MSCs-Derived Decellularised Matrix: Cellular Responses and Regenerative Dentistry

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.


Introduction
The extracellular matrix (ECM) serves as a scaffold, offering support to cells and tissues.It affects cell responses through interactions with receptors and acts as a reservoir for signalling molecules, indirectly governing cell behaviour.Its properties are vital for development, growth, and regeneration. 1The ECM substrate is widely used as a biomaterial 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).][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.

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. 5Matrisome is a proteomic database available for humans, mice, and nematodes (Caenorhabditis elegans) (http://matrisomeproject.mit.edu). 6,7Comprehending composition and function aids targeted interventions, modulating the ECM for tissue repair and regeneration. 8ssentially, 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.2][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. 14The use of 1% Triton X-100 with 20 mM NH 4 OH in PBS preserves the ECM structure. 15A combination of Triton X-100 and freeze-thaw cycles leads to the best-preserved structure and components of the ECM. 11Furthermore, 1% to 5% of sodium dodecyl sulphate (SDS) slightly affects the ECM and increases collagen. 168][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 biomechanical 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. 20,21In regard to physical factors, cyclic loading strain raised elastin and collagen levels in smooth muscle cells, resulting in an improvement in tissue organisation. 22eanwhile, the mechanical loading force enhanced the synthesis of ECM while seeding on scaffold material. 23However, further investigation is needed to understand the impact of  mechanical loading on MSCs' ECM production and its components, 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-b (TGF-b) 24 and bone morphogenetic 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-a (TNF-a) enhance the production of ECM-degrading enzymes, leading to the breakdown of the ECM. 26Culture conditions play a vital role in producing ECM that influences desired cell responses.Under a chondrogenic medium, dECM enhances MSCs viability, spreading, and proliferation, redifferentiation, and anti-inflammatory properties.8][29] Three-dimensional dECM provides a specific environment for specific cell type to retain their differentiation ability but does not affect cell proliferation. 30sing dECM to mimic the in vivo extracellular microenvironment has been shown to be a useful strategy to stimulate cell proliferation and survival. 31The 3-dimensional structures of the ECM support cell growth and serve as a reservoir of growth factors and cytokines that control cell destiny and function. 19,31,32This complexity contributes to the formation of optimal niches for cells to reside in.Furthermore, 3-dimensional culture conditions could lead to an ECM that physiologically resembles native tissue more closely than those derived from the 2-dimensional culture system. 33Together, 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, contributing 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 microenvironments, aiding tissue regeneration.
MSC-derived dECM affects cellular responses diversely, for example, by promoting cell adhesion, migration, and proliferation, 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. 12However, 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 osteogenic differentiation medium. 34Additionally, dECM from ADSCs plays a crucial role in regulating retinal progenitor cell proliferation and differentiation, holding promise for enhancing the effectiveness of retinal progenitor cell treatment in retinal degenerative diseases. 35The microstructure and native constituents preserved in the dECM could provide a useful platform for studying the role of ECM in wound healing. 17Furthermore, it also affects pathologic changes by supporting repopulation in cancer cells during chemotherapy treatment. 36

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. 28urthermore, hSDSCs-dECM promoted self-renewal ability, induced cell proliferation, and enhanced chondrogenic differentiation of SDSCs via the MAPK pathway. 51Therefore, 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. 16Furthermore, dECM-derived from WJ-MSCs rescues cardiac C-kit positive cells from oxidative stress, offering enhanced in vivo transplantation survival and function. 52MSCs 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. 53,54These properties of oxidative stress improvement reduce cellular senescence and promote cell proliferation. 55Placental 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. 57dECM 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. 58Moreover, USCs-dECM reduces proinflammation and boosts antiinflammation in macrophage coculture. 59Therefore, 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 dental treatments (eg, tooth extraction and wisdom tooth removal).Dental MSCs are the promising cell source for regenerative therapy, especially in craniofacial areas. 60Dental tissue-derived MSCs have a faster proliferation rate compared 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. 61,62Sources 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 periodontal 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 (dental follicle stem cells). 60he ability of oral cells to adhere, proliferate, and differentiate into osteogenic differentiation can be significantly influenced 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 appropriate for ex vivo growth of DPSCs. 67Furthermore, dECM derived from hPDLSCs, dental pulp cells, or gingival fibroblasts promotes the proliferation and osteogenic differentiation of hPDLSCs. 68The 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). 29hDPSCs-dECM demonstrated usefulness in modulating dental cell responses in vitro, suggesting 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. 69These dECM improve the formation of dental tissues in vivo without the addition of exogenous factors in subcutaneous implantation. 69dECM derived from hDPSCs on polylactic acid (PLA) scaffolds promotes calvarial bone healing in rats, as determined by increased new bone volume and area compared to PLA alone. 70Interestingly, 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. 71dECM 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. 72otch signalling activation in human dental pulp cells upregulated the ECM organisation pathway, as confirmed by RNA sequencing analysis. 73Proteomic and matrisome analysis of Notch ligands, Jagged-1, stimulated hDPSCs demonstrates the protein component related to osteogenic differentiation. 74Furthermore, 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). 13However, 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 dECMderived 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. 29Cellular 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 differentiation.ECM scaffolds offer the advantage of promoting natural 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. 75Using 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. 77Furthermore, 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. 78However, 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 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.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. 79The 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. 77,78Therefore, 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. 13,29,58,67Additionally, it exhibits immunomodulatory properties that reduce inflammation and enhance tissue repair. 58Despite these positive findings, further research is required to optimise its properties and evaluate its clinical potential.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.

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 colonystimulating factor (M-CSF), granulocyte-macrophage colonystimulating factor, and neurotrophic growth factors.These growth factors induce proliferation and chemotaxis in dental pulp cells. 80In 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-b binding proteins, encompasses growth factor-binding proteins found within glycoproteins. 29ence, 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 antiinflammatory and immunomodulatory properties. 81In 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 colonystimulating factor, MIP-1b, GRO-a, Entoxin, and GRO) and increased anti-inflammatory cytokines (IL-2 and TGF-b). 82In 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. 83Further, 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. 83In addition, dECM derived from DPSC notable release of CXCL12, which could be attributable to the regulation of DPSC homeostasis. 29evertheless, the innovative approach based on cellularderived 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.
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Figure 2 -
Figure2-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.

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Table 1 -
Classification of extracellular matrix proteins by protein structural and proteomic matrisome database.

Table 2 -
(B)The decellularisation methods present specific advantages and disadvantages.

Table 3 -
(A) Effect of the decellularised extracellular matrix derived from dental mesenchymal stem cells on cell responses., 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. ECM

Table 3 -
(B) Effect of the decellularised extracellular matrix derived from mesenchymal stem cells on cell responses.