Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
권호 표지
DOI QR코드

DOI QR Code

Mineralized Polysaccharide Transplantation Modules Supporting Human MSC Conversion into Osteogenic Cells and Osteoid Tissue in a Non-Union Defect

  • Ge, Qing (Division in Anatomy and Developmental Biology, Department of Oral Biology, Oral Science Research Center, BK21 PLUS Project, Yonsei University College of Dentistry) ;
  • Green, David William (Division in Anatomy and Developmental Biology, Department of Oral Biology, Oral Science Research Center, BK21 PLUS Project, Yonsei University College of Dentistry) ;
  • Lee, Dong-Joon (Division in Anatomy and Developmental Biology, Department of Oral Biology, Oral Science Research Center, BK21 PLUS Project, Yonsei University College of Dentistry) ;
  • Kim, Hyun-Yi (Division in Anatomy and Developmental Biology, Department of Oral Biology, Oral Science Research Center, BK21 PLUS Project, Yonsei University College of Dentistry) ;
  • Piao, Zhengguo (Department of Oral and Maxillofacial Surgery, Affiliated Stomatology Hospital of Guangzhou Medical University) ;
  • Lee, Jong-Min (Division in Anatomy and Developmental Biology, Department of Oral Biology, Oral Science Research Center, BK21 PLUS Project, Yonsei University College of Dentistry) ;
  • Jung, Han-Sung (Division in Anatomy and Developmental Biology, Department of Oral Biology, Oral Science Research Center, BK21 PLUS Project, Yonsei University College of Dentistry)
  • Received : 2017.12.28
  • Accepted : 2018.08.23
  • Published : 2018.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

Regenerative orthopedics needs significant devices to transplant human stem cells into damaged tissue and encourage automatic growth into replacements suitable for the human skeleton. Soft biomaterials have similarities in mechanical, structural and architectural properties to natural extracellular matrix (ECM), but often lack essential ECM molecules and signals. Here we engineer mineralized polysaccharide beads to transform MSCs into osteogenic cells and osteoid tissue for transplantation. Bone morphogenic proteins (BMP-2) and indispensable ECM proteins both directed differentiation inside alginate beads. Laminin and collagen IV basement membrane matrix proteins fixed and organized MSCs onto the alginate matrix, and BMP-2 drove differentiation, osteoid tissue self-assembly, and small-scale mineralization. Augmentation of alginate is necessary, and we showed that a few rationally selected small proteins from the basement membrane (BM) compartment of the ECM were sufficient to up-regulate cell expression of Runx-2 and osteocalcin for osteoid formation, resulting in Alizarin red-positive mineral nodules. More significantly, nested BMP-2 and BM beads added to a non-union skull defect, self-generated osteoid expressing osteopontin (OPN) and osteocalcin (OCN) in a chain along the defect, at only four weeks, establishing a framework for complete regeneration expected in 6 and 12 weeks. Alginate beads are beneficial surgical devices for transplanting therapeutic cells in programmed (by the ECM components and alginate-chitosan properties) reaction environments ideal for promoting bone tissue.

Keywords

E1BJB7_2018_v41n12_1016_f0001.png 이미지

Fig. 1. (A) A schematic diagram detailing the fabrication of the mineralized alginate-chitosan beads and analysis. Mineralized polysaccharide beads were cultured in static or dynamic conditions. Transplanted beads were cultured in static conditions for one day before implantation. In this study, we looked at cell osteoinduction in 3-types of alginate microenvironment differing in ECM components. We compared the effectiveness of BM and BMP-2 in alginate beads to differentiate MSCs into osteogenic cells and osteoid tissue. (B) Standardized bead shape, size, shell structure and internal architecture of alginate beads; (i) Single alginate bead measuring 1 mm in diameter; (ii) light microscope image in polarized light to highlight the semi-crystalline structure of the calcium phosphate/ chitosan shell surrounding the alginate core (white arrow). (iii) Histological thin section of a single bead to demonstrate the internal architecture and external morphology; (iv) a single H & E stained sectioned bead to show the distribution of a fully loaded capsule for cell culture; (C) a diagrammatic representation of the four different types of augmented bead environments used in the study; (D) H&E staining of capsules containing hMSCs at different time points in the four individual bead microenvironments (BMP-2 added, BM proteins added, guest, added and normal alginate) to show the changes in cell distribution, density and proliferation. hMSCs inside BMP-2 beads, guest BMP-2 beads, and BM beads increased cell numbers compared to alginate beads. Also, in control group, hMSCs inside alginate beads distributed well separated and did not form clusters compared experiment groups. (E) hMSC numbers increased significantly in the guest BMP-2 beads and the BM beads compared to the MSCs beads and the BMP-2 beads. (* = Guest BMP-2 bead) (Scale bar = 200 μm).

E1BJB7_2018_v41n12_1016_f0002.png 이미지

Fig. 2. (A) Immunolabeling results to identify osteogenic related proteins contrasting between the hMSCs beads, BMP-2 beads, guest BMP-2 beads and BM beads. After two weeks culture, collagen I expression was stronger in the BMP-2 added group and BM added group compared to control group and guest BMP-2 group. The CD 90 expression was absent in all groups after two weeks culture. Thus, all encapsulating hMSCs lost stemness after two weeks culture. MSCs expressed Osterix, the marker for pre-osteoblasts, in alginate, and at higher levels in the BMP-2 and BM beads. Runx-2 staining was positive in the BMP-2 added, guest BMP-2 added and the BM beads, and absent in the control group. The osteocalcin cell expression in the BM beads compared with alginate beads, BMP-2 beads and nested BMP-2 beads; (scale bar = 400 μm); (B) percentages of positively versus negatively stained cells for Runx-2, osteocalcin, and CD90 to show the maturation phase of the hMSCs and their conversion into osteogenic lineage cells. (* = Guest BMP-2 bead). (C) Masson’s trichrome staining shows the in vivo generated structures from the four different kinds of alginate bead transplants within a mouse cranial bone defect. We find a difference between the MSC control beads inside the defect, and the added BMP-2 and BM alginate beads, which both show a narrowing of the pockets and contain osteoid tissue. More significantly, we see that the beads harboring a BMP-2 guest bead generated an extensive series of osteoid pockets across the defect region (C) accompanied by an even tighter narrowing of the regenerated tissue inside the defect. The IHC staining for OCN and OPN is negative among the control MSC beads. In contrast to the MSC beads, BMP-2 and BM laden beads were OPN and OCN positive. The spaces represent the alginate. Among the defects containing nested alginate beads, the staining for OCN and OPN was larger, more widespread and associated with the osteoid pockets (C; * = Guest BMP-2 bead; b

References

  1. Augst, A.D., Kong, H.J., and Mooney, D.J. (2006). Alginate hydrogels as biomaterials. Macromol. Biosci. 7, 623-633.
  2. Bouhadir, K.H., Lee, K.Y., Alsberg, E., Damm, K.L., Anderson, K.W., and Mooney, D.J. (2001). Degradation of partially oxidized alginate and its potential application for tissue engineering. Biotechnol. Prog. 17, 945-950. https://doi.org/10.1021/bp010070p
  3. Burdick, J.A., Mauck, R.L., and Gerecht, S. (2016). To serve and protect: hydrogels to improve stem cell-based therapies. Cell. Stem. Cell. 18, 13-15. https://doi.org/10.1016/j.stem.2015.12.004
  4. Caccavo, D., Cascone, S., Lamberti, G., and Barba, A.A. (2018). Hydrogels: experimental characterization and mathematical modelling of their mechanical and diffusive behaviour. Chem. Soc. Rev. 47, 2357. https://doi.org/10.1039/C7CS00638A
  5. Chaudhuri, O., Koshy, S.T., Branco da Cunha, C., Shin, J.W., Verbeke, C.S., Allison, K.H., and Mooney, D.J. (2014). Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium. Nat. Mater. 13, 970-978. https://doi.org/10.1038/nmat4009
  6. Collier, J.H., and Segura, T. (2011). Evolving the use of peptides as components of biomaterials. Biomaterials 32, 4198-4204. https://doi.org/10.1016/j.biomaterials.2011.02.030
  7. Crane, G.M., Ishaug, S.L., and Mikos, A.G., (1995) Bone tissue engineering. Nat. Med. 1, 1322-1324. https://doi.org/10.1038/nm1295-1322
  8. Fonseca, K.B., Gomes, D.B., Lee, K., Santos, S.G., Sousa, A., Silva, E.A., Mooney, D.J., Granja, P.L., and Barrias, C.C. (2014). Injectable MMP-sensitive alginate hydrogels as hMSC delivery systems. Biomacromolecules 15, 380-390. https://doi.org/10.1021/bm4016495
  9. Engler, A.J., Sen, S., Sweeney, H.L., and Discher DE. (2006). Matrix elasticity directs stem cell lineage specification. Cell 126, 677-689. https://doi.org/10.1016/j.cell.2006.06.044
  10. Gong, T., Xie, J., Liao, J., Zhang, T., Lin, S., and Lin, Y. (2015). Nanomaterials and bone regeneration. Bone Res. 3, 15029. https://doi.org/10.1038/boneres.2015.29
  11. Green, D.W., Leveque, I., Walsh, D., Howard, D., Yang, X.B., Partridge, K.A., Mann, S., and Oreffo, R.O.C. (2005). Biomineralized polysaccharide capsules for encapsulation, organization and delivery of human cell types and growth factors. Adv. Funct. Mater. 15, 917-923. https://doi.org/10.1002/adfm.200400322
  12. Heldin, C.H., Miyazono, K., and ten Dijke, P. (1997). TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature 390, 465-471. https://doi.org/10.1038/37284
  13. Jang, K.I., Chung, H.U., Xu, S., Lee, C.H., Luan, H., Jeong, J., Cheng, H., Kim, G.T., Han, S.Y., Lee, J.W., et al. (2015). Soft network composite materials with deterministic and bio-inspired designs. Nat. Commun. 6, 6566. https://doi.org/10.1038/ncomms7566
  14. Lee, K.Y., and Mooney, D.J. (2012). Alginate: properties and biomedical applications. Prog. Polym. Sci. 37, 106-126. https://doi.org/10.1016/j.progpolymsci.2011.06.003
  15. Luo, Z., Yang, Y., Deng, Y., Sun, Y., Yang, H., and Wei, S. (2016). Peptide-incorporated 3D porous alginate scaffolds with enhanced osteogenesis for bone tissue engineering. Colloids. Surf. B Biointerfaces 143, 243-251. https://doi.org/10.1016/j.colsurfb.2016.03.047
  16. Lv, H., Li, L., Sun, M., Zhang, Y., Chen, L., Rong, Y., and Li, Y. (2005). Mechanism of regulation of stem cell differentiation by matrix stiffness. Stem. Cell Res. Ther. 6, 103.
  17. Ma, W., Tavakoli, T., Derby, E., Serebryakova, Y., Rao, M.S., and Mattson, M.P. (2008). Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells. BMC Dev. Biol. 8, 90. https://doi.org/10.1186/1471-213X-8-90
  18. Moshaverinia, A., Ansari, S., Chen, C., Xu, X., Akiyama, K., Snead, M.L., Zadeh, H.H., and Shi, S. (2013). Co-encapsulation of anti-BMP2 monoclonal antibody and mesenchymal stem cells in alginate microspheres for bone tissue engineering. Biomaterials 34, 6572-6579. https://doi.org/10.1016/j.biomaterials.2013.05.048
  19. Mao, A.S., Shin, J.W., Utech, S., Wang, H., Uzun, O., Li, W., Cooper, M., Hu, Y., Zhang, L., Weitz, D.A., et al. (2017). Deterministic encapsulation of single cells in thin tunable microgels for niche modelling and therapeutic delivery. Nat. Mater. 16, 236-243. https://doi.org/10.1038/nmat4781
  20. Maxhimer, J.B., Bradley, J.P., and Lee, J.C. (2015). Signaling pathways in osteogenesis and osteoclastogenesis: Lessons from cranial sutures and applications to regenerative medicine. Genes. Dis. 2, 57-68. https://doi.org/10.1016/j.gendis.2014.12.004
  21. Murry, C.E., and Keller, G., (2008). Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132, 661-680. https://doi.org/10.1016/j.cell.2008.02.008
  22. Muzzarelli, R.A., El Mehtedi, M., Bottegoni, C., Aquili, A., and Gigante, A. (2015). Genipin-crosslinked chitosan gels and scaffolds for tissue engineering and regeneration of cartilage and bone. Mar. Drugs 13, 7314-7338. https://doi.org/10.3390/md13127068
  23. Perez, R.A., Kim, M., Kim, T.H., Kim, J.H., Lee, J.H., Park, J.H., Knowles, J.C., and Kim, H.W. (2014). Utilizing core-shell fibrous collagen-alginate hydrogel cell delivery system for bone tissue engineering. Tissue Eng. Part A 20, 103-114. https://doi.org/10.1089/ten.tea.2013.0198
  24. Place, E.S., Rojo, L., Gentleman, E., Sardinha, J.P., and Stevens, M.M. (2011). Strontium- and zinc-alginate hydrogels for bone tissue engineering. Tissue Eng. Part A 17, 2713-2722. https://doi.org/10.1089/ten.tea.2011.0059
  25. Pound, J.C., Green, D.W., Chaudhuri, J.B., Mann, S., Roach, H.I., and Oreffo, R.O.C. (2006). Strategies to promote chondrogenesis and osteogenesis from human bone marrow cells and articular chondrocytes encapsulated in polysaccharide templates. Tissue Eng. 12, 2789-2799. https://doi.org/10.1089/ten.2006.12.2789
  26. Rowley, J.A., Madlambayan, G., and Mooney, D.J. (1999). Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 20, 45-53. https://doi.org/10.1016/S0142-9612(98)00107-0
  27. Sajesh, K.M., Jayakumar, R., Nair, S.V., and Chennazhi, K.P. (2013). Biocompatible conducting chitosan/polypyrrole-alginate composite scaffold for bone tissue engineering. Int. J. Biol. Macromol. 62, 465-471. https://doi.org/10.1016/j.ijbiomac.2013.09.028
  28. Shah, K. (2013). Encapsulated stem cells for cancer therapy. Biomatter. 3, pii: e24278.
  29. Shi, Y., and Massague, J. (2003). Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 113, 685-700. https://doi.org/10.1016/S0092-8674(03)00432-X
  30. Sowjanya, J.A., Singh, J., Mohita, T, Sarvanan, S., Moorthi, A., Srinivasan, N., and Selvamurugan, N. (2013). Biocomposite scaffolds containing chitosan/alginate/nano-silica for bone tissue engineering. Colloids and Surfaces B Biointerfaces 1, 294-300.
  31. Stevens, M.M. (2008). Biomaterials for bone tissue engineering. Mats. Today 11, 18-25.
  32. Swioklo, S., Constantinescu, A., and Connon, C.J. (2016). Alginateencapsulation for the improved hypothermic preservation of human adipose-derived stem cells. Stem. Cells Transl. Med. 5, 339-349. https://doi.org/10.5966/sctm.2015-0131
  33. Xia, Y., Mei, F., Duan, Y., Gao, Y., Xiong, Z., Zhang, T., and Zhang, H. (2013). Bone tissue engineering using bone marrow stromal cells and an injectable sodium alginate/gelatin scaffold. Journal of Biomedical Materials Research Part A. 109, 294-300.
  34. Yang, X.B., Whitaker, M.J., Sebald, W., Clarke, N., Howdle, S.M., Shakesheff, K.M., and Oreffo. R.O. (2004). Human osteoprogenitor bone formation using encapsulated bone morphogenetic protein 2 in porous polymer scaffolds. Tissue Eng. 10, 1037-1045. https://doi.org/10.1089/ten.2004.10.1037

Cited by

  1. Oral Bone Tissue Regeneration: Mesenchymal Stem Cells, Secretome, and Biomaterials vol.22, pp.10, 2021, https://doi.org/10.3390/ijms22105236
  2. BMP-2 Delivery through Liposomes in Bone Regeneration vol.12, pp.3, 2022, https://doi.org/10.3390/app12031373