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. 2024 Nov 12;13(12):1620–1626. doi: 10.1021/acsmacrolett.4c00520

Generating Tooth Organoids Using Defined Bioorthogonally Cross-Linked Hydrogels

Xuechen Zhang †, Nicola Contessi Negrini ‡,§, Rita Correia ‡,§, Paul T Sharpe †, Adam D Celiz ‡,§,*, Ana Angelova Volponi †,*
PMCID: PMC11656705  PMID: 39532305

Abstract

graphic file with name mz4c00520_0005.jpg

Generating teeth in vitro requires mimicking tooth developmental processes. Biomaterials are essential to support 3D tooth organoid formation, but their properties must be finely tuned to achieve the required biomimicry for tooth development. For the first time, we used bioorthogonally cross-linked hydrogels as defined 3D matrixes for tooth developmental engineering, and we highlighted how their properties play a pivotal role in enabling 3D tooth organoid formation in vitro. We prepared hydrogels by mixing gelatin precursors modified either with tetrazine (Tz) or norbornene (Nb) moieties. We tuned the hydrogel properties (E = 2–7 kPa; G′ = 500–1500 Pa) by varying the gelatin concentration (8% vs 12% w/V) and stoichiometric ratio (Tz:Nb = 1 vs 0.5). We encapsulated dental epithelial-mesenchymal cell pellets in a library of hydrogels and identified a hydrogel formulation that enabled successful growth kinetics and morphogenesis of tooth germs, introducing a defined tunable platform for tooth organoid engineering and modeling.

Tooth loss is a prevalent oral health issue affecting millions of individuals worldwide, caused by different factors like dental caries, periodontal disease, dental trauma, and some systematic diseases.1 Beyond impairing chewing and speaking, tooth loss also causes aesthetic and psychological issues. Current replacement solutions, including removable and fixed dentures and dental implants, are nonbiological and often fail to fully restore natural tooth form and function. Consequently, research has focused on biological tooth replacement strategies, with tissue engineering emerging as a promising approach. This method leverages cells, biomaterials, and growth factors to engineer tooth structures that mimic the features and functions of natural teeth.2

The goal of regenerative dentistry is to bioengineer an entire tooth, which requires replicating the interactions between the dental epithelium and mesenchyme. In 1987, Mina and Kollar found that early first branchial arch epithelium can stimulate nonodontogenic, neural-crest-derived mesenchymal cells in the second arch to form dental papilla.3 When dissociated dental epithelial and mesenchymal cells derived from mice embryos are reassociated in vitro, tooth germ-like structures were formed.4,5 These tissue-engineered cell-based in vitro structures were shown to recapitulate the developmental aspects of the complex structures of the corresponding in vivo dental tissues and develop into a functional organ4 and, therefore, can be defined as organoids.6

Organoids are formed from embryonic and adult pluripotent or tissue-resident stem cells, as well as progenitor or differentiated cells derived from healthy or diseased tissues.6,7 Tooth organoids are defined as three-dimensional (3D) in vitro structures that replicate the developmental processes and structural complexity of natural teeth. These organoids are derived from dental cells, which can be of either embryonic or adult origin. The development of tooth organoids involves the self-organization of cells into structures that mimic the cellular composition and functional attributes of the actual teeth. When these structures are transplanted in vivo, they can then fully develop into mature teeth.8,9 While these studies used tissues and cells derived from embryonic origin, Ohazama et al. used adult bone marrow-derived mesenchyme cells and embryonic early, inductive, dental epithelium resulting in tooth formation in vitro, demonstrating that adult mesenchymal cells can actively participate in tooth development.10 Similarly, our previous research showed that human gingival epithelial cells respond to embryonic, mouse mesenchyme signals, leading to the formation of teeth.11 These studies demonstrate that bioengineered organ replacements hold promise as a regenerative therapy. To achieve fully functional bioengineered teeth, cells from adequate sources should be gathered in suitable 3D artificial matrixes (biomaterials) that must support cell self-organization and promote appropriate tooth morphogenesis.12

Different biomaterials have been investigated to support mesenchymal-epithelial crosstalk to regenerate teeth, including polyglycolide acid (PGA), poly-l-lactide acid (PLLA), and poly(lactic-co-glycolic acid) (PLGA),13−16 collagen sponges,17 and decellularized tooth buds.18 Cell-laden hydrogels have been also tested to provide a 3D environment supporting cell encapsulation, growth and rearrangement,19 including collagen20,21 and Matrigel.22,23 Despite these biomaterials serving as successful 3D matrixes for cell crosstalk and tooth bud (tooth germ-like structures) formation, the lack of information and limited control and tunability of their physicomechanical properties hinder our capability of fully understanding the pivotal role that biomaterials play in tooth organoid formation in vitro.24,25

In this work, we engineered cell-laden bioorthogonally cross-linked gelatin hydrogels and investigated for the first time their potential for tooth primordia engineering. Compared to other biomaterials, these hydrogels allow fine-tuning of their properties by varying different design parameters, enabling a better understanding on the involvement of biomaterial properties in determining tooth growth kinetics and morphogenesis in vitro. We cross-linked gelatin using the inverse-electron demand Diels–Alder reaction between tetrazine (Tz) and norbornene (Nb) to obtain cell-laden gelatin-based bioorthogonally cross-linked click hydrogels.26 We tuned the physicomechanical properties of the hydrogels by modifying the biomaterial design (i.e., concentration and ratio between Tz and Nb) to obtained controlled and defined 3D matrixes for cell culture, and we investigate their potential in supporting odontogenic interactions between dental epithelial cells and mesenchymal cells to generate tooth organoids (Figure 1).

Figure 1.

Figure 1

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Preparation of the 3D tooth organoids. Dental mesenchyme and epithelium were obtained in green fluorescent protein (GFP) and CD1 mouse embryos at embryonic day 14.5 (E14.5), separately. After digesting to single cell suspensions, the two cell populations were combined to obtain an epithelial-mesenchymal cell pellet, encapsulated in hydrogels (day 0), and cultured in vitro to generate 3D tooth organoids (day 8). Created with BioRender.com.

We chemically modified gelatin with either tetrazine or norbornene moieties (GEL_Tz or GEL_Nb) to obtain the hydrogel precursors, and we verified and quantified the polymer functionalization via 1H NMR. After polymer functionalization, we identified the characteristic peaks of Tz and Nb within the 1H NMR spectra of the functionalized gelatin, compared to nonfunctionalized gelatin (Figure 2B). The DOM for both GEL_Tz and GEL_Nb was 0.10 ± 0.01 mmol g–1 of gelatin.

Figure 2.

Figure 2

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Bioorthogonally cross-linked click gelatin hydrogels. (A) Schematic of hydrogel structures prepared by varying the concentration (8% and 12% w/V) and ratio between gelatin modified with tetrazine (GEL_Tz, blue lines) and gelatin modified with norbornene (GEL_Nb, yellow lines) at 1:1 (R1) or 0.5:1 (R05). (B) Representative 1H NMR spectra of unmodified gelatin (GEL), gelatin modified with norbornene (GEL_Nb), and gelatin modified with tetrazine (GEL_Tz). (C) Representative rheological curves of the frequency response of the cross-linked hydrogels. (D) Swelling and weight variation in culture medium of the cross-linked hydrogels (n = 4). (E) Representative stress–strain curves (σ–ε) of cross-linked swollen hydrogels and elastic modulus (E). (F) Instantaneous modulus calculated from indentation tests (n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001). Created with Biorender.com.

We then prepared hydrogels by mixing the GEL_Tz and GEL_Nb precursors (Figure 2A, Table S1), enabling the bioorthogonal click reaction between tetrazine and norbornene. All hydrogels successfully cross-linked after mixing the hydrogel precursors. We then evaluated the hydrogel rheological properties after cross-linking. Rheological tests confirmed the hydrogel formation and evidenced a higher storage modulus G′ for hydrogels prepared with a higher polymer concentration and stoichiometric ratio (Figure 2C). The estimated mesh sizes ξ were 27, 22, and 17 nm for GEL_8%_R05, GEL_8%_R1, and GEL_12%_R05, respectively. We performed swelling tests to evaluate the stability of the cross-linked hydrogels in in vitro cell culture-like conditions. All hydrogels were stable in culture medium at 37 °C for at least 2 weeks, showing the possibility of using these hydrogels as platforms for cell cultures, and different swelling profiles were identified (Figure 2D): at plateau, GEL_8%_R05 was more swollen than GEL_8%_R1, which was in turn more swollen than GEL_12%_R05. We then investigated the mechanical properties of the hydrogels and the possibility of tuning them by varying the hydrogel design. The swollen hydrogels were characterized by different compressive mechanical properties (Figure 2E, left). All hydrogels showed a hysteresis cycle during the compression test, characterized by an increase in stress during loading and a decrease in stress during unloading. The mechanical properties were tuned by varying the hydrogel concentration and the stoichiometric ratio. The elastic moduli E varied between 2 and 7 kPa approximatively (EGEL_8%_R05 < EGEL_8%_R1 < EGEL_12%_R05; Figure 2E, right). A decrease in E during swelling was observed (Figure S1) and attributed to the absorption of aqueous medium and decrease in polymer density in the 3D hydrogel network (Figure S2). Similarly, the Instantaneous Modulus measured by indentation tests increased by increasing the polymer concentration and by using a 1:1 Tz:Nb ratio (Figure 2F). All hydrogels were successfully used to incorporate viable cells in the biomaterial 3D structure (Figure S3A); after 1 day of culture, the percentage cell viability was >70% for all the tested hydrogels (Figure S3B), confirming their cytocompatibility. These results show the possibility of tuning the hydrogel physicomechanical properties by varying different design parameters, including the hydrogel concentration and the ratio between the hydrogel precursors, to obtain cytocompatible defined hydrogels.

By injecting mouse embryo whole tooth germs at embryonic day 14.5 (E14.5) inside the three different hydrogel formulations (Figure 3A1–3), we assessed the influence of the hydrogel physicomechanical properties on the growth kinetics and morphological development of tooth germs. After comparing with the physiological tooth growth in vivo (Figure S4), we found that increased hydrogel rheological and mechanical properties decreased tooth germ growth rates, indicating that the hydrogel matrix biomechanically regulates tooth germ development. After 8 days of in vitro culture, tooth germ morphology in the GEL_8%_R1 group (Figure 3A2 and Figure 3C2) and GEL_12%_R05 group (Figure 3A3 and Figure 3C3) exhibited absence of characteristic late cap stage structures with typical inner and outer enamel epithelium and stellate reticulum (Figure S4G), as well as more condensed mesenchymal cells, in contrast to the more defined and structured tooth germs in the GEL_8%_R05 group (Figure 3A1 and Figure 3C1). Tooth germs encapsulated in lower stiffness hydrogels displayed typical structural development, displaying an observable inner and outer enamel epithelium and stellate reticulum (Figure S4G).

Figure 3.

Figure 3

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Whole tooth germs and recombination of dental mesenchymal and epithelial cells in bioorthogonally cross-linked hydrogels prepared by varying polymer concentrations and stoichiometric ratios. (A1–3) Tooth germs in GEL_8%_R05 (n = 6), GEL_8%_R1 (n = 6), or GEL_12%_R05 (n = 6). (B1–3) Recombination in GEL_8%_R05 (n = 13), GEL_8%_R1 (n = 9), or GEL_12%_R05 (n = 6). (C1–3) H&E staining images for A1–3. (D1–3) H&E staining images for B1–3. Scale bar: 400 μm.

Observed morphological differences demonstrated that hydrogel properties influence the structural progression of encapsulated tooth germs. As the ratios between the GEL_Tz and GEL_Nb precursors was changed from 0.5 to 1, a higher number of chemical cross-links formed while keeping the gelatin concentration constant (GEL_8%_R05 vs GEL_8%_R1), the hydrogel rheological and mechanical properties increased, resulting in decelerated growth kinetics, accompanied by changes in morphology. Similarly, increasing the hydrogel concentration while maintaining the GEL_Tz and GEL_Nb ratio constant (GEL_8%_R05 vs GEL_12%_R05) resulted in slower tooth growth and morphogenesis.

We also investigated the impact of the hydrogel design on tooth organoid formation in an in vitro model. The formation of tooth organoids was observed exclusively in the GEL_8%_R05 group. The consistent formation of tooth organoids in this group (n = 13), suggests that the hydrogels with lower mechanical and rheological properties facilitate the tooth organoid formation (Table S2). In contrast, tooth organoids formed only once in the GEL_8%_R1 group (n = 9). In the GEL_12%_R05 group, the stiffest of the three hydrogels tested, tooth organoids did not form (n = 6), suggesting that hydrogels with increased stiffness are less favorable in the formation of dental organoids.

Brightfield microscopy did not discern detailed morphological differences across the hydrogel samples. However, compared to the images taken at day 0 (Figure 1 and Figure S5), which show cells instead of structures concentrated inside the hydrogel, images captured after 8 days of culture (Figure 3B1–3) reveal visible round structures indicating self-sorting and cellular crosstalk in each group. To reveal the structural characteristics of formed organoids, histological analysis of tissue sections stained with H&E had been performed. In the GEL_8%_R05 group, histological analysis revealed the presence of well-defined tooth organoid with an epithelium, and condensed mesenchyme (Figure 3D1), which was similar to the entire tooth germs group in the same hydrogel ratio (Figure 3C1), indicating a successful development of tooth organoids within this hydrogel environment. In contrast, we found epithelial cysts and histologically undefined, round structures in the other groups (Figure 3D2–3). Based on these histological differences, hydrogels of higher stiffness showed a decreased ability to enable organoid formation.

Immunofluorescence was used to determine the cellular composition and to clarify the origin and relationship between epithelial and mesenchymal cells within the tooth organoids. We found that tooth organoids in GEL_8%_R05 hydrogel contained epithelial and mesenchymal cells recombined from different origins (Figure 4A1-A6;Video S1, Video S2). After staining with DAPI, both epithelial and mesenchymal cells had been stained and showed blue fluorescence, while only mesenchymal cells derived from GFP mice showed green fluorescence. This can provide evidence that the tooth organoid that formed in the hydrogel is a new-form structure. The dynamic interplay of epithelial and mesenchymal cells in the GEL_8%_R05 hydrogel environment indicated dental organoid formation. The other two hydrogel formulations did not support tooth organoid formation, as evidenced by the absence of organogenesis via fluorescence staining (Figure 4B1–6 and Figure 4C1–6). Specifically, mesenchymal cells (GFP+) were located outside of the round structures which formed by only epithelial cells (GFP–), indicating they had little or no communication with epithelial cells. These results suggest that tuning the hydrogel properties is pivotal in modulating the occurrence of tooth organoids, providing valuable insight for tissue engineering and regenerative dentistry applications in the future.

Figure 4.

Figure 4

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Recombination of GFP dental mesenchymal cells and CD1 dental epithelial cells in bioorthogonally cross-linked hydrogels prepared by varying polymer concentrations and stoichiometric ratios. (A) GEL_8%_R05, (B) GEL_8%_R1, and (C) GEL_12%_R05 (1–3 are in low magnification; 4–6 are the small white squares in A3, B3 and C3 in high magnification.) Scale bar: 200 μm in lower magnification and 50 μm in higher magnification.

Reproducing the biological mechanisms of embryogenesis in vitro is essential to engineer developmental tissue engineering strategies for tooth repair and in vitro platform for dental development pathophysiological studies.12,27 The interaction between the oral epithelium and mesenchyme initiates tooth development, forming tooth buds that progress through bud, cap, and bell stages (Figure S4). These stages lead to the development of the enamel organ, dental papilla, and dental follicle, ultimately forming dental tissues such as enamel, dentin, and supporting structures.28 To regenerate teeth through tissue engineering, epithelial and mesenchymal cells must interact within a scaffold that facilitates these interactions, resulting in tooth organoids that can fully develop into teeth in vivo.4

Producing artificial extracellular matrix (ECM) for tissue engineering and in vitro modeling requires synthesizing cytocompatible hydrogels with customizable and defined characteristics. The choice of polymer and its cross-linking mechanism are crucial in regulating the physical and mechanical characteristics of the hydrogel. This is essential to attain biomimetic and cytocompatible hydrogels that can effectively guide the desired cellular communication.24 Here, we examined gelatin-based hydrogels obtained by the bioorthogonal click reaction between Tz and Nb to mimic ECM and support tooth organoids’ formation.

We selected gelatin as the polymer to form the hydrogel networks. As a collagen derivative, gelatin is characterized by several advantages including versatility of fabrication, availability, presence of cell-adhesive motifs (i.e., RGD sequences that promote integrin-mediated interactions), biodegradability, and lower immunogenicity and antigenicity compared to collagen.29 Moreover, gelatin hydrogel properties can be tuned by varying several design parameters including gelatin source, polymer concentration, cross-linking methods, and cross-linking density.30 We then selected the click reaction between Tz and Nb to prepare our cell-laden hydrogels due to its bioorthogonal nature, enabling the cross-linking reaction to occur in physiological conditions with no interference with native biochemical processes,31 and the tunability of the material properties prepared using this cross-linking chemistry.32 Moreover, this hydrogel formulation can be simply prepared by mixing hydrogel precursors to allow their spontaneous cross-linking. We then altered the biomaterial design parameters to tune the properties of the hydrogels. In our previous study, we fixed the DOM of gelatin at 10%.26 Here, we investigated how varying the GEL_Tz and GEL_Nb ratio and polymer concentration affects hydrogel physicomechanical properties. A nonstoichiometric ratio (i.e., R05 vs R1) and a lower gelatin concentration (i.e., 8% vs 12%) led to lower rheological properties, higher swelling, and softer mechanical properties. These findings align with previous studies using different cross-linkers for gelatin hydrogels.33−35 Fine tuning the hydrogel physicomechanical properties enabled us to investigate their effect on the maturation of tooth buds.

Establishing epithelial-mesenchymal cell communication is critical for generating tooth organoids in hydrogels. In our experiments, we used dissociated mesenchymal and epithelial cells from mice embryos at the E14.5 stage (Figure S4D). At this stage, the odontogenic signal has already been transferred from epithelial cells to mesenchymal cells; thus the mesenchymal cells are expected to drive the induction and formation of the tooth organoids.36 In our previous study, we successfully demonstrated that dispersed dental pulp stem cells (DPSCs) exhibit a more elongated cell morphology within hydrogels characterized by lower mechanical properties.26 Additionally, our results in the whole tooth germ groups demonstrate how hydrogel physicomechanical properties influence tooth germ development. In native tissues, the ECM serves as a mechanical framework and triggers biological signaling in the cells. Several factors influence 3D cell behavior, including ECM stiffness.37,38 Here, we used hydrogels to mimic ECM and the stiffness of the hydrogel plays an important role in the organoid culture. It impacts the cellular communication dynamics, ultimately impacting the formation of tooth organoids.39

Our experimental design examines tooth organoid development at the cellular level in more detail while altering the 3D culture environment. The hydrogels used as scaffolds offer a tunable environment for tooth organoid formation by introducing appropriate polymer concentrations and Nb/Tz ratios. Fresh embryonic mouse cells were used exclusively, as both mesenchymal and epithelial cells lose odontogenic potential when cultured for extended periods of time.40 To potentially preserve these odontogenic signals, future studies could consider incorporating soluble factors within the hydrogel. Although no growth factors were included in this study, the adaptability of this hydrogel allows for the integration of specific molecules, thereby enhancing its functionality and versatility.

In this work, we evidenced the pivotal role that hydrogel properties play in shaping tooth organogenesis in vitro in artificial 3D matrixes. We designed hydrogels by changing their formulation and tuning their swelling, mechanical properties, and rheological properties, to show their involvement in determining the development of tooth germs at both tissue and organoid levels. Our findings highlight the essential role of biomaterials in facilitating tooth organogenesis for applications in developmental tissue engineering, regenerative dentistry, and in vitro tooth modeling.

Acknowledgments

China Scholarship Council (X.Z.). The authors acknowledge Rousselot Biomedical for providing gelatin.

Glossary

Abbreviations

Tz

tetrazine

Nb

norbornene

GEL_Tz

gelatin decorated with tetrazine

GEL_Nb

gelatin decorated with norbornene

3D

three-dimensional

PGA

polyglycolide acid

PLLA

poly-l-lactide acid

PLGA

poly(lactic-co-glycolic acid)

GFP

green fluorescent protein

E14.5

embryonic day 14.5

ECM

extracellular matrix

DPSCs

dental pulp stem cells

DOM

degree of modification

GEL

gelatin hydrogels

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmacrolett.4c00520.

  • Materials and methods, figures of variation of the hydrogel elastic modulus and hydrogel polymer density, staining images, percentage cell viability, H&E staining images, bright field images, and tables of bioorthogonally crosslinked hydrogel samples definitions and success rates of tooth organoid formation (PDF)

  • Tooth organoid in 3D culturing hydrogel (AVI)

  • Tooth organoid in 3D culturing hydrogel (AVI)

Author Contributions

# These authors contributed equally (X.Z. and N.C.N.). The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. CRediT: Xuechen Zhang data curation, formal analysis, methodology, visualization, writing - original draft, writing - review & editing; Nicola Contessi Negrini conceptualization, data curation, formal analysis, investigation, methodology, supervision, visualization, writing - original draft, writing - review & editing; Rita Correia data curation, formal analysis, methodology, writing - review & editing; Paul T. Sharpe conceptualization, funding acquisition, investigation, supervision, writing - review & editing; Adam D. Celiz conceptualization, funding acquisition, investigation, project administration, supervision, writing - review & editing; Ana Angelova Volponi conceptualization, investigation, supervision, writing - review & editing.

UKRI Future Leaders Fellowship (MR/S034757/1; A.DC.).

The authors declare no competing financial interest.

Supplementary Material

mz4c00520_si_001.pdf (682.7KB, pdf)
mz4c00520_si_002.avi (1.8MB, avi)
mz4c00520_si_003.avi (1.1MB, avi)

References

  1. Bernabe E.; Marcenes W.; Hernandez C. R.; Bailey J.; Abreu L. G.; Alipour V.; Amini S.; Arabloo J.; Arefi Z.; Arora A.; et al. Global, Regional, and National Levels and Trends in Burden of Oral Conditions from 1990 to 2017: A Systematic Analysis for the Global Burden of Disease 2017 Study. Journal of Dental Research 2020, 99 (4), 362–373. 10.1177/0022034520908533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Zhang W.; Yelick P. C. Tooth repair and regeneration: potential of dental stem cells. Trends in molecular medicine 2021, 27 (5), 501–511. 10.1016/j.molmed.2021.02.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Mina M.; Kollar E. The induction of odontogenesis in non-dental mesenchyme combined with early murine mandibular arch epithelium. Archives of oral biology 1987, 32 (2), 123–127. 10.1016/0003-9969(87)90055-0. [DOI] [PubMed] [Google Scholar]
  4. Nakao K.; Morita R.; Saji Y.; Ishida K.; Tomita Y.; Ogawa M.; Saitoh M.; Tomooka Y.; Tsuji T. The development of a bioengineered organ germ method. Nat. Methods 2007, 4 (3), 227–230. 10.1038/nmeth1012. [DOI] [PubMed] [Google Scholar]
  5. Yamamoto H.; Kim E.-J.; Cho S.-W.; Jung H.-S. Analysis of tooth formation by reaggregated dental mesenchyme from mouse embryo. Microscopy 2003, 52 (6), 559–566. 10.1093/jmicro/52.6.559. [DOI] [PubMed] [Google Scholar]
  6. Zhao Z.; Chen X.; Dowbaj A. M.; Sljukic A.; Bratlie K.; Lin L.; Fong E. L. S.; Balachander G. M.; Chen Z.; Soragni A. Organoids. Nature Reviews Methods Primers 2022, 2 (1), 94. 10.1038/s43586-022-00174-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Blatchley M. R.; Anseth K. S. Middle-out methods for spatiotemporal tissue engineering of organoids. Nature Reviews Bioengineering 2023, 1 (5), 329–345. 10.1038/s44222-023-00039-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Oshima M.; Mizuno M.; Imamura A.; Ogawa M.; Yasukawa M.; Yamazaki H.; Morita R.; Ikeda E.; Nakao K.; Takano-Yamamoto T. Functional tooth regeneration using a bioengineered tooth unit as a mature organ replacement regenerative therapy. PloS one 2011, 6 (7), e21531 10.1371/journal.pone.0021531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ikeda E.; Morita R.; Nakao K.; Ishida K.; Nakamura T.; Takano-Yamamoto T.; Ogawa M.; Mizuno M.; Kasugai S.; Tsuji T. Fully functional bioengineered tooth replacement as an organ replacement therapy. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (32), 13475–13480. 10.1073/pnas.0902944106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ohazama A.; Modino S.; Miletich I.; Sharpe P. Stem-cell-based tissue engineering of murine teeth. Journal of dental research 2004, 83 (7), 518–522. 10.1177/154405910408300702. [DOI] [PubMed] [Google Scholar]
  11. Angelova Volponi A.; Kawasaki M.; Sharpe P. Adult human gingival epithelial cells as a source for whole-tooth bioengineering. Journal of Dental Research 2013, 92 (4), 329–334. 10.1177/0022034513481041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Negrini N. C.; Volponi A. A.; Higgins C.; Sharpe P.; Celiz A. Scaffold-based developmental tissue engineering strategies for ectodermal organ regeneration. Materials Today Bio 2021, 10, 100107 10.1016/j.mtbio.2021.100107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Young C.; Terada S.; Vacanti J.; Honda M.; Bartlett J.; Yelick P. Tissue engineering of complex tooth structures on biodegradable polymer scaffolds. Journal of dental research 2002, 81 (10), 695–700. 10.1177/154405910208101008. [DOI] [PubMed] [Google Scholar]
  14. Duailibi M.; Duailibi S.; Young C.; Bartlett J.; Vacanti J.; Yelick P. Bioengineered teeth from cultured rat tooth bud cells. Journal of dental research 2004, 83 (7), 523–528. 10.1177/154405910408300703. [DOI] [PubMed] [Google Scholar]
  15. Abukawa H.; Zhang W.; Young C. S.; Asrican R.; Vacanti J. P.; Kaban L. B.; Troulis M. J.; Yelick P. C. Reconstructing mandibular defects using autologous tissue-engineered tooth and bone constructs. Journal of Oral and Maxillofacial Surgery 2009, 67 (2), 335–347. 10.1016/j.joms.2008.09.002. [DOI] [PubMed] [Google Scholar]
  16. van Manen E. H.; Zhang W.; Walboomers X. F.; Vazquez B.; Yang F.; Ji W.; Yu N.; Spear D. J.; Jansen J. A.; Yelick P. C. The influence of electrospun fibre scaffold orientation and nano-hydroxyapatite content on the development of tooth bud stem cells in vitro. Odontology 2014, 102, 14–21. 10.1007/s10266-012-0087-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Honda M. J.; Tsuchiya S.; Sumita Y.; Sagara H.; Ueda M. The sequential seeding of epithelial and mesenchymal cells for tissue-engineered tooth regeneration. Biomaterials 2007, 28 (4), 680–689. 10.1016/j.biomaterials.2006.09.039. [DOI] [PubMed] [Google Scholar]
  18. Zhang W.; Vazquez B.; Oreadi D.; Yelick P. Decellularized tooth bud scaffolds for tooth regeneration. Journal of dental research 2017, 96 (5), 516–523. 10.1177/0022034516689082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Vedadghavami A.; Minooei F.; Mohammadi M. H.; Khetani S.; Kolahchi A. R.; Mashayekhan S.; Sanati-Nezhad A. Manufacturing of hydrogel biomaterials with controlled mechanical properties for tissue engineering applications. Acta biomaterialia 2017, 62, 42–63. 10.1016/j.actbio.2017.07.028. [DOI] [PubMed] [Google Scholar]
  20. Yang L.; Angelova Volponi A.; Pang Y.; Sharpe P. Mesenchymal cell community effect in whole tooth bioengineering. Journal of Dental Research 2017, 96 (2), 186–191. 10.1177/0022034516682001. [DOI] [PubMed] [Google Scholar]
  21. Ono M.; Oshima M.; Ogawa M.; Sonoyama W.; Hara E. S.; Oida Y.; Shinkawa S.; Nakajima R.; Mine A.; Hayano S. Practical whole-tooth restoration utilizing autologous bioengineered tooth germ transplantation in a postnatal canine model. Sci. Rep. 2017, 7 (1), 44522 10.1038/srep44522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Xiao L.; Tsutsui T. Three-dimensional epithelial and mesenchymal cell co-cultures form early tooth epithelium invagination-like structures: Expression patterns of relevant molecules. Journal of cellular biochemistry 2012, 113 (6), 1875–1885. 10.1002/jcb.24056. [DOI] [PubMed] [Google Scholar]
  23. Zhang W.; Ahluwalia I. P.; Yelick P. C. Three dimensional dental epithelial-mesenchymal constructs of predetermined size and shape for tooth regeneration. Biomaterials 2010, 31 (31), 7995–8003. 10.1016/j.biomaterials.2010.07.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Caliari S. R.; Burdick J. A. A practical guide to hydrogels for cell culture. Nat. Methods 2016, 13 (5), 405–414. 10.1038/nmeth.3839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kozlowski M. T.; Crook C. J.; Ku H. T. Towards organoid culture without Matrigel. Communications Biology 2021, 4 (1), 1387. 10.1038/s42003-021-02910-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Contessi Negrini N.; Angelova Volponi A.; Sharpe P. T.; Celiz A. D. Tunable cross-linking and adhesion of gelatin hydrogels via bioorthogonal click chemistry. ACS Biomaterials Science & Engineering 2021, 7 (9), 4330–4346. 10.1021/acsbiomaterials.1c00136. [DOI] [PubMed] [Google Scholar]
  27. Thesleff I. From understanding tooth development to bioengineering of teeth. European Journal of Oral Sciences 2018, 126 (S1), 67–71. 10.1111/eos.12421. [DOI] [PubMed] [Google Scholar]
  28. Berkovitz B. K.; Holland G. R.; Moxham B. J.. Oral Anatomy, Histology and Embryology E-Book: Oral Anatomy, Histology and Embryology E-Book; Elsevier Health Sciences, 2017. [Google Scholar]
  29. Negrini N. C.; Bonnetier M.; Giatsidis G.; Orgill D. P.; Farè S.; Marelli B. Tissue-mimicking gelatin scaffolds by alginate sacrificial templates for adipose tissue engineering. Acta Biomaterialia 2019, 87, 61–75. 10.1016/j.actbio.2019.01.018. [DOI] [PubMed] [Google Scholar]
  30. Michelini L.; Probo L.; Farè S.; Negrini N. C. Characterization of gelatin hydrogels derived from different animal sources. Mater. Lett. 2020, 272, 127865 10.1016/j.matlet.2020.127865. [DOI] [Google Scholar]
  31. Bertozzi C. R. A Decade of Bioorthogonal Chemistry. Acc. Chem. Res. 2011, 44 (9), 651–653. 10.1021/ar200193f. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Arkenberg M. R.; Nguyen H. D.; Lin C.-C. Recent advances in bio-orthogonal and dynamic crosslinking of biomimetic hydrogels. J. Mater. Chem. B 2020, 8 (35), 7835–7855. 10.1039/D0TB01429J. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Contessi Negrini N.; Tarsini P.; Tanzi M.; Farè S. Chemically crosslinked gelatin hydrogels as scaffolding materials for adipose tissue engineering. J. Appl. Polym. Sci. 2019, 136 (8), 47104 10.1002/app.47104. [DOI] [Google Scholar]
  34. Liu Y.; Weng R.; Wang W.; Wei X.; Li J.; Chen X.; Liu Y.; Lu F.; Li Y. Tunable physical and mechanical properties of gelatin hydrogel after transglutaminase crosslinking on two gelatin types. Int. J. Biol. Macromol. 2020, 162, 405–413. 10.1016/j.ijbiomac.2020.06.185. [DOI] [PubMed] [Google Scholar]
  35. Hölzl K.; Fürsatz M.; Göcerler H.; Schädl B.; Žigon-Branc S.; Markovic M.; Gahleitner C.; Hoorick J. V.; Van Vlierberghe S.; Kleiner A. Gelatin methacryloyl as environment for chondrocytes and cell delivery to superficial cartilage defects. Journal of tissue engineering and regenerative medicine 2022, 16 (2), 207–222. 10.1002/term.3273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Jussila M.; Thesleff I. Signaling networks regulating tooth organogenesis and regeneration, and the specification of dental mesenchymal and epithelial cell lineages. Cold Spring Harbor perspectives in biology 2012, 4 (4), a008425 10.1101/cshperspect.a008425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Cruz Walma D.; Yamada K. Extracellular matrix in human craniofacial development. Journal of Dental Research 2022, 101 (5), 495–504. 10.1177/00220345211052982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Saraswathibhatla A.; Indana D.; Chaudhuri O. Cell–extracellular matrix mechanotransduction in 3D. Nat. Rev. Mol. Cell Biol. 2023, 24 (7), 495–516. 10.1038/s41580-023-00583-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Vernerey F. J.; Lalitha Sridhar S.; Muralidharan A.; Bryant S. J. Mechanics of 3D cell–hydrogel interactions: experiments, models, and mechanisms. Chem. Rev. 2021, 121 (18), 11085–11148. 10.1021/acs.chemrev.1c00046. [DOI] [PubMed] [Google Scholar]
  40. Zheng Y.; Cai J.; Hutchins A. P.; Jia L.; Liu P.; Yang D.; Chen S.; Ge L.; Pei D.; Wei S. Remission for loss of odontogenic potential in a new micromilieu in vitro. PloS one 2016, 11 (4), e0152893 10.1371/journal.pone.0152893. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

mz4c00520_si_001.pdf (682.7KB, pdf)
mz4c00520_si_002.avi (1.8MB, avi)
mz4c00520_si_003.avi (1.1MB, avi)

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