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«J. Funct. Biomater. 2015, 6, 634-649; doi:10.3390/jfb6030634 OPEN ACCESS Journal of Functional Biomaterials ISSN 2079-4983 ...»

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We investigated whether our organ germ method could regenerate a bioengineered lacrimal gland and restore its physiological function. The bioengineered lacrimal gland germ, which was reconstituted using the epithelial and mesenchymal cells from the lacrimal gland germ of an ED 16.5 mouse, successfully developed branching morphogenesis followed by stalk elongation and cleft formation in organ culture in vitro. Bioengineered harderian gland germs were also regenerated via the organ germ method (Figure 3a).

J. Funct. Biomater. 2015, 6 640 Figure 3. Functional lacrimal gland regeneration via bioengineered lacrimal gland germ transplant: (a) Phase-contrast images of the bioengineered lacrimal gland germ (upper line) and bioengineered harderian gland germ (lower line) development. Scale bar, 100 µm;

(b) Photographs of the bioengineered lacrimal gland germ after transplanting into a mouse with the extra-orbital lacrimal gland removed (arrowhead) (left panel; Scale bar, 1 mm).

At 30 days after transplantation, the bioengineered lacrimal gland was successfully engrafted and developed (center; Scale bar, 500 µm). The hematoxylin-eosin(H.E.) staining revealed that the bioengineered lacrimal gland achieved a mature secretory gland structure including acini (white arrowhead) and duct (black arrowhead) (right; Scale bar, 50 µm);

(c) Histological analysis of the duct connection between the bioengineered lacrimal gland and recipient lacrimal excretory duct. Bioengineered lacrimal glands regenerated using DsRed transgenic mouse-derived epithelial cells (red) and normal mouse-derived mesenchymal cells developed with the correct duct association in the recipient mouse (arrowhead). Fluorescein isothiocyanate (FITC) -gelatin (green), which was injected from the recipient lacrimal excretory duct, could successfully reach the bioengineered lacrimal gland. 4',6-diamidino-2-phenylindole (DAPI; blue) and the excretory duct (dotted line) are shown. Scale bars, 100 µm; (d) Immunohistochemical analysis of the bioengineered lacrimal gland after transplantation. Aquaporin-5 is red and E-cadherin is green in the left panel.

Calponin is red and E-cadherin is green in the center panel. Calponin is red, neurofilament-H (NF-H) is green, and DAPI is blue in the right panel. Scale bars, 50 µm. Modified and reprinted from Hirayama et al. [23].

7.1. Engraftment of Bioengineered Lacrimal Gland Germ with Duct Association

A duct association between the bioengineered lacrimal gland and the mouse receiving the ocular surface discharge is required for tear secretion from the bioengineered lacrimal gland. The bioengineered lacrimal gland germ and the bioengineered harderian gland germ were successfully engrafted to a mouse from which an extra-orbital lacrimal gland had been removed, and the bioengineered lacrimal gland duct was connected to the recipient lacrimal excretory duct using our thread-guided transplant method (Figure 3b,c). After the transplant, the bioengineered lacrimal and harderian glands formed the appropriate histo-architecture, including acini-expressing aquaporin 5 and myoepithelial cells, duct, and nerve fibers, by reproducing the developmental process that occurs during organogenesis (Figure 3c,d).

Thus, the bioengineered lacrimal gland and harderian gland can develop after in vivo orthotopic or ectopic transplantation.

7.2. Tear Secretion Ability of the Bioengineered Lacrimal Gland

Reconstituting neural pathways between the bioengineered lacrimal gland and the recipient’s neural system is important to protect the ocular surface via restored tear secretion [99–101]. Tearing resulting from a cooling stimulation at the ocular surface that is activated via corneal thermoreceptors and is a representative neural pathway for lacrimal gland function (Figure 4a) [102,103]. We demonstrated that the bioengineered lacrimal gland could secret tears in cooperation with peripheral tissues, including neural systems, because the tear secretion volume from the bioengineered lacrimal gland increased after J. Funct. Biomater. 2015, 6 641 ocular surface cooling stimulation. Tear components secreted from acini in the lacrimal and harderian glands, such as lactoferrin and lipids, respectively, are essential for physiological tear functions such as increased stability, wound healing, and anti-bacterial effects [104–109]. Current therapies for severe lacrimal gland dysfunction include medical treatments such as albumin eye drops and autologous serum eye drops that attempt to substitute tear protein function [54,110–114]. We have shown that tears from the bioengineered lacrimal gland contained major tear proteins, including lactoferrin. In addition, the lipid concentration increased significantly in tears from the bioengineered harderian gland. These results indicated that these bioengineered glands can produce appropriate tear components. Functional replacements of the bioengineered lacrimal gland would be an attractive strategy for treating severe dry eye disease.

7.3. Ocular Surface Protection Effect by the Bioengineered Lacrimal Gland

Protecting the ocular surface is the main purpose of using the bioengineered lacrimal gland to restore lacrimal gland function. Punctate staining of the impaired area on the ocular surface [115,116] and corneal epithelial changes including thinning and stromal fibroblast activation [117,118] were observed in a mouse with an extra-orbital lacrimal gland defect, which mimics the corneal epithelial damage caused by lacrimal gland dysfunction. However, these changes were prevented using a bioengineered lacrimal gland (Figure 4b,c). Our results indicate that the bioengineered lacrimal gland can develop and provide sufficient function to maintain a healthy ocular surface.

J. Funct. Biomater. 2015, 6 642

Figure 4. Tear secretion and ocular surface protection for the bioengineered lacrimal gland:

(a) Schematic representation of the neural reflex loop for tear secretion. Cooling on the ocular surface stimulates tear secretion from the lacrimal gland via the central nervous system; (b) Representative images of the corneal surface of a normal lacrimal gland (upper), a lacrimal gland-defect mouse (center), and a bioengineered lacrimal gland–engrafted mouse (lower). The punctate staining area by fluorescein showed impaired area on corneal surface.

Scale bar, 1 mm. Modified and reprinted from Hirayama et al. [23]; (c) Representative microscopic images of the corneal epithelium, including a normal mouse (upper), lacrimal gland–defective mouse (center), and bioengineered lacrimal gland–transplanted mouse (lower) are shown. Chronic dry eye status in lacrimal gland–defective mouse induced corneal thickening as shown in the center panel, whereas these changes were not observed in the bioengineered lacrimal gland-transplanted mouse. Scale bars, 25 μm. Modified and reprinted from Hirayama et al. [23].

8. Conclusions and Future Directions

Bioengineered lacrimal gland germs exhibit appropriate physiological functions, such as tear secretion, in response to nervous stimulation and ocular surface protection. These studies are a proof-of-concept for bioengineered organs that can functionally restore the lacrimal gland. Our bioengineered organ regeneration concept, which has also been applied to salivary gland regeneration [98], provides substantial advances for regenerative therapies for dry eye disease and xerostomia. Epithelial and mesenchymal stem cells, which have organ-inductive potential for bioengineered organs, have not been reported in adult tissues. To realize the future practical clinical applications of organ replacement regenerative therapy, studies to develop technologies for organ regeneration, such as investigations of available cell sources (e.g., pluripotent stem cells represented as embryonic stem cells and induced pluripotent stem cells) and the efficacy of disease models (e.g., Sjogren syndrome and Stevens-Johnson syndrome) for these methods, technical procedures for culture methods to create bioengineered organs, and appropriate transplantation methods for human patients, are required. Bioengineered organ regenerative therapy is expected to be an essential therapeutic strategy for the next generation of regenerative medicine.


This work was partially supported by a Grant-in-Aid for Scientific Research A (to T.T.) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. T.T. has already acquired patents related to this article’s subject of organ regeneration, and they have been applied for in other countries.

Conflicts of Interest

–  –  –


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