Biomimetic biomaterials for protein and gene delivery for salivary gland regeneration

(NIDCR, 5R01DE022971-07 MPI grant with Dr. Olga Baker)

Stelios T. Andreadis Lab

Introduction & Significance

The Stelios Andreadis laboratory has developed novel methods to deliver growth factors, genes and recombinant viruses for multiple applications ranging from wound healing, vascular tissue engineering, lentiviral arrays and gland regeneration.

For example, they developed a strategy to deliver keratinocyte growth factor (KGF) in excisional wounds using fibrin hydrogels 1. To this end, the lab took advantage of known peptide sequences, which are recognized by factor XIII, a transglutaminase that attaches several molecules e.g., fibronectin, plasminogen in fibrin clots during wound healing. They used genetic engineering to generate a peptide-TGF-b1 fusion protein in order to immobilize it with fibrin hydrogels and improve the function of vascular grafts 2. The lab found that immobilized TGF-b1 prolonged downstream signaling leading to significant enhancement of vascular contractility in 3D tissues, as compared to soluble TGF-b1. They also designed matrices for delivery of plasmid DNA and recombinant lentivirus from these gels for applications in transfection microarrays 3 or lentiviral microarrays 4. 

Salivary Gland Regeneration: According to the American Cancer Society, more than 60,000 people will develop head and neck cancer this year and those patients must receive radiation therapy to survive. This treatment regularly destroys the salivary glands (SG), leading to loss of secretory function which is typically permanent. Hyposalivation causes an altered sense of taste, difficulty in speech and swallowing food as well as oral mucositis. Current treatments remain largely ineffective, with therapeutic interventions limited to use of saliva substitutes with modest effectiveness and medications that provide only temporary relief. In light of the unmet clinical need, development of alternative treatments to restore SG functioning is essential. This is the goal of a NIDCR funded project in collaboration with Olga Baker. 

Research Findings

Zoom image: Fig. 1(A): Schematic depicting SG structure and key factors in SG organogenesis. Confocal images of SG organoids on day 32 of human iPSC differentiation.  4.1.

Fig. 1(A): Schematic depicting SG structure and key factors in SG organogenesis. Confocal images of SG organoids on day 32 of human iPSC differentiation. 

Zoom image: Fig1 (B): Top view 4.2.

Fig1 (B): Top view

Zoom image: Fig 1(C): Depth map. The cells were stained for Mist-1 (red) the key transcription factor of acinar differentiation; actin phalloidin (green) and TO-Pro-III (blue).   4.3.

Fig 1(C): Depth map.

The cells were stained for Mist-1 (red) the key transcription factor of acinar differentiation; actin phalloidin (green) and TO-Pro-III (blue).  

In response to the challenges, the lab have been working for the past seven years to engineer hydrogels that enable three-dimensional (3D) cell assembly into organoids with functional lumen in vitro and salivary gland regeneration in vivo 5-12 . Specifically, our work to date has shown the following:

i) engineering mouse and human SG organoids with function lumens that secrete saliva 6;

ii) identification of the A99 peptide corresponding to the  a1 chain from Laminin-1 (L1) as well as the YIGSR peptide corresponding to the β1 chain from L1, both of which promote formation of functional 3D salivary organoids with mature lumens when immobilized into fibrin hydrogels (FH) (L1p-FH) 9;

iii) enhancement of SG wound healing and secretory function by using L1p-FH in vivo 10,11;

iv) enhancement of branching morphogenesis by controlling the mode of Fibroblast Growth Factor 7 (FGF7) and FGF10 presentation within 3D FH in vitro to control SG cell migration and proliferation 12;  

vi) production of recombinant fusion proteins, containing a FXIII recognition sequence catalyzing their enzymatic conjugation in FH during polymerization to enhance vascularization and functional regeneration of wounded SG in vivo, as evidenced by increased saliva secretion 8.

Future Work

Current and future work funded by a R01 from NIDCR aims at improving acinar differentiation and innervation of SG after radiation treatment. As well as employing cues from developmental biology and developing novel hydrogels with dynamic bonds to engineer SG organoids from human iPSC (Fig. 1). 

Students of the Project

Post-doc: Mohamed Alaa Mohamed, Ph.D.

Graduate students: Ronel Samuel, Laura Sherwood

Collaborators: Olga Baker (University of Missouri, Biochemistry)

References

  1. Geer, D.J., Swartz, D.D. & Andreadis, S.T. Biomimetic delivery of keratinocyte growth factor upon cellular demand for accelerated wound healing in vitro and in vivo. Am J Pathol 167, 1575-1586 (2005).
  2. Liang, M.S. & Andreadis, S.T. Engineering fibrin-binding TGF-beta1 for sustained signaling and contractile function of MSC based vascular constructs. Biomaterials 32, 8684-8693 (2011).
  3. Lei, P., Padmashali, R.M. & Andreadis, S.T. Cell-controlled and spatially arrayed gene delivery from fibrin hydrogels. Biomaterials 30, 3790-3799 (2009).
  4. Padmashali, R.M. & Andreadis, S.T. Engineering fibrinogen-binding VSV-G envelope for spatially- and cell-controlled lentivirus delivery through fibrin hydrogels. Biomaterials 32, 3330-3339 (2011).
  5. Raut, S.D., Lei, P., Padmashali, R.M. & Andreadis, S.T. Fibrin-mediated lentivirus gene transfer: implications for lentivirus microarrays. J Control Release 144, 213-220 (2010).
  6. Brown, C.T., Nam, K., Zhang, Y., Qiu, Y., Dean, S.M., Dos Santos, H.T., Lei, P., Andreadis, S.T. & Baker, O.J. Sex-dependent Regeneration Patterns in Mouse Submandibular Glands. J Histochem Cytochem 68, 305-318 (2020).
  7. Maruyama, C.L., Leigh, N.J., Nelson, J.W., McCall, A.D., Mellas, R.E., Lei, P., Andreadis, S.T. & Baker, O.J. Stem Cell-Soluble Signals Enhance Multilumen Formation in SMG Cell Clusters. J Dent Res 94, 1610-1617 (2015).
  8. McCall, A.D., Nelson, J.W., Leigh, N.J., Duffey, M.E., Lei, P., Andreadis, S.T. & Baker, O.J. Growth factors polymerized within fibrin hydrogel promote amylase production in parotid cells. Tissue Eng Part A 19, 2215-2225 (2013).
  9. Nam, K., Dean, S.M., Brown, C.T., Smith, R.J., Jr., Lei, P., Andreadis, S.T. & Baker, O.J. Synergistic effects of laminin-1 peptides, VEGF and FGF9 on salivary gland regeneration. Acta Biomater 91, 186-194 (2019).
  10. Nam, K., Jones, J.P., Lei, P., Andreadis, S.T. & Baker, O.J. Laminin-111 Peptides Conjugated to Fibrin Hydrogels Promote Formation of Lumen Containing Parotid Gland Cell Clusters. Biomacromolecules 17, 2293-2301 (2016).
  11. Nam, K., Maruyama, C.L., Wang, C.S., Trump, B.G., Lei, P., Andreadis, S.T. & Baker, O.J. Laminin-111-derived peptide conjugated fibrin hydrogel restores salivary gland function. PloS one 12, e0187069 (2017).
  12. Nam, K., Wang, C.S., Maruyama, C.L.M., Lei, P., Andreadis, S.T. & Baker, O.J. L1 Peptide-Conjugated Fibrin Hydrogels Promote Salivary Gland Regeneration. J Dent Res 96, 798-806 (2017).
  13.  Samuel, R.Z., Lei, P., Nam, K., Baker, O.J. & Andreadis, S.T. Engineering the mode of morphogenetic signal presentation to promote branching from salivary gland spheroids in 3D hydrogels. Acta Biomater 105, 121-130 (2020).