948812, under the terms of the Creative Commons Attribution License (CC BY),¹³ https://doi.org/10.3389/fcell.2022.948812.</figcaption></figure>

promote tissue regeneration in the oral cavity, especially dECM derived from dental MSCs. Dental pulp tissue typically exhibits patterns of expression and distribution of ECM proteins like those of hDPSCs. The characteristics of the donor tooth affect the ECM proteins of DPSCs.⁷⁹ The main issue with dental tissue decellularisation is that it can leave behind residual cellular debris that can trigger an immune response or cause complications during the decellularisation process.⁷⁷,⁷⁸ Therefore, dECM from culture dental MSCs can eliminate those limitations of ECM derived

from tissues, potentially leading to novel and effective therapies for repairing and regenerating damaged or lost dental tissues. dECM derived from dental MSCs has shown promise in tissue engineering and regenerative medicine. It was shown to promote the regeneration of bone, cartilage, and dental tissues.¹³,²⁹,⁵⁸,⁶⁷ Additionally, it exhibits immunomodulatory properties that reduce inflammation and enhance tissue repair.⁵⁸ Despite these positive findings, further research is required to optimise its properties and evaluate its clinical potential.
<table><caption>Table 3 – (A) Effect of the decellularised extracellular matrix derived from dental mesenchymal stem cells on cell responses.</caption><thead><tr><th>No.</th><th>ECM derived from MSCs</th><th>ECM production medium</th><th>Decellularisation methods</th><th>Cell reseeded</th><th>Effects</th><th>Signalling pathway related</th><th>Study</th></tr></thead><tbody><tr><td>1</td><td>hPDLSCs and SHEDs</td><td>10% αMEM supplement with 50 µg/mL ascorbic acid (250 uM) for 7 days</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hDPSCs</td><td>Promote cell adhesion, cell proliferation, and osteogenic differentiation in vitro</td><td>-</td><td>Heng et al<sup>67</sup></td></tr><tr><td>2</td><td>hPDLSCs and hUSCs</td><td>10% αMEM for 8 days, 80-90% confluence add 50 µg/mL ascorbic acid</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL-1 DNase I</td><td>hPDLSCs</td><td>Promote cell attachment, osteogenic differentiation, adipogenic differentiation, and angiogenesis in vitro</td><td>-</td><td>Xiong et al<sup>58</sup></td></tr><tr><td>3</td><td>hDPSCs</td><td>Pretreat TCP with 0.2% gelatin, culture in 10% DMEM for 21 days, add 50 µg/mL ascorbic acid on day 14 and culture in osteogenic induction medium for 21 days</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS, DNase I</td><td>hGFs</td><td>Promote mineralisation in vitro</td><td>-</td><td>Nowwarote et al<sup>29</sup></td></tr><tr><td>4</td><td>hDPSCs</td><td>Pretreat TCP with 0.2% gelatin, culture in 10% DMEM for 21 days, add 50 µg/mL ascorbic acid at day 14 and culture in osteogenic induction medium for 21 days</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS, DNase I</td><td>hSCAPs</td><td>Promoted odonto/osteogenic differentiation in vitro</td><td>NOTCH</td><td>Phothichailert et al<sup>13</sup></td></tr></tbody></table>

ECM, extracellular matrix; hDPSCs, human dental pulp stem cells; hGF, human gingival fibroblast; hPDLSCs, human periodontal stem cells; hUSCs, human urine derived stem cells; SCAPs, stem cells isolated from apical papilla; SHEDs, stem cell derived from human exfoliated deciduous teeth.

### The effect of dECM-secreted growth factors in regenerative dentistry

Growth factors are molecules that can stimulate cell activities and modulate tissue repair and regeneration. dECM-secreted growth factors hold immense potential for promoting various tissue regeneration processes in dentistry. Growth factors released from the dentine matrix by mineral trioxide solutions include vascular endothelial growth factor, insulin-like growth factor I and II (IGF-I, IGFBP-1), macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor, and neurotrophic growth factors. These growth factors induce proliferation and chemotaxis in dental pulp cells.⁸⁰ In a recent investigation, it was demonstrated that dECM derived from DPSCs, enriched with IGF-binding proteins 2, 4, and 5, TGF-binding proteins 1, 2, 3, and 4, and TGF-β binding proteins, encompasses growth factor-binding proteins found within glycoproteins.²⁹

Hence, the direct accumulation and functional implications of growth factors secreted by dECM derived from MSCs remain to be explored further. Understanding the signalling pathways governing oral tissue and dentin homeostasis, as well as their role in regeneration, necessitates further investigation.

### The effects of dECM on immunomodulation in regenerative dentistry

Cell-free biomaterials, having either inflammation-boosting or calming effects, effectively control the immediate response to injury after implantation. In a xenotransplantation model, dECM derived from skeleton muscle tissue promotes the M2 macrophage phenotype and has anti-inflammatory and immunomodulatory properties.⁸¹ In addition, the reseeding of CD34⁺ bone marrow mononuclear cells on decellularised aortic scaffolds creates an immunomodulatory microenvironment by reduced proinflammatory cytokines (IL-8, granulocyte-macrophage colony-stimulating factor, MIP-1β, GRO-α, Entoxin, and GRO) and increased anti-inflammatory cytokines (IL-2 and TGF-β).⁸² In a study in a periodontal defect model, decellularised porcine cancellous bone treated with an anti-inflammatory substance demonstrated that the immune response in periodontal tissue began with the engagement of macrophages and giant cells. The presence of these components influenced the local environment and stimulated the release of regenerative factors, which in turn enhanced tissue regeneration.⁸³ Further, a 3D bioprinting of dECM from dental follicle tissue powder with hydrogel demonstrates immunomodulatory effects by reducing the release of inflammatory cytokines from M1 macrophages and alleviating local inflammation in periodontal defects.⁸³ In addition, dECM derived from DPSC notable release of CXCL12, which could be attributable to the regulation of DPSC homeostasis.²⁹

Nevertheless, the innovative approach based on cellular-derived dECM and its unique function in maintaining MSC properties, particularly regarding immunoregulation and matrix formation, is not well explored in the current state of study. How this approach relates to tissue regeneration and its therapeutic applications is a crucial area of unmet research need.
<table><caption>Table 3 – (A) Effect of the decellularised extracellular matrix derived from dental mesenchymal stem cells on cell responses.</caption><thead><tr><th>No.</th><th>ECM derived from MSCs</th><th>ECM production medium</th><th>Decellularisation methods</th><th>Cell reseeded</th><th>Effects</th><th>Signalling pathway related</th><th>Study</th></tr></thead><tbody><tr><td>1</td><td>hPDLSCs and SHEDs</td><td>10% αMEM supplement with 50 µg/mL ascorbic acid (250 uM) for 7 days</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hDPSCs</td><td>Promote cell adhesion, cell proliferation, and osteogenic differentiation in vitro</td><td>-</td><td>Heng et al<sup>67</sup></td></tr><tr><td>2</td><td>hPDLSCs and hUSCs</td><td>10% αMEM for 8 days, 80-90% confluence add 50 µg/mL ascorbic acid</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL-1 DNase I</td><td>hPDLSCs</td><td>Promote cell attachment, osteogenic differentiation, adipogenic differentiation, and angiogenesis in vitro</td><td>-</td><td>Xiong et al<sup>58</sup></td></tr><tr><td>3</td><td>hDPSCs</td><td>Pretreat TCP with 0.2% gelatin, culture in 10% DMEM for 21 days, add 50 µg/mL ascorbic acid on day 14 and culture in osteogenic induction medium for 21 days</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS, DNase I</td><td>hGFs</td><td>Promote mineralisation in vitro</td><td>-</td><td>Nowwarote et al<sup>29</sup></td></tr><tr><td>4</td><td>hDPSCs</td><td>Pretreat TCP with 0.2% gelatin, culture in 10% DMEM for 21 days, add 50 µg/mL ascorbic acid at day 14 and culture in osteogenic induction medium for 21 days</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS, DNase I</td><td>hSCAPs</td><td>Promoted odonto/osteogenic differentiation in vitro</td><td>NOTCH</td><td>Phothichailert et al<sup>13</sup></td></tr></tbody></table>

ECM, extracellular matrix; hDPSCs, human dental pulp stem cells; hGF, human gingival fibroblast; hPDLSCs, human periodontal stem cells; hUSCs, human urine derived stem cells; SCAPs, stem cells isolated from apical papilla; SHEDs, stem cell derived from human exfoliated deciduous teeth.

### The effect of dECM-secreted growth factors in regenerative dentistry

Growth factors are molecules that can stimulate cell activities and modulate tissue repair and regeneration. dECM-secreted growth factors hold immense potential for promoting various tissue regeneration processes in dentistry. Growth factors released from the dentine matrix by mineral trioxide solutions include vascular endothelial growth factor, insulin-like growth factor I and II (IGF-I, IGFBP-1), macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor, and neurotrophic growth factors. These growth factors induce proliferation and chemotaxis in dental pulp cells.⁸⁰ In a recent investigation, it was demonstrated that dECM derived from DPSCs, enriched with IGF-binding proteins 2, 4, and 5, TGF-binding proteins 1, 2, 3, and 4, and TGF-β binding proteins, encompasses growth factor-binding proteins found within glycoproteins.²⁹

Hence, the direct accumulation and functional implications of growth factors secreted by dECM derived from MSCs remain to be explored further. Understanding the signalling pathways governing oral tissue and dentin homeostasis, as well as their role in regeneration, necessitates further investigation.

### The effects of dECM on immunomodulation in regenerative dentistry

Cell-free biomaterials, having either inflammation-boosting or calming effects, effectively control the immediate response to injury after implantation. In a xenotransplantation model, dECM derived from skeleton muscle tissue promotes the M2 macrophage phenotype and has anti-inflammatory and immunomodulatory properties.⁸¹ In addition, the reseeding of CD34⁺ bone marrow mononuclear cells on decellularised aortic scaffolds creates an immunomodulatory microenvironment by reduced proinflammatory cytokines (IL-8, granulocyte-macrophage colony-stimulating factor, MIP-1β, GRO-α, Entoxin, and GRO) and increased anti-inflammatory cytokines (IL-2 and TGF-β).⁸² In a study in a periodontal defect model, decellularised porcine cancellous bone treated with an anti-inflammatory substance demonstrated that the immune response in periodontal tissue began with the engagement of macrophages and giant cells. The presence of these components influenced the local environment and stimulated the release of regenerative factors, which in turn enhanced tissue regeneration.⁸³ Further, a 3D bioprinting of dECM from dental follicle tissue powder with hydrogel demonstrates immunomodulatory effects by reducing the release of inflammatory cytokines from M1 macrophages and alleviating local inflammation in periodontal defects.⁸³ In addition, dECM derived from DPSC notable release of CXCL12, which could be attributable to the regulation of DPSC homeostasis.²⁹

Nevertheless, the innovative approach based on cellular-derived dECM and its unique function in maintaining MSC properties, particularly regarding immunoregulation and matrix formation, is not well explored in the current state of study. How this approach relates to tissue regeneration and its therapeutic applications is a crucial area of unmet research need.
<table><caption>Table 3 – (B) Effect of the decellularised extracellular matrix derived from mesenchymal stem cells on cell responses.</caption><thead><tr><th>No.</th><th>ECM derived from MSCs</th><th>ECM production medium</th><th>Decellularisation methods</th><th>Cell reseeded</th><th>Effects</th><th>Signalling pathway related</th><th>Study</th></tr></thead><tbody><tr><td>1</td><td>hADSCs</td><td>(1) 10%DMEM for 28 days; (2) Osteogenic medium for 28 days; (3) Adipose medium for 28 days</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL DNase I, RNase I, and lipase</td><td>hADSCs</td><td>Change cell fate in vitro</td><td>-</td><td>Guneta et al<sup>34</sup></td></tr><tr><td>2</td><td>hADSCs</td><td>10%DMEM for 15 days, add ascorbic acid (50 µM) at day 8</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL DNase I</td><td>Retinal progenitor cells</td><td>Promote cell proliferation and neuronal differentiation in vitro</td><td>AKT/ERK</td><td>Ji et al<sup>35</sup></td></tr><tr><td>3</td><td>hADSCs</td><td>Pretreat TCP with poly-L-Lysine 10% αMEM for 10 days supplement 0.2 mM ascorbic acid</td><td>1% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 10,20, 100 U/mL</td><td>Human fibroblast and Dermal microvascular endothelial cells</td><td>Promote cell proliferation and cell adhesion in vitro</td><td>-</td><td>Riis et al<sup>17</sup></td></tr><tr><td>4</td><td>hADSCs</td><td>10%DMEM for 21 days</td><td>0.5% Triton X-100 containing 25 mM NH<sub>4</sub>OH in PBS, 300 µg/mL DNase I</td><td>Cancer cell line (HT29 and SW480)</td><td>Successfully repopulate with cancer 3D structure and greater doxorubicin resistance in vitro</td><td>-</td><td>Rubi-Sans et al<sup>36</sup></td></tr><tr><td>5</td><td>hBMSCs</td><td>Pretreat TCP with 0.2% gelatin crosslink with 1% glutaraldehyde in 1M ethanolamine, culture in 10% αMEM supplement with 100 uM ascorbic acid for 8 days</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL DNase I</td><td>hBMSCs</td><td>Promote cell proliferation, osteogenic differentiation and chondrogenic differentiation. Decrease ROS and adipogenic differentiation in vitro</td><td>ERK1/2 and CYCLIND1</td><td>Pei et al<sup>38</sup></td></tr><tr><td>6</td><td>hBMSCs</td><td>10,000 cells/cm<sup>2</sup> in 6 well plate and serum free starvation for 3 days</td><td>0.25% Triton X-100 containing 0.25% sodium-deoxycholate in PSB, 100 µg/mL RNase, 10 U DNase</td><td>Human endothelial cells</td><td>Promote tubular morphogenesis and cell migration in vitro</td><td>-</td><td>Burns et al<sup>39</sup></td></tr><tr><td>7</td><td>hBMSCs</td><td>10% αMEM supplement with 50 uM ascorbic acid for 14 days</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL DNase I and digest with urea. Coat dECM on TCP</td><td>hBMSCs</td><td>Promote cell proliferation, cell attachment, cell spreading, cell migration, osteogenic differentiation, and adipogenic differentiation in vitro</td><td>-</td><td>Lin et al<sup>54</sup></td></tr><tr><td>8</td><td>hBMSCs</td><td>Pretreat TCP with 0.2% gelatin crosslink with 1% glutaraldehyde in 1M ethanolamine, culture in 10% αMEM supplement with 100 uM ascorbic acid for 8 days</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL DNase I</td><td>hBMSCs</td><td>Promote cell proliferation, mineralisation, and antioxidant effect of melatonin in vitro</td><td>-</td><td>He et al<sup>40</sup></td></tr><tr><td>9</td><td>hBMSCs</td><td>Pretreat TCP with POAM and FB culture for 10 days in: (1) 10% DMEM; (2) 10% DMEM supplement with ascorbic acid; (3) Osteogenic differentiation medium</td><td>20 mM NH<sub>4</sub>OH in DDW, 1000 U/mL DNase I</td><td>Hematopoietic stem and progenitor cell (HSPCs)</td><td>Promote cell expansion in vitro</td><td>-</td><td>Prewitz et al<sup>41</sup></td></tr><tr><td>10</td><td>hBMSCs</td><td>10%DMEM supplement with ascorbic acid for 100% confluence</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>mADSCs</td><td>Promote cell proliferation, colony-forming unit ability, osteogenic differentiation, adipogenic differentiation, and chondrogenic differentiation in vitro.</td><td>-</td><td>Xiong et al<sup>85</sup></td></tr></tbody></table>

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<table><caption>Table 3 – (B) Effect of the decellularised extracellular matrix derived from mesenchymal stem cells on cell responses.</caption><thead><tr><th>No.</th><th>ECM derived from MSCs</th><th>ECM production medium</th><th>Decellularisation methods</th><th>Cell reseeded</th><th>Effects</th><th>Signalling pathway related</th><th>Study</th></tr></thead><tbody><tr><td>1</td><td>hADSCs</td><td>(1) 10%DMEM for 28 days; (2) Osteogenic medium for 28 days; (3) Adipose medium for 28 days</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL DNase I, RNase I, and lipase</td><td>hADSCs</td><td>Change cell fate in vitro</td><td>-</td><td>Guneta et al<sup>34</sup></td></tr><tr><td>2</td><td>hADSCs</td><td>10%DMEM for 15 days, add ascorbic acid (50 µM) at day 8</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL DNase I</td><td>Retinal progenitor cells</td><td>Promote cell proliferation and neuronal differentiation in vitro</td><td>AKT/ERK</td><td>Ji et al<sup>35</sup></td></tr><tr><td>3</td><td>hADSCs</td><td>Pretreat TCP with poly-L-Lysine 10% αMEM for 10 days supplement 0.2 mM ascorbic acid</td><td>1% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 10,20, 100 U/mL</td><td>Human fibroblast and Dermal microvascular endothelial cells</td><td>Promote cell proliferation and cell adhesion in vitro</td><td>-</td><td>Riis et al<sup>17</sup></td></tr><tr><td>4</td><td>hADSCs</td><td>10%DMEM for 21 days</td><td>0.5% Triton X-100 containing 25 mM NH<sub>4</sub>OH in PBS, 300 µg/mL DNase I</td><td>Cancer cell line (HT29 and SW480)</td><td>Successfully repopulate with cancer 3D structure and greater doxorubicin resistance in vitro</td><td>-</td><td>Rubi-Sans et al<sup>36</sup></td></tr><tr><td>5</td><td>hBMSCs</td><td>Pretreat TCP with 0.2% gelatin crosslink with 1% glutaraldehyde in 1M ethanolamine, culture in 10% αMEM supplement with 100 uM ascorbic acid for 8 days</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL DNase I</td><td>hBMSCs</td><td>Promote cell proliferation, osteogenic differentiation and chondrogenic differentiation. Decrease ROS and adipogenic differentiation in vitro</td><td>ERK1/2 and CYCLIND1</td><td>Pei et al<sup>38</sup></td></tr><tr><td>6</td><td>hBMSCs</td><td>10,000 cells/cm<sup>2</sup> in 6 well plate and serum free starvation for 3 days</td><td>0.25% Triton X-100 containing 0.25% sodium-deoxycholate in PSB, 100 µg/mL RNase, 10 U DNase</td><td>Human endothelial cells</td><td>Promote tubular morphogenesis and cell migration in vitro</td><td>-</td><td>Burns et al<sup>39</sup></td></tr><tr><td>7</td><td>hBMSCs</td><td>10% αMEM supplement with 50 uM ascorbic acid for 14 days</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL DNase I and digest with urea. Coat dECM on TCP</td><td>hBMSCs</td><td>Promote cell proliferation, cell attachment, cell spreading, cell migration, osteogenic differentiation, and adipogenic differentiation in vitro</td><td>-</td><td>Lin et al<sup>54</sup></td></tr><tr><td>8</td><td>hBMSCs</td><td>Pretreat TCP with 0.2% gelatin crosslink with 1% glutaraldehyde in 1M ethanolamine, culture in 10% αMEM supplement with 100 uM ascorbic acid for 8 days</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL DNase I</td><td>hBMSCs</td><td>Promote cell proliferation, mineralisation, and antioxidant effect of melatonin in vitro</td><td>-</td><td>He et al<sup>40</sup></td></tr><tr><td>9</td><td>hBMSCs</td><td>Pretreat TCP with POAM and FB culture for 10 days in: (1) 10% DMEM; (2) 10% DMEM supplement with ascorbic acid; (3) Osteogenic differentiation medium</td><td>20 mM NH<sub>4</sub>OH in DDW, 1000 U/mL DNase I</td><td>Hematopoietic stem and progenitor cell (HSPCs)</td><td>Promote cell expansion in vitro</td><td>-</td><td>Prewitz et al<sup>41</sup></td></tr><tr><td>10</td><td>hBMSCs</td><td>10%DMEM supplement with ascorbic acid for 100% confluence</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>mADSCs</td><td>Promote cell proliferation, colony-forming unit ability, osteogenic differentiation, adipogenic differentiation, and chondrogenic differentiation in vitro.</td><td>-</td><td>Xiong et al<sup>85</sup></td></tr></tbody></table>

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<table><caption>Table 3. (Continued)</caption><thead><tr><th>No.</th><th>ECM derived from MSCs</th><th>ECM production medium</th><th>Decellularisation methods</th><th>Cell reseeded</th><th>Effects</th><th>Signalling pathway related</th><th>Study</th></tr></thead><tbody><tr><td>11</td><td>hBMSCs</td><td>10% MEM for 10 days, after 100% confluence add 50 µg/mL of ascorbic acid</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>Chondrocyte from the human knee articular</td><td>Promote cell proliferation, cell adhesion, cell migration, and osteogenic differentiation in vitro</td><td>-</td><td>Yang et al<sup>47</sup></td></tr><tr><td>12</td><td>hBMSCs</td><td>Culture in Primer-XV MSC expansion XSFM for 4 days</td><td>detach ECM, then freeze-thaw cycling with 25 mM NH<sub>4</sub>ON in PBS</td><td>hBMSCs</td><td>Promote new bone formation in mouse in vivo</td><td>-</td><td>Motoike et al<sup>12</sup></td></tr><tr><td>13</td><td>hBMSCs</td><td>10%DMEM supplement with 50 µg/mL ascorbic acid for 10 days</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 200 U/mL DNase, RNase-free</td><td>HUVEC</td><td>Promote cell proliferation in vitro</td><td>Proangiogenic signalling</td><td>Sears and Ghosh<sup>18</sup></td></tr><tr><td>14</td><td>hBMSCs</td><td>Pretreat TCP with 0.2% gelatin crosslink with 1% glutaraldehyde in 1M ethanolamine, culture in 10% DMEM for 8 days</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>UCB-MSCs</td><td>Promote cell proliferation and migration in vitro</td><td>-</td><td>Xu et al<sup>48</sup></td></tr><tr><td>15</td><td>hBMSCs</td><td>15% αMEM for 15 days, add ascorbic acid (50 µM) at day 8</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hBMSCs</td><td>Promote small cell size, cell proliferation, MSC surface marker, colony formation and osteogenic differentiation in vitro</td><td>-</td><td>Rakian et al<sup>32</sup></td></tr><tr><td>16</td><td>hBMSCs</td><td>Pretreat TCP with POAM and FB culture in 10% DMEM for 10 days</td><td>20 mM NH<sub>4</sub>OH in DDW</td><td>Hematopoietic stem and progenitor cell (HSPCs)</td><td>Promote cell expansion, cell adhesion, and cell migration in vitro</td><td>Integrin BETA 3</td><td>Krater et al<sup>42</sup></td></tr><tr><td>17</td><td>hBMSCs</td><td>15% αMEM for 15 days, add ascorbic acid (100 µM) at day 8</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL DNase I</td><td>hDPSCs</td><td>Promote cell adhesion and neurogenic differentiation in vitro</td><td>-</td><td>Laudani et al<sup>19</sup></td></tr><tr><td>18</td><td>hBMSCs and hADSCs</td><td>15% αMEM for 15 days, add ascorbic acid (50 µM) at day 8</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hBMSCs, hADSCs, HeLa, MCF7 and MDA-MB-231 cancer cell lines</td><td>Promote MSCs proliferation, cell spreading, osteogenic differentiation, adipogenic differentiation and no effect on cancer cells in vitro</td><td>-</td><td>Marinkovic et al<sup>86</sup></td></tr><tr><td>19</td><td>hBMSCs and HUVECs</td><td>10%DMEM for 7-10 days, add ascorbic acid (50 µM) at day 8</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hBMSCs</td><td>Promote cell proliferation, osteogenic differentiation, adipogenic differentiation, and angiogenesis in vitro</td><td>-</td><td>Carvalho et al<sup>50</sup></td></tr><tr><td>20</td><td>hMSCs</td><td>10% αMEM for 15 days</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL DNase I</td><td>hMSCs</td><td>Promote cell proliferation, colony-forming unit ability, osteogenic differentiation, and adipogenic differentiation in vitro</td><td>ERK1/2</td><td>Decaris et al<sup>49</sup></td></tr></tbody></table>

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<table><caption>**Table 3. (Continued)**</caption><thead><tr><th>No.</th><th>ECM derived from MSCs</th><th>ECM production medium</th><th>Decellularisation methods</th><th>Cell reseeded</th><th>Effects</th><th>Signalling pathway related</th><th>Study</th></tr></thead><tbody><tr><td>11</td><td>hBMSCs</td><td>10% MEM for 10 days, after 100% confluence add 50 µg/mL of ascorbic acid</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>Chondrocyte from the human knee articular</td><td>Promote cell proliferation, cell adhesion, cell migration, and osteogenic differentiation in vitro</td><td>-</td><td>Yang et al<sup>47</sup></td></tr><tr><td>12</td><td>hBMSCs</td><td>Culture in Primer-XV MSC expansion XSFM for 4 days</td><td>detach ECM, then freeze-thaw cycling with 25 mM NH<sub>4</sub>ON in PBS</td><td>hBMSCs</td><td>Promote new bone formation in mouse in vivo</td><td>-</td><td>Motoike et al<sup>12</sup></td></tr><tr><td>13</td><td>hBMSCs</td><td>10%DMEM supplement with 50 µg/mL ascorbic acid for 10 days</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 200 U/mL DNase, RNase-free</td><td>HUVEC</td><td>Promote cell proliferation in vitro</td><td>Proangiogenic signalling</td><td>Sears and Ghosh<sup>18</sup></td></tr><tr><td>14</td><td>hBMSCs</td><td>Pretreat TCP with 0.2% gelatin crosslink with 1% glutaraldehyde in 1M ethanolamine, culture in 10% DMEM for 8 days</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>UCB-MSCs</td><td>Promote cell proliferation and migration in vitro</td><td>-</td><td>Xu et al<sup>48</sup></td></tr><tr><td>15</td><td>hBMSCs</td><td>15% αMEM for 15 days, add ascorbic acid (50 µM) at day 8</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hBMSCs</td><td>Promote small cell size, cell proliferation, MSC surface marker, colony formation and osteogenic differentiation in vitro</td><td>-</td><td>Rakian et al<sup>32</sup></td></tr><tr><td>16</td><td>hBMSCs</td><td>Pretreat TCP with POAM and FB culture in 10% DMEM for 10 days</td><td>20 mM NH<sub>4</sub>OH in DDW</td><td>Hematopoietic stem and progenitor cell (HSPCs)</td><td>Promote cell expansion, cell adhesion, and cell migration in vitro</td><td>Integrin BETA 3</td><td>Krater et al<sup>42</sup></td></tr><tr><td>17</td><td>hBMSCs</td><td>15% αMEM for 15 days, add ascorbic acid (100 µM) at day 8</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL DNase I</td><td>hDPSCs</td><td>Promote cell adhesion and neurogenic differentiation in vitro</td><td>-</td><td>Laudani et al<sup>19</sup></td></tr><tr><td>18</td><td>hBMSCs and hADSCs</td><td>15% αMEM for 15 days, add ascorbic acid (50 µM) at day 8</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hBMSCs, hADSCs, HeLa, MCF7 and MDA-MB-231 cancer cell lines</td><td>Promote MSCs proliferation, cell spreading, osteogenic differentiation, adipogenic differentiation and no effect on cancer cells in vitro</td><td>-</td><td>Marinkovic et al<sup>86</sup></td></tr><tr><td>19</td><td>hBMSCs and HUVECs</td><td>10%DMEM for 7-10 days, add ascorbic acid (50 µM) at day 8</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hBMSCs</td><td>Promote cell proliferation, osteogenic differentiation, adipogenic differentiation, and angiogenesis in vitro</td><td>-</td><td>Carvalho et al<sup>50</sup></td></tr><tr><td>20</td><td>hMSCs</td><td>10% αMEM for 15 days</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL DNase I</td><td>hMSCs</td><td>Promote cell proliferation, colony-forming unit ability, osteogenic differentiation, and adipogenic differentiation in vitro</td><td>ERK1/2</td><td>Decaris et al<sup>49</sup></td></tr></tbody><tfoot><tr><td colspan="8" style="text-align: right;">(continued on next page)</td></tr></tfoot></table>
<table><caption>Table 3. (Continued)</caption><thead><tr><th>No.</th><th>ECM derived from MSCs</th><th>ECM production medium</th><th>Decellularisation methods</th><th>Cell reseeded</th><th>Effects</th><th>Signalling pathway related</th><th>Study</th></tr></thead><tbody><tr><td>21</td><td>hMSCs</td><td>10% αMEM for 15 days, add ascorbic acid (50 µM) at day 8</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL DNase I</td><td>hMSCs</td><td>Promote cell proliferation, colony-forming unit ability, osteogenic differentiation, and adipogenic differentiation in vitro</td><td>ERK1/2 and FGF-2</td><td>Kim and Ma 2013<sup>43</sup></td></tr><tr><td>22</td><td>hUC-MSCs</td><td>Pretreat TCP with 0.2% gelatin and crosslink with 1% glutaraldehyde in 1M ethanolamine, culture in 10% αMEM supplement with 100 µM ascorbic acid for 8 days</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL DNase I</td><td>hUC-MSCs</td><td>Promote cell proliferation, anti-oxidative enzyme activity, and osteogenic differentiation in vitro</td><td>-</td><td>Liu et al<sup>53</sup></td></tr><tr><td>23</td><td>hUC-MSCs</td><td>Pretreat TCP with 0.2% gelatin and crosslink with 1% glutaraldehyde in 1M ethanolamine, culture in 10% αMEM supplement with 100 µM ascorbic acid for 8 days</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL DNase I</td><td>hUC-MSCs</td><td>Promote cell proliferation, osteogenic differentiation, and resistance to oxidative stress-induced premature senescence in vitro</td><td>SIRT1</td><td>Zhou et al<sup>88</sup></td></tr><tr><td>24</td><td>hUC-MSCs</td><td>Pretreat TCP with 25 g/mL fibroblast, culture in 10% αMEM for 15 days, add ascorbic acid (50 µM) at day 8</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>Rabbit chondrocytes</td><td>Promote cell proliferation and chondrogenic differentiation in vitro</td><td>-</td><td>Zhang et al<sup>30</sup></td></tr><tr><td>25</td><td>Immortal cell line from placenta derived MSCs</td><td>10% αMEM for 14 days, add ascorbic acid (50 µM) at day 3</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hMSCs</td><td>Promote cell proliferation and osteogenic differentiation in vitro</td><td>-</td><td>Kusuma et al<sup>56</sup></td></tr><tr><td>26</td><td>Immortal cell line from placenta derived MSCs</td><td>10% αMEM for 14 days, add ascorbic acid (50 µM) at day 7</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hMSCs from placenta</td><td>Promote cell proliferation and adipogenic differentiation in vitro</td><td>-</td><td>Yang et al<sup>10</sup></td></tr><tr><td>27</td><td>hNHDF, hNHAc and hMSCs</td><td>10%DMEM supplement with ascorbic acid (50 mg/mL) for 7 days</td><td>(1) 0.025% trypsin and 0.002% EDTA in PBS; (2) 0.1% Triton X-100, 10 mM Tris-HCl, pH 8.0, and 5 mM EDTA) containing 100 µg/mL DNase I and 100 µg/mL RNase A<br>(1) 1% Triton X-100 in dH<sub>2</sub>O; (2) 1% Triton X-100 in dH<sub>2</sub>O and followed by 150 U/mL DNase I; (3) 1% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hNHAc<br>hNHDF</td><td>Promote cell proliferation and adhesion in vitro</td><td>-</td><td>Hoshiba et al<sup>84</sup></td></tr><tr><td>28</td><td>hBMSCs, hNHDF and iPS hNHDF</td><td>Pretreat TCP with Cell Matrix™, culture in 10%DMEM for 100% confluence</td><td>(1) 1% Triton X-100 in dH<sub>2</sub>O; (2) 1% Triton X-100 in dH<sub>2</sub>O and followed by 150 U/mL DNase I; (3) 1% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hNHDF</td><td>Promote cell proliferation and enhance somatic, multipotent and pluripotent lineage-specific mRNA expression in vitro</td><td>-</td><td>Parmaksiz et al<sup>15</sup></td></tr><tr><td>29</td><td>Human foetal MSCs, human adult MSCs, and human neonatal dermal fibroblast</td><td>10% αMEM for 14 days, add 50 µM ascorbic acid on day 7 or 8</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL-1 DNase I</td><td>hMSCs</td><td>Promote cell proliferation, osteogenic differentiation, chondrogenic differentiation, and adipogenic differentiation ex vivo</td><td>-</td><td>Ng et al<sup>45</sup></td></tr></tbody></table>

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<table><caption>**Table 3. (Continued)**</caption><thead><tr><th>No.</th><th>ECM derived from MSCs</th><th>ECM production medium</th><th>Decellularisation methods</th><th>Cell reseeded</th><th>Effects</th><th>Signalling pathway related</th><th>Study</th></tr></thead><tbody><tr><td>21</td><td>hMSCs</td><td>10% αMEM for 15 days, add ascorbic acid (50 µM) at day 8</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL DNase I</td><td>hMSCs</td><td>Promote cell proliferation, colony-forming unit ability, osteogenic differentiation, and adipogenic differentiation in vitro</td><td>ERK1/2 and FGF-2</td><td>Kim and Ma 2013<sup>43</sup></td></tr><tr><td>22</td><td>hUC-MSCs</td><td>Pretreat TCP with 0.2% gelatin and crosslink with 1% glutaraldehyde in 1M ethanolamine, culture in 10% αMEM supplement with 100 µM ascorbic acid for 8 days</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL DNase I</td><td>hUC-MSCs</td><td>Promote cell proliferation, anti-oxidative enzyme activity, and osteogenic differentiation in vitro</td><td>-</td><td>Liu et al<sup>53</sup></td></tr><tr><td>23</td><td>hUC-MSCs</td><td>Pretreat TCP with 0.2% gelatin and crosslink with 1% glutaraldehyde in 1M ethanolamine, culture in 10% αMEM supplement with 100 µM ascorbic acid for 8 days</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL DNase I</td><td>hUC-MSCs</td><td>Promote cell proliferation, osteogenic differentiation, and resistance to oxidative stress-induced premature senescence in vitro</td><td>SIRT1</td><td>Zhou et al<sup>88</sup></td></tr><tr><td>24</td><td>hUC-MSCs</td><td>Pretreat TCP with 25 g/mL fibroblast, culture in 10% αMEM for 15 days, add ascorbic acid (50 µM) at day 8</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>Rabbit chondrocytes</td><td>Promote cell proliferation and chondrogenic differentiation in vitro</td><td>-</td><td>Zhang et al<sup>30</sup></td></tr><tr><td>25</td><td>Immortal cell line from placenta derived MSCs</td><td>10% αMEM for 14 days, add ascorbic acid (50 µM) at day 3</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hMSCs</td><td>Promote cell proliferation and osteogenic differentiation in vitro</td><td>-</td><td>Kusuma et al<sup>56</sup></td></tr><tr><td>26</td><td>Immortal cell line from placenta derived MSCs</td><td>10% αMEM for 14 days, add ascorbic acid (50 µM) at day 7</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hMSCs from placenta</td><td>Promote cell proliferation and adipogenic differentiation in vitro</td><td>-</td><td>Yang et al<sup>10</sup></td></tr><tr><td>27</td><td>hNHDF, hNHAc and hMSCs</td><td>10%DMEM supplement with ascorbic acid (50 mg/mL) for 7 days</td><td>(1) 0.025% trypsin and 0.002% EDTA in PBS; (2) 0.1% Triton X-100, 10 mM Tris-HCl, pH 8.0, and 5 mM EDTA) containing 100 µg/mL DNase I and 100 µg/mL RNase A<br>(1) 1% Triton X-100 in dH<sub>2</sub>O; (2) 1% Triton X-100 in dH<sub>2</sub>O and followed by 150 U/mL DNase I; (3) 1% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hNHAc<br>hNHDF</td><td>Promote cell proliferation and adhesion in vitro</td><td>-</td><td>Hoshiba et al<sup>84</sup></td></tr><tr><td>28</td><td>hBMSCs, hNHDF and iPS hNHDF</td><td>Pretreat TCP with Cell Matrix™, culture in 10%DMEM for 100% confluence</td><td>(1) 1% Triton X-100 in dH<sub>2</sub>O; (2) 1% Triton X-100 in dH<sub>2</sub>O and followed by 150 U/mL DNase I; (3) 1% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hNHDF</td><td>Promote cell proliferation and enhance somatic, multipotent and pluripotent lineage-specific mRNA expression in vitro</td><td>-</td><td>Parmaksiz et al<sup>15</sup></td></tr><tr><td>29</td><td>Human foetal MSCs, human adult MSCs, and human neonatal dermal fibroblast</td><td>10% αMEM for 14 days, add 50 µM ascorbic acid on day 7 or 8</td><td>0.5% Triton X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL-1 DNase I</td><td>hMSCs</td><td>Promote cell proliferation, osteogenic differentiation, chondrogenic differentiation, and adipogenic differentiation ex vivo</td><td>-</td><td>Ng et al<sup>45</sup></td></tr></tbody></table>

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<table><caption>**Table 3. (Continued)**</caption><thead><tr><th>No.</th><th>ECM derived from MSCs</th><th>ECM production medium</th><th>Decellularisation methods</th><th>Cell reseeded</th><th>Effects</th><th>Signalling pathway related</th><th>Study</th></tr></thead><tbody><tr><td>30</td><td>Human Wharton's jelly MSCs</td><td>10% DMEM for 14 days, add 50 µg/ mL ascorbic acid at day 6</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS, DNase I</td><td>human cardiac c kit cells (CCs)</td><td>Inhibit oxidative stress, promote cardiogenic differentiation in vitro</td><td>-</td><td>Ng et al<sup>52</sup></td></tr><tr><td>31</td><td>hSDSCs and human Wharton's jelly MSCs</td><td>Pretreat TCP with 0.2% gelatin, culture in 10% MEM:F12 for 7 days, after 90% confluence add 250 uM L-ascorbic acid phosphate</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hSDSCs and human Wharton's jelly MSCs</td><td>Promote cell adhesion, proliferation, and chondrogenic differentiation in vitro</td><td>MAPK</td><td>Wang et al<sup>16</sup></td></tr><tr><td>32</td><td>hSDSCs</td><td>Pretreat TCP with 0.2% gelatin, culture in 10% αMEM supplement with ascorbic acid (250 uM) for 8 days</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hSDSCs</td><td>Promoted cell proliferation, chondrogenic differentiation in vitro</td><td>MAPK</td><td>Zhang et al<sup>51</sup></td></tr><tr><td>33</td><td>hUSCs</td><td>10%DMEM supplement with ascorbic acid (50 uM) for 8 days</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hBMSCs</td><td>Promoted chondrogenic differentiation in vitro</td><td>WNT</td><td>Pei et al<sup>59</sup></td></tr><tr><td>34</td><td>mBMSCs</td><td>Pretreat TCP with 0.2% gelatin crosslink with 1% glutaraldehyde in 1M ethanolamine, culture in 10% MEM for 8 days, after 90% confluence, add 100 uM L-ascorbic acid</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL-1 DNase I</td><td>mBMM</td><td>Promote cell proliferation and inhibit osteoclast differentiation in vitro</td><td>-</td><td>Li et al<sup>46</sup></td></tr><tr><td>35</td><td>mMSCs</td><td>10% DMEM for 3 days</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>Hepatocarcinoma cells</td><td>Promote cell adhesion and detoxification activity in vitro</td><td>-</td><td>Park et al<sup>87</sup></td></tr><tr><td>36</td><td>Horse adipose MSCs and horse BMSCs</td><td>10%DMEM supplement with 50 uM ascorbic and chondrogenic medium for 15 days</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL-1 DNase I</td><td>Horse adipose MSCs and horse bone marrow MSCs</td><td>Promote cell proliferation, osteogenic differentiation, and chondrogenic differentiation in vitro</td><td>-</td><td>Perez-Castrillo et al<sup>27</sup></td></tr><tr><td>37</td><td>Rabbit synovium MSCs</td><td>Pretreat TCP with 0.2% gelatin, culture in 10%MEM for 8 days after 90% confluence, add 100 uM L-ascorbic acid</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL-1 DNase I</td><td>Rabbit synovium MSCs</td><td>Promote cell proliferation, anti-inflammatory properties of chondrocyte in vitro</td><td>SIRT1</td><td>Yan et al<sup>28</sup></td></tr></tbody></table>

hADSCs, human adipose stem cells; hBMSCs, human bone marrow stem cells; hMSCs, human mesenchymal stem cells; hNHA, human articular chondrocytes; hNHDF, human dermal fibroblasts; hSDSCs, human synovium derived stem cells; hUC-MSCs, human umbilical cord derived mesenchymal stem cells; hUSCs, human urine derived stem cells; HUVECs, human umbilical vein endothelial cells; mBMM, mouse bone marrow monocyte; UCB-MSCs, human umbilical cord blood mesenchymal stem cell.
<table><caption>**Table 3. (Continued)**</caption><thead><tr><th>No.</th><th>ECM derived from MSCs</th><th>ECM production medium</th><th>Decellularisation methods</th><th>Cell reseeded</th><th>Effects</th><th>Signalling pathway related</th><th>Study</th></tr></thead><tbody><tr><td>30</td><td>Human Wharton's jelly MSCs</td><td>10% DMEM for 14 days, add 50 µg/ mL ascorbic acid at day 6</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS, DNase I</td><td>human cardiac c kit cells (CCs)</td><td>Inhibit oxidative stress, promote cardiogenic differentiation in vitro</td><td>-</td><td>Ng et al<sup>52</sup></td></tr><tr><td>31</td><td>hSDSCs and human Wharton's jelly MSCs</td><td>Pretreat TCP with 0.2% gelatin, culture in 10% MEM:F12 for 7 days, after 90% confluence add 250 uM L-ascorbic acid phosphate</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hSDSCs and human Wharton's jelly MSCs</td><td>Promote cell adhesion, proliferation, and chondrogenic differentiation in vitro</td><td>MAPK</td><td>Wang et al<sup>16</sup></td></tr><tr><td>32</td><td>hSDSCs</td><td>Pretreat TCP with 0.2% gelatin, culture in 10% αMEM supplement with ascorbic acid (250 uM) for 8 days</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hSDSCs</td><td>Promoted cell proliferation, chondrogenic differentiation in vitro</td><td>MAPK</td><td>Zhang et al<sup>51</sup></td></tr><tr><td>33</td><td>hUSCs</td><td>10%DMEM supplement with ascorbic acid (50 uM) for 8 days</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>hBMSCs</td><td>Promoted chondrogenic differentiation in vitro</td><td>WNT</td><td>Pei et al<sup>59</sup></td></tr><tr><td>34</td><td>mBMSCs</td><td>Pretreat TCP with 0.2% gelatin crosslink with 1% glutaraldehyde in 1M ethanolamine, culture in 10% MEM for 8 days, after 90% confluence, add 100 uM L-ascorbic acid</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL-1 DNase I</td><td>mBMM</td><td>Promote cell proliferation and inhibit osteoclast differentiation in vitro</td><td>-</td><td>Li et al<sup>46</sup></td></tr><tr><td>35</td><td>mMSCs</td><td>10% DMEM for 3 days</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS</td><td>Hepatocarcinoma cells</td><td>Promote cell adhesion and detoxification activity in vitro</td><td>-</td><td>Park et al<sup>87</sup></td></tr><tr><td>36</td><td>Horse adipose MSCs and horse BMSCs</td><td>10%DMEM supplement with 50 uM ascorbic and chondrogenic medium for 15 days</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL-1 DNase I</td><td>Horse adipose MSCs and horse bone marrow MSCs</td><td>Promote cell proliferation, osteogenic differentiation, and chondrogenic differentiation in vitro</td><td>-</td><td>Perez-Castrillo et al<sup>27</sup></td></tr><tr><td>37</td><td>Rabbit synovium MSCs</td><td>Pretreat TCP with 0.2% gelatin, culture in 10%MEM for 8 days after 90% confluence, add 100 uM L-ascorbic acid</td><td>0.5% Trion X-100 containing 20 mM NH<sub>4</sub>OH in PBS, 100 U/mL-1 DNase I</td><td>Rabbit synovium MSCs</td><td>Promote cell proliferation, anti-inflammatory properties of chondrocyte in vitro</td><td>SIRT1</td><td>Yan et al<sup>28</sup></td></tr></tbody></table>

hADSCs, human adipose stem cells; hBMSCs, human bone marrow stem cells; hMSCs, human mesenchymal stem cells; hNHA, human articular chondrocytes; hNHDF, human dermal fibroblasts; hSDSCs, human synovium derived stem cells; hUC-MSCs, human umbilical cord derived mesenchymal stem cells; hUSCs, human urine derived stem cells; HUVECs, human umbilical vein endothelial cells; mBMM, mouse bone marrow monocyte; UCB-MSCs, human umbilical cord blood mesenchymal stem cell.
## Conclusions

In summary, the utilisation of MSCs-derived decellularised matrices in regenerative dentistry offers a promising avenue for promoting tissue repair and regeneration in various oral and dental applications. On-going research and advancements in this field may lead to innovative therapies that enhance oral health and improve patient outcomes.

## Conflict of interest

None disclosed.

## Author contributions

SP, NN, BPF, and TO created a concept. SP and SS wrote the original draft of the manuscript. JI, NN, and TO edited the manuscript. BPF, JI, SCN, NN, and TO participated in the discussions. NN and TO revised the manuscript. NN and TO contributed equally to this work.

## Funding

This study was funded by the Thailand Science Research and Innovation Fund Chulalongkorn University (HEA663200065) to T.O.

## REFERENCES

1. Bonnans C, Chou J, Werb Z. Remodelling the extracellular matrix in development and disease. Nat Rev Mol Cell Biol 2014;15(12):786-801.

2. Zhong Y, Jiang A, Sun F, et al. A comparative study of the effects of different decellularization methods and genipin-cross-linking on the properties of tracheal matrices. Tissue Eng Regen Med 2019;16(1):39-50.

3. Faulk DM, Wildemann JD, Badylak SF. Decellularization and cell seeding of whole liver biologic scaffolds composed of extracellular matrix. J Clin Exp Hepatol 2015;5(1):69-80.

4. Gilpin A, Yang Y. Decellularization strategies for regenerative medicine: from processing techniques to applications. Biomed Res Int 2017;2017:9831534.

5. Byron A, Humphries JD, Humphries MJ. Defining the extracellular matrix using proteomics. Int J Exp Pathol 2013;94(2):75-92.

6. Naba A, Clauser KR, Hynes RO. Enrichment of extracellular matrix proteins from tissues and digestion into peptides for mass spectrometry analysis. J Vis Exp 2015:e53057.

7. Naba A, Clauser KR, Ding H, Whittaker CA, Carr SA, Hynes RO. The extracellular matrix: tools and insights for the "omics" era. Matrix Biol 2016;49:10-24.

8. Shao X, Gomez CD, Kapoor N, et al. MatrisomeDB 2.0: 2023 updates to the ECM-protein knowledge database. Nucleic Acids Res 2023;51(D1):1519-30.

9. Hoshiba T. Cultured cell-derived decellularized extracellular matrix (cultured cell-derived dECM): future applications and problems—a mini review. Curr Opin Biomed Eng 2021; 17:100256.

10. Yang MC, O'Connor AJ, Kalionis B, Heath DE. Improvement of mesenchymal stromal cell proliferation and differentiation via decellularized extracellular matrix on substrates with a range of surface chemistries. Front Med Technol 2022; 4:834123.

11. Li M, Zhang T, Jiang J, Mao Y, Zhang A, Zhao J. ECM coating modification generated by optimized decellularization process improves functional behavior of BMSCs. Mater Sci Eng C Mater Biol Appl 2019;105:110039.

12. Motoike S, Kajiya M, Komatsu N, et al. Clumps of mesenchymal stem cell/extracellular matrix complexes generated with xeno-free conditions facilitate bone regeneration via direct and indirect osteogenesis. Int J Mol Sci 2019;20(16):3970.

13. Phothichailert S, Nowwarote N, Fournier BPJ, et al. Effects of decellularized extracellular matrix derived from Jagged1-treated human dental pulp stem cells on biological responses of stem cells isolated from apical papilla. Front Cell Dev Biol 2022;10:948812.

14. Jeon J, Lee MS, Yang HS. Differentiated osteoblasts derived decellularized extracellular matrix to promote osteogenic differentiation. Biomater Res 2018;22:4.

15. Parmaksiz M, Elcin AE, Elcin YM. Decellularized cell culture ECMs act as cell differentiation inducers. Stem Cell Rev Rep 2020;16(3):569-84.

16. Wang Y, Jiang C, Cong S, Guo C, Yan Z. Extracellular matrix deposited by Wharton's jelly mesenchymal stem cells enhances cell expansion and tissue specific lineage potential. Am J Transl Res 2018;10(11):3465-80.

17. Riis S, Hansen AC, Johansen L, et al. Fabrication and characterization of extracellular matrix scaffolds obtained from adipose-derived stem cells. Methods 2020;171:68-76.

18. Sears V, Ghosh G. Harnessing mesenchymal stem cell秘toe: effect of extracellular matrices on proangiogenic signaling. Biotechnol Bioeng 2020;117(4):1159-71.

19. Laudani S, La Cognata V, Iemmolo R, et al. Effect of a bone marrow-derived extracellular matrix on cell adhesion and neural induction of dental pulp stem cells. Front Cell Dev Biol 2020;8:100.

20. Chiquet M. Regulation of extracellular matrix gene expression by mechanical stress. Matrix Biol 1999;18(5):417-26.

21. Schultz GS, Wysocki A. Interactions between extracellular matrix and growth factors in wound healing. Wound Repair Regen 2009;17(2):153-62.

22. Kim B-S, Nikolovski J, Bonadio J, Mooney DJ. Cyclic mechanical strain regulates the development of engineered smooth muscle tissue. Nat Biotechnol 1999;17(10):979-83.

23. Zhang P, Liu X, Guo P, et al. Effect of cyclic mechanical loading on immunoinflammatory microenvironment in biofabricating hydroxyapatite scaffold for bone regeneration. Bioact Mater 2021;6(10):3097-108.

24. Roberts AB, McCune BK, Sporn MB. TGF-β: regulation of extracellular matrix. Kidney Int 1992;41(3):557-9.

25. Krase A, Abedian R, Steck E, Hurschler C, Richter W. BMP activation and Wnt-signalling affect biochemistry and functional biomechanical properties of cartilage tissue engineering constructs. Osteoarthritis Cartilage 2014;22(2):284-92.

26. Yang S, Li L, Zhu L, et al. Aucubin inhibits IL-1beta- or TNF-alpha-induced extracellular matrix degradation in nucleus pulposus cell through blocking the miR-140-5p/CREB1 axis. J Cell Physiol 2019;234(8):13639-48.

27. Perez-Castrillo S, Gonzalez-Fernandez ML, Lopez-Gonzalez ME, Villar-Suarez V. Effect of ascorbic and chondrogenic derived decellularized extracellular matrix from mesenchymal stem cells on their proliferation, viability and differentiation. Ann Anat 2018;220:60-9.

28. Yan J, Chen X, Pu C, et al. Synovium stem cell-derived matrix enhances anti-inflammatory properties of rabbit articular chondrocytes via the SIRT1 pathway. Mater Sci Eng C Mater Biol Appl 2020;106:110286.

29. Nowwarote N, Petit S, Ferre FC, et al. Extracellular matrix derived from dental pulp stem cells promotes mineralization. Front Bioeng Biotechnol 2021;9:740712.
## Conclusions

In summary, the utilisation of MSCs-derived decellularised matrices in regenerative dentistry offers a promising avenue for promoting tissue repair and regeneration in various oral and dental applications. On-going research and advances in this field may lead to innovative therapies that enhance oral health and improve patient outcomes.

## Conflict of interest

None disclosed.

## Author contributions

SP, NN, BPF, and TO created a concept. SP and SS wrote the original draft of the manuscript. JI, NN, and TO edited the manuscript. BPF, JI, SCN, NN, and TO participated in the discussions. NN and TO revised the manuscript. NN and TO contributed equally to this work.

## Funding

This study was funded by the Thailand Science Research and Innovation Fund Chulalongkorn University (HEA663200065) to T.O.

## REFERENCES

1. Bonnans C, Chou J, Werb Z. Remodelling the extracellular matrix in development and disease. Nat Rev Mol Cell Biol 2014;15(12):786-801.

2. Zhong Y, Jiang A, Sun F, et al. A comparative study of the effects of different decellularization methods and genipin-cross-linking on the properties of tracheal matrices. Tissue Eng Regen Med 2019;16(1):39-50.

3. Faulk DM, Wildemann JD, Badylak SF. Decellularization and cell seeding of whole liver biologic scaffolds composed of extracellular matrix. J Clin Exp Hepatol 2015;5(1):69-80.

4. Gilpin A, Yang Y. Decellularization strategies for regenerative medicine: from processing techniques to applications. Biomed Res Int 2017;2017:9831534.

5. Byron A, Humphries JD, Humphries MJ. Defining the extracellular matrix using proteomics. Int J Exp Pathol 2013;94(2):75-92.

6. Naba A, Clauser KR, Hynes RO. Enrichment of extracellular matrix proteins from tissues and digestion into peptides for mass spectrometry analysis. J Vis Exp 2015:e53057.

7. Naba A, Clauser KR, Ding H, Whittaker CA, Carr SA, Hynes RO. The extracellular matrix: tools and insights for the "omics" era. Matrix Biol 2016;49:10-24.

8. Shao X, Gomez CD, Kapoor N, et al. MatrisomeDB 2.0: 2023 updates to the ECM-protein knowledge database. Nucleic Acids Res 2023;51(D1):1519-30.

9. Hoshiba T. Cultured cell-derived decellularized extracellular matrix (cultured cell-derived dECM): future applications and problems—a mini review. Curr Opin Biomed Eng 2021; 17:100256.

10. Yang MC, O'Connor AJ, Kalionis B, Heath DE. Improvement of mesenchymal stromal cell proliferation and differentiation via decellularized extracellular matrix on substrates with a range of surface chemistries. Front Med Technol 2022; 4:834123.

11. Li M, Zhang T, Jiang J, Mao Y, Zhang A, Zhao J. ECM coating modification generated by optimized decellularization process improves functional behavior of BMSCs. Mater Sci Eng C Mater Biol Appl 2019;105:110039.

12. Motoike S, Kajiya M, Komatsu N, et al. Clumps of mesenchymal stem cell/extracellular matrix complexes generated with xeno-free conditions facilitate bone regeneration via direct and indirect osteogenesis. Int J Mol Sci 2019;20(16):3970.

13. Phothichailert S, Nowwarote N, Fournier BPJ, et al. Effects of decellularized extracellular matrix derived from Jagged1-treated human dental pulp stem cells on biological responses of stem cells isolated from apical papilla. Front Cell Dev Biol 2022;10:948812.

14. Jeon J, Lee MS, Yang HS. Differentiated osteoblasts derived decellularized extracellular matrix to promote osteogenic differentiation. Biomater Res 2018;22:4.

15. Parmaksiz M, Elcin AE, Elcin YM. Decellularized cell culture ECMs act as cell differentiation inducers. Stem Cell Rev Rep 2020;16(3):569-84.

16. Wang Y, Jiang C, Cong S, Guo C, Yan Z. Extracellular matrix deposited by Wharton's jelly mesenchymal stem cells enhances cell expansion and tissue specific lineage potential. Am J Transl Res 2018;10(11):3465-80.

17. Riis S, Hansen AC, Johansen L, et al. Fabrication and characterization of extracellular matrix scaffolds obtained from adipose-derived stem cells. Methods 2020;171:68-76.

18. Sears V, Ghosh G. Harnessing mesenchymal stem cell秘toe: effect of extracellular matrices on proangiogenic signaling. Biotechnol Bioeng 2020;117(4):1159-71.

19. Laudani S, La Cognata V, Iemmolo R, et al. Effect of a bone marrow-derived extracellular matrix on cell adhesion and neural induction of dental pulp stem cells. Front Cell Dev Biol 2020;8:100.

20. Chiquet M. Regulation of extracellular matrix gene expression by mechanical stress. Matrix Biol 1999;18(5):417-26.

21. Schultz GS, Wysocki A. Interactions between extracellular matrix and growth factors in wound healing. Wound Repair Regen 2009;17(2):153-62.

22. Kim B-S, Nikolovski J, Bonadio J, Mooney DJ. Cyclic mechanical strain regulates the development of engineered smooth muscle tissue. Nat Biotechnol 1999;17(10):979-83.

23. Zhang P, Liu X, Guo P, et al. Effect of cyclic mechanical loading on immunoinflammatory microenvironment in biofabricating hydroxyapatite scaffold for bone regeneration. Bioact Mater 2021;6(10):3097-108.

24. Roberts AB, McCune BK, Sporn MB. TGF-β: regulation of extracellular matrix. Kidney Int 1992;41(3):557-9.

25. Krase A, Abedian R, Steck E, Hurschler C, Richter W. BMP activation and Wnt-signalling affect biochemistry and functional biomechanical properties of cartilage tissue engineering constructs. Osteoarthritis Cartilage 2014;22(2):284-92.

26. Yang S, Li L, Zhu L, et al. Aucubin inhibits IL-1beta- or TNF-alpha-induced extracellular matrix degradation in nucleus pulposus cell through blocking the miR-140-5p/CREB1 axis. J Cell Physiol 2019;234(8):13639-48.

27. Perez-Castrillo S, Gonzalez-Fernandez ML, Lopez-Gonzalez ME, Villar-Suarez V. Effect of ascorbic and chondrogenic derived decellularized extracellular matrix from mesenchymal stem cells on their proliferation, viability and differentiation. Ann Anat 2018;220:60-9.

28. Yan J, Chen X, Pu C, et al. Synovium stem cell-derived matrix enhances anti-inflammatory properties of rabbit articular chondrocytes via the SIRT1 pathway. Mater Sci Eng C Mater Biol Appl 2020;106:110286.

29. Nowwarote N, Petit S, Ferre FC, et al. Extracellular matrix derived from dental pulp stem cells promotes mineralization. Front Bioeng Biotechnol 2021;9:740712.
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42. Krater M, Jacobi A, Otto O, et al. Bone marrow niche-mimetics modulate HSPC function via integrin signaling. Sci Rep 2017;7(1):2549.

43. Kim J, Ma T. Autocrine fibroblast growth factor 2-mediated interactions between human mesenchymal stem cells and the extracellular matrix under varying oxygen tension. J Cell Biochem 2013;114(3):716-27.

44. Zhang ZY, Teoh SH, Chong MS, et al. Superior osteogenic capacity for bone tissue engineering of fetal compared with perinatal and adult mesenchymal stem cells. Stem Cells 2009;27(1):126-37.

45. Ng CP, Sharif AR, Heath DE, et al. Enhanced ex vivo expansion of adult mesenchymal stem cells by fetal mesenchymal stem cell ECM. Biomaterials 2014;35(13):4046-57.

46. Li M, Chen X, Yan J, et al. Inhibition of osteoclastogenesis by stem cell-derived extracellular matrix through modulation of intracellular reactive oxygen species. Acta Biomater 2018;71:118-31.

47. Yang Y, Lin H, Shen H, Wang B, Lei G, Tuan RS. Mesenchymal stem cell-derived extracellular matrix enhances chondrogenic phenotype of and cartilage formation by encapsulated chondrocytes in vitro and in vivo. Acta Biomater 2018;69:71-82.

48. Xu Y, Yan M, Gong Y, Chen L, Zhao F, Zhang Z. Response of endothelial cells to decellularized extracellular matrix

deposited by bone marrow mesenchymal stem cells. Int J Clin Exp Med 2014;7(12):4997-5003.

49. Decaris ML, Mojadedi A, Bhat A, Leach JK. Transferable cell-secreted extracellular matrices enhance osteogenic differentiation. Acta Biomater 2012;8(2):744-52.

50. Carvalho MS, Silva JC, Cabral JMS, da Silva CL, Vashishth D. Cultured cell-derived extracellular matrices to enhance the osteogenic differentiation and angiogenic properties of human mesenchymal stem/stromal cells. J Tissue Eng Regen Med 2019;13(9):1544-58.

51. Zhang Y, Pizzute T, Li J, He F, Pei M. sb203580 preconditioning recharges matrix-expanded human adult stem cells for chondrogenesis in an inflammatory environment—a feasible approach for autologous stem cell based osteoarthritic cartilage repair. Biomaterials 2015;64:88-97.

52. Ng WH, Ramasamy R, Yong YK, et al. Extracellular matrix from decellularized mesenchymal stem cells improves cardiac gene expressions and oxidative resistance in cardiac C-kit cells. Regen Ther 2019;11:8-16.

53. Liu X, Zhou L, Chen X, et al. Culturing on decellularized extracellular matrix enhances antioxidant properties of human umbilical cord-derived mesenchymal stem cells. Mater Sci Eng C Mater Biol Appl 2016;61:437-48.

54. Lin H, Yang G, Tan J, Tuan RS. Influence of decellularized matrix derived from human mesenchymal stem cells on their proliferation, migration and multi-lineage differentiation potential. Biomaterials 2012;33(18):4480-9.

55. Torres CA, Perez VI. Proteasome modulates mitochondrial function during cellular senescence. Free Radic Biol Med 2008;44(3):403-14.

56. Kusuma GD, Brennecke SP, O'Connor AJ, Kalonis B, Heath DE. Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion. PLoS One 2017;12(2):e0171488.

57. Huang YZ, He T, Cui J, et al. Urine-derived stem cells for regenerative medicine: basic biology, applications, and challenges. Tissue Eng Part B Rev 2022;28(5):978-94.

58. Xiong X, Yang X, Dai H, et al. Extracellular matrix derived from human urine-derived stem cells enhances the expansion, adhesion, spreading, and differentiation of human periodontal ligament stem cells. Stem Cell Res Ther 2019;10(1):396.

59. Pei M, Li J, Zhang Y, Liu G, Wei L, Zhang Y. Expansion on a matrix deposited by nonchondrogenic urine stem cells strengthens the chondrogenic capacity of repeated-passage bone marrow stromal cells. Cell Tissue Res 2014;356(2):391-403.

60. Egusa H, Sonoyama W, Nishimura M, Atsuta I, Akiyama K. Stem cells in dentistry—part II: clinical applications. J Prosthodont Res 2012;56(4):229-48.

61. Stanko P, Kaiserova K, Altanerova V, Altaner C. Comparison of human mesenchymal stem cells derived from dental pulp, bone marrow, adipose tissue, and umbilical cord tissue by gene expression. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2014;158(3):373-7.

62. Davies OG, Cooper PR, Shelton RM, Smith AJ, Scheven BA. A comparison of the in vitro mineralisation and dentinogenic potential of mesenchymal stem cells derived from adipose tissue, bone marrow and dental pulp. J Bone Miner Metab 2015;33(4):371-82.

63. Osathanon T, Nowwarote N, Pavasant P. Basic fibroblast growth factor inhibits mineralization but induces neuronal differentiation by human dental pulp stem cells through a FGFR and PLCgamma signaling pathway. J Cell Biochem 2011;112(7):1807-16.

64. Nowwarote N, Pavasant P, Osathanon T. Role of endogenous basic fibroblast growth factor in stem cells isolated from
30. Zhang W, Yang J, Zhu Y, et al. Extracellular matrix derived by human umbilical cord-deposited mesenchymal stem cells accelerates chondrocyte proliferation and differentiation potential in vitro. Cell Tissue Bank 2019;20(3):351-65.

31. Aubin H, Kranz A, Hulsmann J, Lichtenberg A, Akhyari P. Decellularized whole heart for bioartificial heart. Methods Mol Biol 2013;1036:163-78.

32. Rakian R, Block TJ, Johnson SM, et al. Native extracellular matrix preserves mesenchymal stem cell "stemness" and differentiation potential under serum-free culture conditions. Stem Cell Res Ther 2015;6:235.

33. Bhumiratana S, Bernhard JC, Alfi DM, et al. Tissue-engineered autologous grafts for facial bone reconstruction. Sci Transl Med 2016;8(343):343-83.

34. Guneta V, Zhou Z, Tan NS, Sugii S, Wong MTC, Choong C. Recellularization of decellularized adipose tissue-derived stem cells: role of the cell-secreted extracellular matrix in cellular differentiation. Biomater Sci 2017;6(1):168-78.

35. Ji J, Zhang D, Wei W, et al. Decellularized matrix of adipose-derived mesenchymal stromal cells enhanced retinal progenitor cell proliferation via the Akt/Erk pathway and neuronal differentiation. Cytotherapy 2018;20(1):74-86.

36. Rubi-Sans G, Nyga A, Rebollo E, et al. Development of cell-derived matrices for three-dimensional in vitro cancer cell models. ACS Appl Mater Interfaces 2021;13(37):44108-23.

37. Yang L, Chang N, Liu X, et al. Bone marrow-derived mesenchymal stem cells differentiate to hepatic myofibroblasts by transforming growth factor-beta1 via sphingosine kinase/ sphingosine 1-phosphate (S1P)/S1P receptor axis. Am J Pathol 2012;181(1):85-97.

38. Pei M, He F, Kish VL. Expansion on extracellular matrix deposited by human bone marrow stromal cells facilitates stem cell proliferation and tissue-specific lineage potential. Tissue Eng Part A 2011;17(23-24):3067-76.

39. Burns JS, Kristiansen M, Kristensen LP, et al. Decellularized matrix from tumorigenic human mesenchymal stem cells promotes neovascularization with galectin-1 dependent endothelial interaction. PLoS One 2011;6(7):e21888.

40. He F, Liu X, Xiong K, et al. Extracellular matrix modulates the biological effects of melatonin in mesenchymal stem cells. J Endocrinol 2014;223(2):167-80.

41. Prewitz M, Stiel A, Friedrichs J, et al. Extracellular matrix deposition of bone marrow stroma enhanced by macromolecular crowding. Biomaterials 2015;73:60-9.

42. Krater M, Jacobi A, Otto O, et al. Bone marrow niche-mimetics modulate HSPC function via integrin signaling. Sci Rep 2017;7(1):2549.

43. Kim J, Ma T. Autocrine fibroblast growth factor 2-mediated interactions between human mesenchymal stem cells and the extracellular matrix under varying oxygen tension. J Cell Biochem 2013;114(3):716-27.

44. Zhang ZY, Teoh SH, Chong MS, et al. Superior osteogenic capacity for bone tissue engineering of fetal compared with perinatal and adult mesenchymal stem cells. Stem Cells 2009;27(1):126-37.

45. Ng CP, Sharif AR, Heath DE, et al. Enhanced ex vivo expansion of adult mesenchymal stem cells by fetal mesenchymal stem cell ECM. Biomaterials 2014;35(13):4046-57.

46. Li M, Chen X, Yan J, et al. Inhibition of osteoclastogenesis by stem cell-derived extracellular matrix through modulation of intracellular reactive oxygen species. Acta Biomater 2018;71:118-31.

47. Yang Y, Lin H, Shen H, Wang B, Lei G, Tuan RS. Mesenchymal stem cell-derived extracellular matrix enhances chondrogenic phenotype of and cartilage formation by encapsulated chondrocytes in vitro and in vivo. Acta Biomater 2018;69:71-82.

48. Xu Y, Yan M, Gong Y, Chen L, Zhao F, Zhang Z. Response of endothelial cells to decellularized extracellular matrix

deposited by bone marrow mesenchymal stem cells. Int J Clin Exp Med 2014;7(12):4997-5003.

49. Decaris ML, Mojadedi A, Bhat A, Leach JK. Transferable cell-secreted extracellular matrices enhance osteogenic differentiation. Acta Biomater 2012;8(2):744-52.

50. Carvalho MS, Silva JC, Cabral JMS, da Silva CL, Vashishth D. Cultured cell-derived extracellular matrices to enhance the osteogenic differentiation and angiogenic properties of human mesenchymal stem/stromal cells. J Tissue Eng Regen Med 2019;13(9):1544-58.

51. Zhang Y, Pizzute T, Li J, He F, Pei M. sb203580 preconditioning recharges matrix-expanded human adult stem cells for chondrogenesis in an inflammatory environment—a feasible approach for autologous stem cell based osteoarthritic cartilage repair. Biomaterials 2015;64:88-97.

52. Ng WH, Ramasamy R, Yong YK, et al. Extracellular matrix from decellularized mesenchymal stem cells improves cardiac gene expressions and oxidative resistance in cardiac C-kit cells. Regen Ther 2019;11:8-16.

53. Liu X, Zhou L, Chen X, et al. Culturing on decellularized extracellular matrix enhances antioxidant properties of human umbilical cord-derived mesenchymal stem cells. Mater Sci Eng C Mater Biol Appl 2016;61:437-48.

54. Lin H, Yang G, Tan J, Tuan RS. Influence of decellularized matrix derived from human mesenchymal stem cells on their proliferation, migration and multi-lineage differentiation potential. Biomaterials 2012;33(18):4480-9.

55. Torres CA, Perez VI. Proteasome modulates mitochondrial function during cellular senescence. Free Radic Biol Med 2008;44(3):403-14.

56. Kusuma GD, Brennecke SP, O'Connor AJ, Kalonis B, Heath DE. Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion. PLoS One 2017;12(2):e0171488.

57. Huang YZ, He T, Cui J, et al. Urine-derived stem cells for regenerative medicine: basic biology, applications, and challenges. Tissue Eng Part B Rev 2022;28(5):978-94.

58. Xiong X, Yang X, Dai H, et al. Extracellular matrix derived from human urine-derived stem cells enhances the expansion, adhesion, spreading, and differentiation of human periodontal ligament stem cells. Stem Cell Res Ther 2019;10(1):396.

59. Pei M, Li J, Zhang Y, Liu G, Wei L, Zhang Y. Expansion on a matrix deposited by nonchondrogenic urine stem cells strengthens the chondrogenic capacity of repeated-passage bone marrow stromal cells. Cell Tissue Res 2014;356(2):391-403.

60. Egusa H, Sonoyama W, Nishimura M, Atsuta I, Akiyama K. Stem cells in dentistry—part II: clinical applications. J Prosthodont Res 2012;56(4):229-48.

61. Stanko P, Kaiserova K, Altanerova V, Altaner C. Comparison of human mesenchymal stem cells derived from dental pulp, bone marrow, adipose tissue, and umbilical cord tissue by gene expression. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2014;158(3):373-7.

62. Davies OG, Cooper PR, Shelton RM, Smith AJ, Scheven BA. A comparison of the in vitro mineralisation and dentinogenic potential of mesenchymal stem cells derived from adipose tissue, bone marrow and dental pulp. J Bone Miner Metab 2015;33(4):371-82.

63. Osathanon T, Nowwarote N, Pavasant P. Basic fibroblast growth factor inhibits mineralization but induces neuronal differentiation by human dental pulp stem cells through a FGFR and PLCgamma signaling pathway. J Cell Biochem 2011;112(7):1807-16.

64. Nowwarote N, Pavasant P, Osathanon T. Role of endogenous basic fibroblast growth factor in stem cells isolated from
human exfoliated deciduous teeth. Arch Oral Biol 2015;60
(3):408-15.

65. Osathanon T, Ritprajak P, Nowwarote N, Manokawinchoke J, Giachelli C, Pavasant P. Surface-bound orientated Jagged-1 enhances osteogenic differentiation of human periodontal ligament-derived mesenchymal stem cells. J Biomed Mater Res A 2013;101(2):358-67.

66. Fournier BP, Ferre FC, Couty L, et al. Multipotent progenitor cells in gingival connective tissue. Tissue Eng Part A 2010;16 (9):2891-9.

67. Heng BC, Zhu S, Xu J, Yuan C, Gong T, Zhang C. Effects of decellularized matrices derived from periodontal ligament stem cells and SHED on the adhesion, proliferation and osteogenic differentiation of human dental pulp stem cells in vitro. Tissue Cell 2016;48(2):133-43.

68. Huang JP, Wu YM, Liu JM, et al. Decellularized matrix could affect the proliferation and differentiation of periodontal ligament stem cells in vitro. J Periodontal Res 2021;56(5):929-39.

69. Ravindran S, Zhang Y, Huang CC, George A. Odontogenic induction of dental stem cells by extracellular matrix-inspired three-dimensional scaffold. Tissue Eng Part A 2014;20(1-2):92-102.

70. Alksne M, Kalvaityte M, Simoliunas E, et al. Dental pulp stem cell-derived extracellular matrix: autologous tool boosting bone regeneration. Cytotherapy 2022;24(6):597-607.

71. Narazaki G, Uosaki H, Teranishi M, et al. Directed and systematic differentiation of cardiovascular cells from mouse induced pluripotent stem cells. Circulation 2008;118(5):498-506.

72. Alsanea R, Ravindran S, Fayad MI, et al. Biomimetic approach to perforation repair using dental pulp stem cells and dentin matrix protein 1. J Endod 2011;37(8):1092-7.

73. Manokawinchoke J, Nattasit P, Thongngam T, et al. Indirect immobilized Jagged1 suppresses cell cycle progression and induces odonto/osteogenic differentiation in human dental pulp cells. Sci Rep 2017;7(1):10124.

74. Chansaenroj A, Kornsuthisopon C, Roytrakul S, et al. Indirect immobilised Jagged-1 enhances matrisome proteins associated with osteogenic differentiation of human dental pulp stem cells: a proteomic study. Int J Mol Sci 2022;23(22):13897.

75. Alqahtani Q, Zaky SH, Patil A, Beniash E, Ray H, Sfeir C. Decellularized swine dental pulp tissue for regenerative root canal therapy. J Dent Res 2018;97(13):1460-7.

76. Prescott RS, Alsanea R, Fayad MI, et al. In vivo generation of dental pulp-like tissue by using dental pulp stem cells, a collagen scaffold, and dentin matrix protein 1 after subcutaneous transplantation in mice. J Endod 2008;34(4):421-6.

77. Nie Z, Wang X, Ren L, Kang Y. Development of a decellularized porcine bone matrix for potential applications in bone tissue regeneration. Regen Med 2020;15(4):1519-34.

78. Son H, Jeon M, Choi HJ, et al. Decellularized human periodontal ligament for periodontium regeneration. PLoS One 2019;14 (8):e0221236.

79. Harumi Miyagi SP, Kerkis I, da Costa Maranduba CM, Gomes CM, Martins MD, Marques MM. Expression of extracellular matrix proteins in human dental pulp stem cells depends on the donor tooth conditions. J Endod 2010;36(5):826-31.

80. Tomson PL, Lumley PJ, Smith AJ, Cooper PR. Growth factor release from dentine matrix by pulp-capping agents promotes pulp tissue repair-associated events. Int Endod J 2017;50 (3):281-92.

81. Fishman JM, Lowdell MW, Urbani L, et al. Immunomodulatory effect of a decellularized skeletal muscle scaffold in a discordant xenotransplantation model. Proc Natl Acad Sci U S A 2013;110(35):14360-5.

82. Gao L, Feng A, Li C, Schmull S, Sun H. Decellularized aortic scaffold alleviates H(2)O(2)-induced inflammation and apoptosis in CD34+ progenitor cells while driving neovasculogenesis. Biomed Res Int 2020;2020:6782072.

83. Yang X, Ma Y, Wang X, et al. A 3D-bioprinted functional module based on decellularized extracellular matrix bioink for periodontal regeneration. Adv Sci (Weinh) 2023;10(5): e2205041.

84. Hoshiba T, Lu H, Yamada T, Kawazoe N, Tateishi T, Chen G. Effects of extracellular matrices derived from different cell sources on chondrocyte functions. Biotechnol Prog 2011;27 (3):788-95.

85. Xiong Y, He J, Zhang W, Zhou G, Cao Y, Liu W. Retention of the stemness of mouse adipose-derived stem cells by their expansion on human bone marrow stromal cell-derived extracellular matrix. Tissue Eng Part A 2015;21(11-12): 1886-94.

86. Marinkovic M, Block T, Rakian R, et al. One size does not fit all: developing a cell-specific niche for in vitro study of cell behavior. Matrix Biol 2016;52:426-41.

87. Park J, Kim J, Sullivan KM, et al. Decellularized matrix produced by mesenchymal stem cells modulates growth and metabolic activity of hepatic cell cluster. ACS Biomater Sci Eng 2018;4(2):456-62.

88. Zhou L, Chen X, Liu T, et al. SIRT1-dependent anti-senescence effects of cell-deposited matrix on human umbilical cord mesenchymal stem cells. J Tissue Eng Regen Med 2018;12 (2):1008-21.
human exfoliated deciduous teeth. Arch Oral Biol 2015;60
(3):408-15.

65. Osathanon T, Ritprajak P, Nowwarote N, Manokawinchoke J, Giachelli C, Pavasant P. Surface-bound orientated Jagged-1 enhances osteogenic differentiation of human periodontal ligament-derived mesenchymal stem cells. J Biomed Mater Res A 2013;101(2):358-67.

66. Fournier BP, Ferre FC, Couty L, et al. Multipotent progenitor cells in gingival connective tissue. Tissue Eng Part A 2010;16 (9):2891-9.

67. Heng BC, Zhu S, Xu J, Yuan C, Gong T, Zhang C. Effects of decellularized matrices derived from periodontal ligament stem cells and SHED on the adhesion, proliferation and osteogenic differentiation of human dental pulp stem cells in vitro. Tissue Cell 2016;48(2):133-43.

68. Huang JP, Wu YM, Liu JM, et al. Decellularized matrix could affect the proliferation and differentiation of periodontal ligament stem cells in vitro. J Periodontal Res 2021;56(5):929-39.

69. Ravindran S, Zhang Y, Huang CC, George A. Odontogenic induction of dental stem cells by extracellular matrix-inspired three-dimensional scaffold. Tissue Eng Part A 2014;20(1-2):92-102.

70. Alksne M, Kalvaityte M, Simoliunas E, et al. Dental pulp stem cell-derived extracellular matrix: autologous tool boosting bone regeneration. Cytotherapy 2022;24(6):597-607.

71. Narazaki G, Uosaki H, Teranishi M, et al. Directed and systematic differentiation of cardiovascular cells from mouse induced pluripotent stem cells. Circulation 2008;118(5):498-506.

72. Alsanea R, Ravindran S, Fayad MI, et al. Biomimetic approach to perforation repair using dental pulp stem cells and dentin matrix protein 1. J Endod 2011;37(8):1092-7.

73. Manokawinchoke J, Nattasit P, Thongngam T, et al. Indirect immobilized Jagged1 suppresses cell cycle progression and induces odonto/osteogenic differentiation in human dental pulp cells. Sci Rep 2017;7(1):10124.

74. Chansaenroj A, Kornsuthisopon C, Roytrakul S, et al. Indirect immobilised Jagged-1 enhances matrisome proteins associated with osteogenic differentiation of human dental pulp stem cells: a proteomic study. Int J Mol Sci 2022;23(22):13897.

75. Alqahtani Q, Zaky SH, Patil A, Beniash E, Ray H, Sfeir C. Decellularized swine dental pulp tissue for regenerative root canal therapy. J Dent Res 2018;97(13):1460-7.

76. Prescott RS, Alsanea R, Fayad MI, et al. In vivo generation of dental pulp-like tissue by using dental pulp stem cells, a collagen scaffold, and dentin matrix protein 1 after subcutaneous transplantation in mice. J Endod 2008;34(4):421-6.

77. Nie Z, Wang X, Ren L, Kang Y. Development of a decellularized porcine bone matrix for potential applications in bone tissue regeneration. Regen Med 2020;15(4):1519-34.

78. Son H, Jeon M, Choi HJ, et al. Decellularized human periodontal ligament for periodontium regeneration. PLoS One 2019;14 (8):e0221236.

79. Harumi Miyagi SP, Kerkis I, da Costa Maranduba CM, Gomes CM, Martins MD, Marques MM. Expression of extracellular matrix proteins in human dental pulp stem cells depends on the donor tooth conditions. J Endod 2010;36(5):826-31.

80. Tomson PL, Lumley PJ, Smith AJ, Cooper PR. Growth factor release from dentine matrix by pulp-capping agents promotes pulp tissue repair-associated events. Int Endod J 2017;50 (3):281-92.

81. Fishman JM, Lowdell MW, Urbani L, et al. Immunomodulatory effect of a decellularized skeletal muscle scaffold in a discordant xenotransplantation model. Proc Natl Acad Sci U S A 2013;110(35):14360-5.

82. Gao L, Feng A, Li C, Schmull S, Sun H. Decellularized aortic scaffold alleviates H(2)O(2)-induced inflammation and apoptosis in CD34+ progenitor cells while driving neovasculogenesis. Biomed Res Int 2020;2020:6782072.

83. Yang X, Ma Y, Wang X, et al. A 3D-bioprinted functional module based on decellularized extracellular matrix bioink for periodontal regeneration. Adv Sci (Weinh) 2023;10(5): e2205041.

84. Hoshiba T, Lu H, Yamada T, Kawazoe N, Tateishi T, Chen G. Effects of extracellular matrices derived from different cell sources on chondrocyte functions. Biotechnol Prog 2011;27 (3):788-95.

85. Xiong Y, He J, Zhang W, Zhou G, Cao Y, Liu W. Retention of the stemness of mouse adipose-derived stem cells by their expansion on human bone marrow stromal cell-derived extracellular matrix. Tissue Eng Part A 2015;21(11-12): 1886-94.

86. Marinkovic M, Block T, Rakian R, et al. One size does not fit all: developing a cell-specific niche for in vitro study of cell behavior. Matrix Biol 2016;52:426-41.

87. Park J, Kim J, Sullivan KM, et al. Decellularized matrix produced by mesenchymal stem cells modulates growth and metabolic activity of hepatic cell cluster. ACS Biomater Sci Eng 2018;4(2):456-62.

88. Zhou L, Chen X, Liu T, et al. SIRT1-dependent anti-senescence effects of cell-deposited matrix on human umbilical cord mesenchymal stem cells. J Tissue Eng Regen Med 2018;12 (2):1008-21.