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Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Faculty of Dentistry, Chulalongkorn University, Bangkok, ThailandDepartment of Research Affairs, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Faculty of Dentistry, Chulalongkorn University, Bangkok, ThailandDepartment of Oral Pathology, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand
Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Faculty of Dentistry, Chulalongkorn University, Bangkok, ThailandFaculty of Dentistry, National University of Singapore, Singapore
Dysfunction and damage of the lacrimal gland (LG) results in ocular discomfort and dry eye disease (DED). Current therapies for DED do not fully replenish the necessary lubrication to rescue optimal vision. New drug discovery for DED has been limited perhaps because in vitro models cannot mimic the biology of the native LG. The existing platforms for LG organoid culture are scarce and still not ready for consistency and scale up production towards drug screening. The magnetic three-dimensional (3D) bioprinting (M3DB) is a novel system for 3D in vitro biofabrication of cellularized tissues using magnetic nanoparticles to bring cells together. M3DB provides a scalable platform for consistent handling of spheroid-like cell cultures facilitating consistent biofabrication of organoids. Previously, we successfully generated innervated secretory epithelial organoids from human dental pulp stem cells with M3DB and found that this platform is feasible for epithelial organoid bioprinting. Research targeting LG organogenesis, drug discovery for DED has extensively used mouse models. However, certain inter-species differences between mouse and human must be considered. Porcine LG appear to have more similarities to human LG than the mouse counterparts. We have conducted preliminary studies with the M3DB for fabricating LG organoids from primary cells isolated from murine and porcine LG, and found that this platform provides robust LG organoids for future potential high-throughput analysis and drug discovery. The LG organoid holds promise to be a functional model of tearing, a platform for drug screening, and may offer clinical applications for DED.
The lacrimal gland (LG) produces an ocular lubricating secretion to maximize the visual acuity and support homeostasis of the eye. Dysfunction of the LG can be commonly caused by trauma, age-related degeneration, graft versus host disease, Sjögren's syndrome, and from chemotherapy secondary effects, resulting in LG morbidities such as ocular discomfort and dry eye disease (DED)[
]. Mild manifestations of DED can be partially tamed with artificial tears or anti-inflammatory drugs, though these are just palliative management approaches that do not fully replenish the ocular lubrication and vision [
]. Hence, recent and potentially promising therapies to rescue LG secretion have been focusing on three approaches by: (1) stimulation of the remaining epithelial cells and stem cell niche; (2) transplantation of stem cells for gland homing, differentiation and generation of new secretory units; and (3) transplantation of ex vivo epithelial secretory cells or mini-organs/organoids into the damaged glands [
]. The latter approach has led to the development of organoid platforms to recapitulate the epithelial architecture and functional properties of the native LG organ. This LG organoid holds potential to be a functional model of tearing, a platform of drug screening and discovery, and a clinical approach for an effective management of DED [
Recent research into human LG organogenesis, platform for drug screening, and DED therapy application has been based extensively in mouse and porcine model investigations [
]. A comprehensive understanding of LG organogenesis, biology and physiology upon epithelial maturation are necessary for developing the LG organ in vitro [
]. Herein, LG organogenesis steps will be discussed in detail in order to apply such foundational steps towards the development of LG organoid platforms for multiple applications including bioprinting and high-throughput screening.
1. Features of the mouse, porcine, and human lacrimal glands
In the mouse, the development of LG is initiated by the invagination of conjunctival epithelium from the temporal extremity into the periorbital mesenchyme at embryonic day (E) 13.517. Two cell lineages start deriving from multipotent stem cells during this stage, a SRY (sex-determining region Y)-box 10 (Sox10) positive and a Sox10 negative, which will mature towards an acinar and ductal epithelial progeny, respectively [
]. At E15.5, Sox10 negative cells divide and differentiate to form a ductal network, and at E16.5, this structure branches into two different lobes and ultimately two organs in different locations, the intraorbital and exorbital LG (iLG and eLG). At postnatal day (P) 30, these two glands reach epithelial maturation and can produce three layers of tear film including the mucous layer, aqueous layer, and superficial lipid layer to maintain ocular homeostasis [
Fig. 1Anatomy of the mouse, porcine, and human lacrimal glands. Mouse possesses two pairs of major LGs including intraorbital and exorbital LGs and the lipid producing Harderian gland while porcine and human has single LG. Each gland produces its tear component into the ocular surface via a specific duct. Created with BioRender.com
] can identify the gland primordium at the 50th day of gestation. Next, the gland forms 5-8 lobes within the dense connective tissue at the 94th day and divides the parenchyma into a distinct lobe by the connective tissue trabeculae with complete vascularization at the 112th day of gestation.
The human LG development is initiated since the palpebral primordium is observed at E 18 or O'Rahilly stage 8. The epithelial bud is clearly formed in the superior conjunctival fornix and epithelial lumenization can be observed at E52 and E56, respectively. Then, the epithelial bud expansion and invagination are initiated at the 9th-12th week of development and the gland organization, vascularization, and stromal condensation, continues to be observed as a highly branched gland at the 15th fetal week [
]. Next, we will compare different features of the mouse, porcine and human LG, including anatomical and histological architecture, stem cell populations and their progeny, and physiological regulation.
1.1 Anatomical and histological architecture
Like humans, the porcine LG is located anteriorly in the superolateral aspect of the orbit [
]. Meanwhile, the mouse LG comprises two main glands, the intraorbital LG and exorbital LG. The exorbital LG is a primary lobe that is superficially situated in the anterior aspect of the ear with a duct stretching to the eye at the lateral rim (Fig. 1) [
Regarding their histological architecture, LG is classified as a tubuloacinar exocrine gland that can produce tear fluid in acini and then drain it into the ocular surface through the ductal network [
]. As with other tubuloacinar exocrine glands, the functional unit of the adult human, porcine, and murine LG is composed of acinar, ductal, and myoepithelial compartments working reciprocally to produce and secrete tear fluid into the ocular surface [
During murine LG organogenesis, the multipotent stem cells give rise to two progenitor populations, Sox9+/Sox10- and Sox9+/Sox10+ cells, one will mature into ductal and the other into acinar epithelial cells, respectively [
]. In early adulthood, differentiated acinar cells continue to express Sox9 and Sox10 and cytokeratin proteins are commonly co-expressed, particularly cytokeratin 5 and 1418. Human and mouse LG acinar secretory compartment is classified as seromucous [
]. Both types of acinar cells are arranged in a mosaic pattern as a secretory unit with a narrow lumen surrounded by myoepithelial cells and connective tissue [
]. The different acinar cell types in the LG can produce a tear fluid with a variable composition depending on the status/condition, for example aging-related diseases of the ocular surface [
]. Herein, we demonstrated, for the first time, the majority of acinar cells in porcine and human LG are highlighted by Periodic Acid Schiff (PAS) and mucicarmine stains, indicative of predominance of mucous cell type. In contrast, mice LG acinar cells are negative for PAS and mucicarmine stains, indicative of majority of serous cell type (Fig. 2).
Fig. 2Histology of murine and porcine LG when compared to human exocrine glands. LG tissues from ICR mice (upper panel) and porcine (middle panel) were stained with hematoxylin and eosin (H&E), Periodic acid–Schiff (PAS), and Mucicarmine stains and microscopically observed at 200X magnification. The acinar cells (a) are organized in lobules separated by connective tissue (c). The interlobular ducts (d) are lined by the cuboidal epithelium that exhibits more hyperchromatic nuclei when compared to the acinar cells. Human salivary gland (SG) tissue is the positive control for PAS and mucicarmine stains. Scale bar=200 μm.
The secretion of water, protein and electrolyte from LG is tightly regulated by parasympathetic and sympathetic nerves that innervate the secretory cells, and the excretory ducts [
]. The nerves release neurotransmitters to activate the acinar cells on their basolateral transmembrane membrane receptors including muscarinic acetylcholine receptors (e.g. M3AChR) and vasoactive intestinal peptide receptor (VIPR) for parasympathetic control and adrenergic receptor type alpha 1D (AR-α1D) for sympathetic control [
]. If the EGF is anchored on basolateral membrane, it would interact with the acinar EGF receptors whereas if it is present in the apical membranes, it would be released into the tear fluid and then interact with the receptors on the ocular epithelium [
Detection of transforming growth factor-alpha mRNA and protein in rat lacrimal glands and characterization of transforming growth factor-alpha in human tears.
]. These suggest that tear protein secretion derived by EGF interaction can be stimulated by both sympathetic and parasympathetic neurotransmitters.
1.1.2 Ductal compartment
Tear fluid is produced in secretory acini in both human and mouse LG and it is drained into the ocular cavity through three different types of ducts: intercalated, intralobular, and interlobular [
]. Thus, the characterization of basal and luminal epithelium by using protein markers Krt14+ and Krt19+, respectively, is essential to investigate the ability of these progenitors to develop a newly mature LG ductal network [
Myoepithelial cells (MEC) are also derived from the same multipotent distal stem cell lineage that differentiates into the acinar epithelial progeny, the Sox9+/Sox10+ population; though MEC exclusively express the smooth muscle actin (SMA) protein [
. During mouse LG organogenesis, the MEC compartment can be observed in the periphery of the LG since P3 and becomes flattened between the basal membrane of the epithelial layer and the extracellular matrix (ECM) with the persistence of SOX9, SOX10, and SMA [
]. The roles of MEC on LG development have been more extensively studied including the involvement on epithelium mesenchymal cell signaling and the niche for the LG stem/progenitor cells [
. In terms of morphology, the mature MEC displays a cuboidal shape instead of a stellate shape and localizes in the external layer of the developing adult LG bud [
]. Humans and mice MEC progenitor cells express the paired box protein-6 (Pax6) and filament protein nestin which can be used as specific protein markers [
Characterization of the LG progenitor cells by lineage tracing is scarce and thus the LG progeny is not well understood when compared to other exocrine glands like pancreases and salivary gland. In embryonic LG development, there are multipotent stem cells and progenitor cells that can commit to all epithelial cell types [
]. Two hypotheses are suggested to support adult epithelial cell renewal and differentiation to all epithelial lineages: First, the epithelial progeny originates from a common multipotent stem/progenitor cell or be delivered from their own lineage-specified progenitor cells [
]. Based on previous studies, progenitor markers such as Sox9, Pax6, Runx1, c-kit, Krt5, and Krt14 are expressed in the early LG stem/progenitor cells and can be used as hallmarks to identify multipotent cells [
Transcription factors Runx1 to 3 are expressed in the lacrimal gland epithelium and are involved in regulation of gland morphogenesis and regeneration.
. Earlier in 2017, a LG progenitor cell population was characterized by Gromova and colleagues as c-kit+dim/EpCAM+/Sca1−/CD34−/CD45−4. This cell population can form acini and ducts in three-dimensional (3D) cultures and rescued LG function after transplantation into LG defective mice [
]. More recently, in 2020, Basova et al. traced these progenitor cells in postnatal mice LG and found that there are αSMA+/Krt5+/Krt14+/Sox9+/Sox10+ long-lived progenitor cells within the basal layer of the duct can promote epithelial regeneration in IL-1α injured mice [
]. These findings suggest that this progenitor population may be important for epithelial tissue repair and functional restoration in the LG during adulthood.
2 Signaling pathways between ECM, mesenchyme and epithelial cells during LG development
The importance of mesenchymal-epithelial interactions in LG development was first established in 2004, when it was observed that the embryonic LG continued to expand even though the rate of cell proliferation had decreased after the first branching [
. Currently, there are several signaling cascades that regulate LG morphogenesis events including cell expansion, differentiation, ductal formation, and ductal lumenization through multi-reciprocal actions between the parenchymal epithelium and the periocular mesenchyme (Table 2) [
. Fibroblast growth factor (FGF) families, particularly FGF7 and its closed homolog FGF10, are expressed in the periocular mesenchyme and play important roles in epithelial outgrowth and branching morphogenesis [
Transcription factors Runx1 to 3 are expressed in the lacrimal gland epithelium and are involved in regulation of gland morphogenesis and regeneration.
]. FGF10 is expressed in the mesenchyme throughout LG development while FGF7 does not have a primary role in LG organogenesis but may compensate for the absence of FGF10 signaling [
. Mesenchyme-derived FGF10 regulates the lacrimal cell fate by inducing Ras/mitogen-activated protein kinase (MAPK) signaling via transmembrane FGF receptor-2 (FGFR2) IIIb isoform on the conjunctival epithelium [
]. Studies have reported that FGF10 can also trigger downstream Sox10 molecular targets in the acinar epithelial cell via FGFR2 to promote the epithelial branching [
. Likewise, the impairment of LG-associated Sox10 reduction was also observed in Sox9 conditional knockout mice suggesting that Sox9 is located upstream of Sox10 and can regulate its expression during LG morphogenesis [
]. FGF10 also stimulates the epithelial migration and elongation through the ECM by cooperating with the protein barH‑like homeobox 2 (Barx2) as a functional network for activating MMPs particularly MMP2 and MMP956. Consequently, the MMPs induce ECM remodeling and promote FGF10 release from the ECM [
]. BMP7 appears to control LG mesenchymal proliferation and condensation mediated by the transcription factor Foxc157. Foxc1 is expressed in both LG mesenchyme and epithelial cells, but it does not promote epithelial proliferation [
]. In summary, the mesenchymal BMP7 signals can regulate LG branching morphogenesis by promoting mesenchymal proliferation/condensation and epithelial proliferation/elongation. It has been proposed that such mechanisms are suppressed by canonical Wnt signaling in the periorbital mesenchyme [
]. However, it does not restore LG function and it is not sustainable in the mid- to long-term since the biochemical composition of artificial tears is less complex and lacks similarities with the original tear composition [
]. Thus, tissue engineering and regenerative medicine have been considered as an approach to permanently rescue LG function and reverse DED. Next, we will describe emerging in vitro and in vivo cell-based technologies for drug discovery in DED and potential human transplantation techniques aiming to regenerate LG tissues.
3.1 Three-dimensional cultures to generate organoids for LG transplantation
Cell culture systems in 3D were established to fabricate cellularized matrices that retain morphology and a phenotype comparable with in vivo tissues [
]. In 3D co-cultures of rabbit conjunctival epithelium and LG, cells can retain their functional and secretory markers including aquaporin-5 (AQP5), lysozyme, and lactotransferrin mimicking in vivo conditions [
]. Furthermore, these properties make 3D cell culture suitable as a model for studying LG biology and investigate such 3D cell matrices in DED models to rescue LG secretion. Next, we will discuss platforms for 3D LG culture using scaffold-free and scaffold-based cultures, and bioprinting systems.
3.1.1 Scaffold-free 3D culture systems
Scaffold-free tissue engineering was first established in the 1980s by Rheinwald and Green [
]. This culture system offers a simple way to fabricate spheroid-like cultures, although it is difficult to consistently control the formation and shape of the spheroids. In LG tissue engineering, many scaffold-free methods were established including non-adherent surface culture system [
]. Based on our preliminary data, the lacrispheres can be fabricated in an ultra-low attachment culture plate after 48h of culture time (Fig. 3A). However, these lacrispheres display a large variation in size and shape which is a limitation of this conventional technique (Fig. 3B). Human lacrisphere cultures using this technique have also been reported [
. Human lacrispheres formed in an ultra-low attachment surfaces or cell-repellent surfaces provide the architecture and proliferation profile similar to native lacrimal cells [
]. Nevertheless, the lacrispheres could be maintained and propagated for only 3-4 weeks. Another group of researchers attempted to develop longer human lacrisphere cultures by using a non-adhesive micro-mold for 3D cell biofabrication [
], and subsequently, they formed a mini-gland-like structure and showed cell proliferation and differentiation capabilities. However, cells lost their differentiation potential after four passages [
]. This latter event was perhaps caused by the lack of mesenchymal-epithelial interactions during the expansion and differentiation. To overcome these issues, a scaffold-based culture was proposed as an in vitro 3D platform for tissue regeneration to mimic the phenotype and functional features of the native primary organ [
Fig. 3The size variation of lacrispheres cultured by non-adherent 3D cell culture system. (A) Murine and (B) porcine lacrispheres morphology shown by brightfield micrograph at 48h. (C) Lacrisphere size was measured from total 100 lacrispheres and express in a scatter plot. Scale bar: 100 μm.
Scaffold-based 3D culture systems focus on the application of cytocompatible biomaterials (natural or synthetic) to support the cell proliferation, differentiation and function and allow for nutrient and metabolite exchange. There are commercially available natural biomaterials for 3D cell culture including Matrigel, ECM gel, and Maxgel. These generally provide high biocompatibility, yet their undefined degradation rate and the existence of xenogenic components limit their human applications [
]. Collagen type I-based scaffolds were previously used to engineer the murine LG and Harderian gland by organ-germ method, demonstrating that the glands can be grown, differentiated and undergo branching morphogenesis [
]. Moreover, the bioengineered glands can be transplanted and restore the physiological functions of the LG in defected mice. This demonstrated that collagen type I plays an important role as ECM component for LG development and differentiation. Furthermore, the commercial Matrigel compound mainly composed of laminin, collagen IV, proteoglycans, matrix metalloproteinases, and specific growth factors, is widely used for LG 3D cell culture [
. Coating decellularized LG with Matrigel prior the recellularization improves and prolongs the viability and secretory function of LG cells within the decellularized scaffold [
]. Upon 3D co-culture of LG-derived MSC with epithelial cells derived from porcine LG and human dermal endothelial cells (hDMEC) derived from human foreskin, lacrispheres were formed in Matrigel and then transferred to decellularized matrices of porcine jejunum [
]. After this transfer, lacrispheres repopulated the mucosal crypt of the tissue, displayed specific markers of all three cell types (epithelial, endothelial and MSC), and responded to parasympathetic stimulation [
]. This is in agreement with previous reports, where the existing serum-free culture conditions can only support the 3D LG cell culture systems for a limited time [
]. More recent studies can overcome this challenge. Matrigel-based 3D culture systems supplemented with FGF10, EGF, ROCK Inhibitor Y-27632, and Wnt signaling activator Wnt3A prolonged the viability of LG cells which can be passaged up to 40 times every 7 days [
]. Another long-term serum free culture condition was then established in Matrigel-based 3D cultures supplemented with nicotinamide, TGF-β inhibitor A83, and BMPs inhibitor Noggin [
]. This later study showed that the LG organoid can recapitulate the native LG tissue, consistently increasing organoid size, and cells could be expanded until passage 1912. Overall, these studies demonstrated success in obtaining a reliable serum-free culture for long-term LG organoids.
A comprehensive profiling to determine LG heterogeneity in 3D cultures is essential in long-term LG 3D culture systems [
]. In addition, this study used mouse-based BME as a scaffold which provided a long-term LG organoid when supplemented with FGF10, r-spondin 3, noggin, TGF-β inhibitor A83-01, prostaglandin E2 (PGE2) and forskolin (FSK) [
]. The organoids could proliferate and expand for 40 passages. Withdrawal of small molecules induced the organoid differentiation leading to upregulation of functional proteins and receptors for important cholinergic neurotransmitters [
]. The study offered an experimental platform to test LG function. Overall, both mouse-based Matrigel and BME matrices can create LG organoids with polarized epithelial architecture, secretory function, and responsive neurostimulation. Main drawbacks of these 3D matrices consist of the following: (1) poor ability to control the final organoid size making its mature architecture unpredictable; (2) presence of xenogeneic components; (3) time-consuming biofabrication process; and (4) the poorly predictable matrix biodegradation. Novel 3D bioprinting platforms will be discussed next, as these can surpass certain limitations of the above-mentioned 3D culture systems by for example allowing cells to synthesize their own matrices and by offering more scale up solutions for more consistent organoid morphologies.
3.1.3 Bioprinting 3D culture systems for LG biofabrication
Recently, 3D bioprinting has been recognized as a promising tissue engineering technique for the biofabrication and recapitulation of complex biological tissues [
]. Herein, a novel 3D cell culture system, the magnetic 3D bioprinting (M3DB), will be discussed based on preliminary data. This system is xenogenic-free, highly scalable and reproducible 3D culture platform for exocrine glands like the salivary glands [
. This technique employs biocompatible magnetic nanoparticles (MNP) to tag cells and print them into a 3D spatially organized structure (according to the magnetic field used). This allows the labeled cells to freely assemble into controllable sizes within a shorter culture time (Fig. 4) [
]. Cells in suspension can be magnetized with the biocompatible NanoShuttleTM MNP, which will electrostatically bind to the cell membrane during a short incubation time [
Effect of lyso-phosphatidylcholine and Schnurri-3 on osteogenic transdifferentiation of vascular smooth muscle cells to calcifying vascular cells in 3D culture.
]. The magnetized cells can be printed into vascular or spheroid 3D structures by placing the culture plate on top of a ring type or dot type of magnet, respectively. Thus, this technology surpasses the limitations of other high-throughput platforms by being rapid, reproducible, scalable, and biocompatible (in vitro and in vivo) [
. In addition, these bioprinted organoids could rescue epithelial damage in ex vivo irradiated glands. Using NanoShuttleTM MNP for 3D bioprinting increases cell viability in the organoid and does not generate deleterious processes such as inflammation and oxidative stress [
]. Herein, our research group is displaying preliminary data to validate the M3DB culture system towards the biofabrication of lacrispheres from porcine primary LG cells (Fig. 5). LG cells at a density of 25,000 per well were tagged with 1 μl NanoShuttleTM MNP solution (Greiner Bio-One, Germany) and spatially arranged with the use of a drive with magnet dots in order to generate 3D LG spheroids. Magnetization of LG cells can fabricate the lacrispheres within 24h upon the placement of the magnetic drive (overnight), while the lacrispheres in non-magnetized LG cells can be observed later at 48h of culture. The lacrispheres were continuously maintained in the expansion medium until 120h (Fig. 5B and C), and lacrisphere size significantly increase from 48h to 96h and then became stable (Fig. 5B). At 96h (4 days) and 120h (5 days), lacrispheres produced from the M3DB platform showed a significant improvement in cell viability (more metabolically active cells) over non-magnetized LG cells (control) in the same culture medium (Fig. 5C).
Fig. 5Effects of magnetic 3D bioprinting on porcine LG cells. (A) Porcine LG cells at 96h with or without magnetization by NanoShuttle™ are imaged under a light microscope at 400X high power microscope field. (B) The lacrisphere size is measured at 48h, 96h, and 120h of incubation by using ImageJ analysis software (NIH, USA) (n=5). (C) Cell viability was determined by measuring ATP luciferase activity with CellTiter-Glo® 3D (Promega Corporation, Madison, WI, USA) cell viability assay (n=5). Unpaired and paired student t-tests were performed: *p<0.05 and ****p<0.0001. Scale bar: 100 mm.
]. Herein, we demonstrated that LG cells can be bio-assembled into a spheroid-like morphology by a M3DB platform. Whereas, a previous study, the ovarian carcinoma cell assembled by this same platform reflected their papillary morphology [
]. This indicated that M3DB system allows magnetized cells to assemble not only by the presence of a magnetic field, but also through the spontaneous self-arrangement ability of each cell type. Thus, assessing the histology, ultrastructure, and expression of epithelial and functional markers of M3DB-produced epithelial-like organs is critical and can be performed while validating the morphogenetic recapitulation to the native organ [
Our preliminary data demonstrates the first successful step towards the use of M3DB culture system for a consistent lacrisphere formation and expansion leading to LG organoid biofabrication. The transitioning of SG organoid to high-throughput application has been demonstrated by our previous study but not yet in the LG. Cell-cell interactions and lacrisphere production efficiency can also be quantified in a 96-well plate by measuring their diameter size from a 6,400dpi optical resolution image taken by a high-resolution Perfection V550 Epson flatbed scanner (Seiko Epson Corporation, Japan). Further studies are ongoing to determine whether the M3DB culture system can recapitulate the LG native function for high-throughput analysis, and to validate the LG organoid M3DB platform towards drug screening applications aimed for DED.
Thus, M3DB culture technology offers a consistent approach towards exocrine gland organoid biofabrication, particularly for the salivary glands [
and potentially for the LG as well. Hence, M3DB can be a feasible in vitro platform for future high-throughput drug screening in exocrine gland organoids. (Table 1)
Table 1Protein markers expressed in the different mature LG compartments.
Transcription factors Runx1 to 3 are expressed in the lacrimal gland epithelium and are involved in regulation of gland morphogenesis and regeneration.
Conditional disruption of mouse Klf5 results in defective eyelids with malformed meibomian glands, abnormal cornea and loss of conjunctival goblet cells.
The authors declared no conflicts of interest to the research, contribution, and publication of this review article.
This article is being reproduced in print post-publication in a sponsored print collection for distribution. The company sponsoring the print collection was not involved in the editorial selection or review of this article.
Acknowledgments
This project is funded by: National Research Council of Thailand (NRCT) (grant number NRCT5-RSA63001-12) to J.N.F. and supported by Faculty Research Grant (grant number DRF64014) Faculty of Dentistry Chulalongkorn University provided to J.N.F.. Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit is funded by the Ratchadaphiseksomphot Endowment Fund, Chulalongkorn University. T.R. is funded by a Postdoctoral Fellowship, Ratchadapisek Somphot Fund, Chulalongkorn University. Also, the authors are grateful to Somchai Yodsanga from the Department of Oral Pathology, Faculty of Dentistry, Chulalongkorn University for conducting histological slides and supporting histological techniques.
Detection of transforming growth factor-alpha mRNA and protein in rat lacrimal glands and characterization of transforming growth factor-alpha in human tears.
Transcription factors Runx1 to 3 are expressed in the lacrimal gland epithelium and are involved in regulation of gland morphogenesis and regeneration.
Effect of lyso-phosphatidylcholine and Schnurri-3 on osteogenic transdifferentiation of vascular smooth muscle cells to calcifying vascular cells in 3D culture.
Conditional disruption of mouse Klf5 results in defective eyelids with malformed meibomian glands, abnormal cornea and loss of conjunctival goblet cells.
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