Native dental pulp extracellular matrix (DPEM) has proven to be an effective biomaterial for dental pulp regeneration. However, as a significant extracellular matrix glycoprotein, partial laminins were lost during the decellularization process, which were essential for odontoblast differentiation. Thereby, this study investigated the feasibility of LN supplementation to improve the surface of DPEM for odontoblast layer regeneration. The influences of laminin on cell adhesion and odontogenic differentiation were evaluated in vitro. Then, we fabricated laminin-modified DPEM based on the physical coating strategy and observed the location and persistency of laminin coating by immunofluorescent staining. Finally, laminin-modified DPEM combined with treated dentin matrix (TDM) was transplanted in orthotopic jaw bone of beagles (n = 3) to assess the effect of LNs on dental pulp tissue regeneration. The in vitro results showed that laminins could improve the adhesion of dental pulp stem cells (DPSCs) and promoted DPSCs toward odontogenic differentiation. Continuous odontoblastic layer-like structure was observed in laminin-modified DPEM group, expressing the markers for odontoblastogenesis, dentine matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP). Overall, these studies demonstrate that the supplementation of laminins to DPEM contributes to the odontogenic differentiation of cells and to the formation of odontoblast layer in dental pulp regeneration.

Introduction: Decellularized extracellular matrix has been recognized as an optimal scaffold for dental pulp regeneration. However, the limited amount of native dental pulp tissue restricts its clinical applications. The submandibular gland shares some basic extracellular matrix components and characteristics with dental pulp. However, whether decellularized submandibular gland extracellular matrix (DSMG) can be used as an alternative scaffold for dental pulp regenerative medicine is unclear. Methods: Thus, we successfully decellularized the whole rat submandibular gland and human dental pulp, and then conducted in vitro and in vivo studies to compare the properties of these two scaffolds for dental pulp regeneration. Results: Our results showed that extracellular matrix of the submandibular gland had great similarities in structure and composition with that of dental pulp. Furthermore, it was confirmed that the DSMG could support adhesion and proliferation of dental pulp stem cells in vitro. In vivo findings revealed that implanted cell-seeded DSMG formed a vascularized dental pulp-like tissue and expressed markers involved in dentinogenesis and angiogenesis. Discussion: In summary, we introduced a novel accessible biological scaffold and validated its effectiveness as an extracellular matrix-based tissue engineering scaffold for dental pulp regenerative therapy.

There is an age-dependent decline of pulp regeneration, due to the decline of migration, proliferation, and cell survival of resident stem cells. Trypsin is a proteolytic enzyme clinically used for tissue repair. Here, we investigated the effects of trypsin pretreatment of pulpectomized teeth prior to cell transplantation on pulp regeneration in aged dogs. The amount of regenerated pulp was significantly higher in trypsin-pretreated teeth compared to untreated teeth. Trypsin pretreatment increased the number of cells attached to the dentinal wall that differentiated into odontoblast-like cells. The trypsin receptor, PAR2, was higher in vitro expression in the periodontal ligament cells (PDLCs) from aged dogs compared to those from young. The direct effects of trypsin on aged PDLCs were increased expression of genes related to immunomodulation, cell survival, and extracellular matrix degradation. To examine the indirect effects on microenvironment, highly extracted proteins from aged cementum were identified by proteomic analyses. Western blotting demonstrated that significantly increased fibronectin was released by the trypsin treatment of aged cementum compared to young cementum. The aged cementum extract (CE) and dentin extract (DE) by trypsin treatment increased angiogenesis, neurite extension and migration activities as elicited by fibronectin. Furthermore, the DE significantly increased the mRNA expression of immunomodulatory factors and pulp markers in the aged DPSCs. These results demonstrated the effects of trypsin on the microenvironment in addition to the resident cells including PDLCs in the aged teeth. In conclusion, the potential utility of trypsin pretreatment to stimulate pulp regeneration in aged teeth and the underlying mechanisms were demonstrated.

Failure of corneal endothelium cell monolayer is the main cause leading to corneal transplantation. Autologous cell-based therapies are required to reconstruct in vitro the cell monolayer. Several strategies have been proposed using embryonic stem cells and induced pluripotent stem cells, although their use has ethical issues as well as limited clinical applications. For this purpose, we propose the use of dental pulp stem cells isolated from the third molars to form the corneal endothelium cell monolayer. We hypothesize that using dental pulp stem cells that share an embryological origin with corneal endothelial cells, as they both arise from the neural crest, may allow a direct differentiation process avoiding the use of reprogramming techniques, such as induced pluripotent stem cells. In this work, we report a two-step differentiation protocol, where dental pulp stem cells are derived into neural crest stem-like cells and, then, into corneal endothelial-like cells. Initially, for the first-step we used an adhesion culture and compared two initial cell sources: a direct formation from dental pulp stem cells with the differentiation from induced pluripotent stem cells. Results showed significantly higher levels of early stage marker AP2 for the dental pulp stem cells compared to induced pluripotent stem cells. In order to provide a better environment for neural crest stem cells generation, we performed a suspension method, which induced the formation of neurospheres. Results showed that neurosphere formation obtained the peak of neural crest stem cell markers expression after 4 days, showing overexpression of AP2, Nestin, and p75 markers, confirming the formation of neural crest stem-like cells. Furthermore, pluripotent markers Oct4, Nanog, and Sox2 were as well-upregulated in suspension culture. Neurospheres were then directly cultured in corneal endothelial conditioned medium for the second differentiation into corneal endothelial-like cells. Results showed the conversion of dental pulp stem cells into polygonal-like cells expressing higher levels of ZO-1, ATP1A1, COL4A2, and COL8A2 markers, providing a proof of the conversion into corneal endothelial-like cells. Therefore, our findings demonstrate that patient-derived dental pulp stem cells may represent an autologous cell source for corneal endothelial therapies that avoids actual transplantation limitations as well as reprogramming techniques.

Objectives: Fallopian tube (FT) injury is an important factor that can lead to tubal infertility. Stem-cell-based therapy shows great potential for the treatment of injured fallopian tube. However, little research has shown that mesenchymal stem cells (MSCs) can be used to treat fallopian tube damage by in situ injection. In this study, we in situ transplanted PF127 hydrogel encapsulating dental pulp stem cells (DPSCs) into the injured sites to promote the repair and regeneration of fallopian tube injury. Materials and methods: The properties of dental pulp stem cells were evaluated by flow cytometry, immunofluorescence analysis, and multi-differentiation detection. The immunomodulatory and angiogenic characteristics of dental pulp stem cells were analyzed on the basis of the detection of inflammatory factor expression and the formation of capillary-like structures, respectively. The biocompatibility of PF127 hydrogel was evaluated by using Live/Dead and CCK-8 assays. The effects of PF127 hydrogel containing dental pulp stem cells on the repair and regeneration of fallopian tube injury were evaluated by histological analysis [e.g., hematoxylin and eosin (H&E) and Masson's trichrome staining, TUNEL staining, immunofluorescence staining, and immunohistochemistry], Enzyme-linked immunosorbent assay (ELISA), and RT-PCR detections. Results: Dental pulp stem cells had MSC-like characteristics and great immunomodulatory and angiogenic properties. PF127 hydrogel had a thermosensitive feature and great cytocompatibility with dental pulp stem cells. In addition, our results indicated that PF127 hydrogel containing dental pulp stem cells could promote the repair and regeneration of fallopian tube damage by inhibiting cell apoptosis, stimulating the secretion of angiogenic factors, promoting cell proliferation, modulating the secretion of inflammatory factors, and restoring the secretion of epithelial cells. Conclusion: In this study, our results reported that in situ injection of PF127 hydrogel encapsulating dental pulp stem cells into the injured sites could provide an attractive strategy for the future treatment of fallopian tube injury in clinical settings.

Sclerostin (Sost) and dickkopf (Dkk)-1 are inhibitors of the Wnt signaling pathway that plays a role in regenerative processes. Hypoxia-based strategies are used for regenerative approaches, but the influence of hypoxia on Sost and Dkk-1 production in a pro-inflammatory environment is unclear. The aim of this study was to assess if pro-inflammatory molecules have an influence on Sost and Dkk-1 production in dental pulp cells (DPC) under normoxia and hypoxia. Human DPC were treated with interleukin (IL)-1β, tumor necrosis factor (TNF)α or transforming growth factor (TGF)β, with L-mimosine (L-MIM) or hypoxia or a combination. Sost and Dkk-1 mRNA and protein levels were measured with qPCR and western blot, respectively. TNFα, TGFβ, L-MIM, or combined treatment did not modulate Sost and Dkk-1. IL-1β downregulated Sost at the mRNA level. Hypoxia alone and together with inflammatory markers downregulated Dkk-1 at the mRNA level. Sost and Dkk-1 protein production was below the detection limit. In conclusion, there is a differential effect of hypoxia and IL-1β on the mRNA production of Sost and Dkk-1. Pro-inflammatory molecules do not further modulate the effects of L-MIM or hypoxia on Sost and Dkk-1 production in DPC.

The ability to effectively repair craniomaxillofacial (CMF) bone defects in a fully functional and aesthetically pleasing manner is essential to maintain physical and psychological health. Current challenges for CMF repair therapies include the facts that craniofacial bones exhibit highly distinct properties as compared to axial and appendicular bones, including their unique sizes, shapes and contours, and mechanical properties that enable the ability to support teeth and withstand the strong forces of mastication. The study described here examined the ability for tyrosine-derived polycarbonate, E1001(1K)/β-TCP scaffolds seeded with human dental pulp stem cells (hDPSCs) and human umbilical vein endothelial cells (HUVECs) to repair critical sized alveolar bone defects in an in vivo rabbit mandible defect model. Human dental pulp stem cells are uniquely suited for use in CMF repair in that they are derived from the neural crest, which naturally contributes to CMF development. E1001(1k)/β-TCP scaffolds provide tunable mechanical and biodegradation properties, and are highly porous, consisting of interconnected macro- and micropores, to promote cell infiltration and attachment throughout the construct. Human dental pulp stem cells/HUVECs seeded and acellular E1001(1k)/β-TCP constructs were implanted for one and three months, harvested and analyzed by micro-computed tomography, then demineralized, processed and sectioned for histological and immunohistochemical analyses. Our results showed that hDPSC seeded E1001(1k)/β-TCP constructs to support the formation of osteodentin-like mineralized jawbone tissue closely resembling that of natural rabbit jaw bone. Although unseeded scaffolds supported limited alveolar bone regeneration, more robust and homogeneous bone formation was observed in hDPSC/HUVEC-seeded constructs, suggesting that hDPSCs/HUVECs contributed to enhanced bone formation. Importantly, bioengineered jaw bone recapitulated the characteristic morphology of natural rabbit jaw bone, was highly vascularized, and exhibited active remodeling by the presence of osteoblasts and osteoclasts on newly formed bone surfaces. In conclusion, these results demonstrate, for the first time, that E1001(1K)/ β-TCP scaffolds pre-seeded with human hDPSCs and HUVECs contributed to enhanced bone formation in an in vivo rabbit mandible defect repair model as compared to acellular E1001(1K)/β-TCP constructs. These studies demonstrate the utility of hDPSC/HUVEC-seeded E1001(1K)/β-TCP scaffolds as a potentially superior clinically relevant therapy to repair craniomaxillofacial bone defects.

Adipose tissue-derived stem cells (ADSCs) and dental pulp stem cells (DPSCs) have become promising sources for bone tissue engineering. Our study aimed at evaluating bone regeneration potential of cryopreserved ADSCs and DPSCs combined with bovine-derived xenografts with 10% porcine collagen. In vitro studies revealed that although DPSCs had higher proliferative abilities, ADSCs exhibited greater mineral depositions and higher osteogenic-related gene expression, indicating better osteogenic differentiation potential of ADSCs. After applying cryopreserved ADSCs and DPSCs in a critical-sized calvarial defect model, both cryopreserved mesenchymal stem cells significantly improved bone volume density and new bone area at 2, 4, and 8 weeks. Furthermore, the combined treatment with ADSCs and xenografts was more efficient in enhancing bone repair processes compared to combined treatment with DPCSs at all-time points. We also evaluated the sequential early bone healing process both histologically and radiographically, confirming a high agreement between these two methods. Based on these results, we propose grafting of the tissue-engineered construct seeded with cryopreserved ADSCs as a useful strategy in accelerating bone healing processes.

Stem cell-based developmental engineering has been considered as a promising strategy for tissue/organ regeneration. Tooth is formed by sequential reciprocal interactions between epithelium derived from surface ectoderm and mesenchymal cells derived from cranial neural crest. The neural crest cell is an appealing cell source for tooth development and regeneration research. In this study, we investigated the odontogenic differentiation and dentin-pulp complex regeneration potential of neural crest cells. Our results showed that neural crest cells (O9-1 mouse cranial neural crest cell line) can sequentially differentiate into dentin matrix acidic phosphoprotein 1 (DMP-1)-positive odontoblasts within a developing tooth germ in vitro. Moreover, O9-1 cells and induced pluripotent stem cell (iPSC)-derived neural crest-like cells (iNCLCs) can form well-organized vascularized dentin-pulp complex when transplanted in vivo with tooth scaffold. Furthermore, both O9-1 cells and iNCLCs can be differentiated into odontoblast-like cells, positive staining with odontogenic-related markers DMP-1 and dentin sialophosphoprotein (DSPP), under odontogenic induction with the administration of bone morphogenetic protein 4 (BMP-4). These results demonstrated that neural crest cells, especially the unlimited iNCLCs, are a promising cell source for tooth development and dental tissue/tooth organ regeneration studies.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative pathogen for coronavirus disease-2019 (COVID-19), which has posed an increasing serious public health threat. However, still there are no approved antiviral agents or vaccines available yet. Mesenchymal stem cells (MSCs) are emerging as a novel promising adjuvant therapy for the attenuation of COVID-19 based on its putative pathogenesis. MSCs may exert anti-inflammatory, immunomodulatory, anti-apoptotic, as well as regenerative effects through a series of mechanisms. Remarkably, MSCs may be resistant to virus infection, which is fundamental for the treatment of COVID-19. The beneficial therapeutic effects of MSCs have been preliminarily proved to be safe and efficacious for the treatment of COVID-19 in current clinical trials. This work aims to review the beneficial effects of MSCs in treating ALI/ARDS, which provides novel insight into the potential therapeutic strategies against COVID-19. However, further research is warranted regarding both safety and efficacy of MSCs.

There is a substantial global market for orthopedic implants, but these implants still face the problem of a high failure rate in the short and long term after implantation due to the complex physiological conditions in the body. The use of multifunctional coatings on orthopedic implants has been proposed as an effective way to overcome a range of difficulties. Here, a multifunctional (TA@HA/Lys)n coating composed of tannic acid (TA), hydroxyapatite (HA), and lysozyme (Lys) was fabricated in a layer-by-layer (LBL) manner, where TA deposited onto HA firmly stuck Lys and HA together. The deposition of TA onto HA, the growth of (TA@HA/Lys)n, and multiple related biofunctionalities were thoroughly investigated. Our data demonstrated that such a hybrid coating displayed antibacterial and antioxidant effects, and also facilitated the rapid attachment of cells [both mouse embryo osteoblast precursor cells (MC3T3-E1) and dental pulp stem cells (DPSCs)] in the early stage and their proliferation over a long period. This accelerated osteogenesis in vitro and promoted bone formation in vivo. We believe that our findings and the developed strategy here could pave the way for multifunctional coatings not only on orthopedic implants, but also for additional applications in catalysts, sensors, tissue engineering, etc.

Human endometrial stem cells (hEnSCs), dental pulp stem cells (hDPSCs) and adipose tissue-derived stem cells (hADSCs) are considered to be the promising candidates for the treatment of pancreas diseases. The prognosis is better with in situ injection of mesenchymal stem cells (MSCs) to the damaged pancreas compared with intravenous injection. However, the clinical application of these cells are limited, due to poor engraftment of transplanted cells after delivery. On the other hand, understanding the role of the biomaterials in cell therapy is essential to promote the therapeutic effects of MSCs. Matrigel, a basement membrane matrix biomaterial, is rich in laminin and collagen IV. The aim of this study is to investigate the difference of biological characteristics of hEnSCs, hDPSCs and hADSCs in vitro and their survival situation with Matrigel post intrapancreatic transplantation in vivo. Our findings showed, firstly, there was no significant difference in morphology and immunophenotype of these MSCs. Secondly, the biological properties, including cell proliferation, the ability of adipogenic and osteogenic differentiation and the mRNA expression levels of pancreas development-related genes, have been showed distinct difference among these MSCs. Thirdly, Matrigel can improve the survival of MSCs in vivo, especially for Matrigel-based hDPSCs and Matrigel-based hEnSCs in pancreas parenchyma of SD rats. These results suggest that hDPSCs and hEnSCs are with the greater inherent therapeutic potential for pancreas diseases compared with hADSCs.

Critical-size bone defects are those that will not heal without intervention and can arise secondary to trauma, infection, and surgical resection of tumors. Treatment options are currently limited to filling the defect with autologous bone, of which there is not always an abundant supply, or ceramic pastes that only allow for limited osteo-inductive and -conductive capacity. In this study we investigate the repair of bone defects using a 3D printed LayFomm scaffold. LayFomm is a polymer blend of polyvinyl alcohol (PVA) and polyurethane (PU). It can be printed using the most common method of 3D printing, fused deposition modeling, before being washed in water-based solutions to remove the PVA. This leaves a more compliant, micro-porous PU elastomer. In vitro analysis of dental pulp stem cells seeded onto macro-porous scaffolds showed their ability to adhere, proliferate and form mineralized matrix on the scaffold in the presence of osteogenic media. Subcutaneous implantation of LayFomm in a rat model showed the formation of a vascularized fibrous capsule, but without a chronic inflammatory response. Implantation into a mandibular defect showed significantly increased mineralized tissue production when compared to a currently approved bone putty. While their mechanical properties are insufficient for use in load-bearing defects, these findings are promising for the use of polyurethane scaffolds in craniofacial bone regeneration.

Mesenchymal stromal cells (MSCs) are multipotent cells found in different tissues: bone marrow, peripheral blood, adipose tissues, skeletal muscle, perinatal tissues, and dental pulp. MSCs are able to self-renew and to differentiate into multiple lineages, and they have been extensively used for cell therapy mostly owing to their anti-fibrotic and immunoregulatory properties that have been suggested to be at the basis for their regenerative capability. MSCs exert their effects by releasing a variety of biologically active molecules such as growth factors, chemokines, and cytokines, either as soluble proteins or enclosed in extracellular vesicles (EVs). Analyses of MSC-derived secretome and in particular studies on EVs are attracting great attention from a medical point of view due to their ability to mimic all the therapeutic effects produced by the MSCs (i.e., endogenous tissue repair and regulation of the immune system). MSC-EVs could be advantageous compared with the parental cells because of their specific cargo containing mRNAs, miRNAs, and proteins that can be biologically transferred to recipient cells. MSC-EV storage, transfer, and production are easier; and their administration is also safer than MSC therapy. The skeletal muscle is a very adaptive tissue, but its regenerative potential is altered during acute and chronic conditions. Recent works demonstrate that both MSCs and their secretome are able to help myofiber regeneration enhancing myogenesis and, interestingly, can be manipulated as a novel strategy for therapeutic interventions in muscular diseases like muscular dystrophies or atrophy. In particular, MSC-EVs represent promising candidates for cell free-based muscle regeneration. In this review, we aim to give a complete picture of the therapeutic properties and advantages of MSCs and their products (MSC-derived EVs and secreted factors) relevant for skeletal muscle regeneration in main muscular diseases.

In the last two decades, alginate scaffolds have been variously studied as extracellular matrix analogs for tissue engineering. However, relevant evidence is still lacking concerning their ability to mimic the microenvironment of hierarchical tissues such as bone. Hence, an increasing amount of attention has recently been devoted to the fabrication of macro/microporous sponges with pore anisotropy able to more accurately replicate the cell niche structure as a trigger for bioactive functionalities. This paper presents an in vivo study of alginate sponges with anisotropic microporous domains (MAS) formed by ionic crosslinking in the presence of different fractions (30 or 50% v) of hydroxyapatite (HA). In comparison with unloaded sponges (MAS0), we demonstrated that HA confers peculiar physical and biological properties to the sponge, depending upon the inorganic fraction used, enabling the sponge to bio-mimetically support the regeneration of newly formed bone. Scanning electron microscopy analysis showed a preferential orientation of pores, ascribable to the physical constraints exerted by HA particles during the pore network formation. Energy dispersive spectroscopy (EDS) and X-Ray diffraction (XRD) confirmed a chemical affinity of HA with the native mineral phase of the bone. In vitro studies via WST-1 assay showed good adhesion and proliferation of human Dental Pulp-Mesenchymal Stem Cells (hDP-MSC) that increased in the presence of the bioactive HA signals. Moreover, in vivo studies via micro-CT and histological analyses of a bone model (e.g., a rat calvaria defect) confirmed that the maximum osteogenic response after 90 days was achieved with MAS30, which supported good regeneration of the calvaria defect without any evidence of inflammatory reaction. Hence, all of the results suggested that MAS is a promising scaffold for supporting the regeneration of hard tissues in different body compartments.