A popular approach to make neocartilage in vitro is to immobilize cells with chondrogenic potential in hydrogels. However, functional cartilage cannot be obtained by control of cells only, as function of cartilage is largely dictated by architecture of extracellular matrix (ECM). Therefore, characterization of the cells, coupled with structural and biochemical characterization of ECM, is essential in understanding neocartilage assembly to create functional implants in vitro. We focused on mesenchymal stem cells (MSC) immobilized in alginate hydrogels, and used immunohistochemistry (IHC) and gene expression analysis combined with advanced microscopy techniques to describe properties of cells and distribution and organization of the forming ECM. In particular, we used second harmonic generation (SHG) microscopy and focused ion beam/scanning electron microscopy (FIB/SEM) to study distribution and assembly of collagen. Samples with low cell seeding density (1e7 MSC/ml) showed type II collagen molecules distributed evenly through the hydrogel. However, SHG microscopy clearly indicated only pericellular localization of assembled fibrils. Their distribution was improved in hydrogels seeded with 5e7 MSC/ml. In those samples, FIB/SEM with nm resolution was used to visualize distribution of collagen fibrils in a three dimensional network extending from the pericellular region into the ECM. In addition, distribution of enzymes involved in procollagen processing were investigated in the alginate hydrogel by IHC. It was discovered that, at high cell seeding density, procollagen processing and fibril assembly was also occurring far away from the cell surface, indicating sufficient transport of procollagen and enzymes in the intercellular space. At lower cell seeding density, the concentration of enzymes involved in procollagen processing was presumably too low. FIB/SEM and SHG microscopy combined with IHC localization of specific proteins were shown to provide meaningful insight into ECM assembly of neocartilage, which will lead to better understanding of cartilage formation and development of new tissue engineering strategies.
References
[1]
Huey DJ, Hu JC, Athanasiou KA (2012) Unlike bone, cartilage regeneration remains elusive. Science 338: 917–921. doi: 10.1126/science.1222454
[2]
Balakrishnan B, Banerjee R (2011) Biopolymer-based hydrogels for cartilage tissue engineering. Chem Rev 111: 4453–4474. doi: 10.1021/cr100123h
[3]
Erickson IE, Kestle SR, Zellars KH, Farrell MJ, Kim M, et al. (2012) High mesenchymal stem cell seeding densities in hyaluronic acid hydrogels produce engineered cartilage with native tissue properties. Acta Biomater 8: 3027–3034. doi: 10.1016/j.actbio.2012.04.033
[4]
Guo Y, Yuan T, Xiao Z, Tang P, Xiao Y, et al. (2012) Hydrogels of collagen/chondroitin sulfate/hyaluronan interpenetrating polymer network for cartilage tissue engineering. J Mater Sci: Mater Med 23: 2267–2279. doi: 10.1007/s10856-012-4684-5
[5]
Nicodemus GD, Skaalure SC, Bryant SJ (2011) Gel structure has an impact on pericellular and extracellular matrix deposition, which subsequently alters metabolic activities in chondrocyte-laden PEG hydrogels. Acta Biomater 7: 492–504. doi: 10.1016/j.actbio.2010.08.021
Chung C, Beecham M, Mauck RL, Burdick JA (2009) The influence of degradation characteristics of hyaluronic acid hydrogels on in vitro neocartilage formation by mesenchymal stem cells. Biomaterials 30: 4287–4296. doi: 10.1016/j.biomaterials.2009.04.040
[8]
Wong M, Siegrist M, Wang XH, Hunziker E (2001) Development of mechanically stable alginate/chondrocyte constructs: effects of guluronic acid content and matrix synthesis. J Orthop Res 19: 493–499. doi: 10.1016/s0736-0266(00)90023-8
[9]
Anderson SB, Lin C-C, Kuntzler DV, Anseth KS (2011) The performance of human mesenchymal stem cells encapsulated in cell-degradable polymer-peptide hydrogels. Biomaterials 32: 3564–3574. doi: 10.1016/j.biomaterials.2011.01.064
[10]
Klein TJ, Rizzi SC, Schrobback K, Reichert JC, Jeon JE, et al. (2010) Long-term effects of hydrogel properties on human chondrocyte behavior. Soft Matter 6: 5175–5183. doi: 10.1039/c0sm00229a
[11]
Kundu J, Shim J-H, Jang J, Kim S-W and Cho D-W (2013) An additive manufacturing-based PCL-alginate-chondrocyte bioprinted scaffold for cartilage tissue engineering. J Tissue Eng Regen Med DOI: 10.1002/term.1682.
[12]
Canty EG, Kadler KE (2005) Procollagen trafficking, processing and fibrillogenesis. J Cell Sci 118: 1341–1353. doi: 10.1242/jcs.01731
[13]
Prockop DJ, Sieron AL, Li SW (1998) Procollagen N-proteinase and procollagen C-proteinase. Two unusual metalloproteinases that are essential for procollagen processing probably have important roles in development and cell signaling. Matrix Biol 16: 399–408. doi: 10.1016/s0945-053x(98)90013-0
[14]
Kalamajski S, Oldberg A (2010) The role of small leucine-rich proteoglycans in collagen fibrillogenesis. Matrix Biol 29: 248–253. doi: 10.1016/j.matbio.2010.01.001
[15]
Eyre D (2002) Collagen of articular cartilage. Arthritis Res 4: 30–35.
[16]
Bouhadir KH, Lee KY, Alsberg E, Damm KL, Anderson KW, et al. (2001) Degradation of partially oxidized alginate and its potential application for tissue engineering. Biotechnol Prog 17: 945–950. doi: 10.1021/bp010070p
[17]
Rice MA, Anseth KS (2007) Controlling cartilaginous matrix evolution in hydrogels with degradation triggered by exogenous addition of an enzyme. Tissue Eng 13: 683–691. doi: 10.1089/ten.2006.0142
[18]
Ashton RS, Banerjee A, Punyani S, Schaffer DV, Kane RS (2007) Scaffolds based on degradable alginate hydrogels and poly(lactide-co-glycolide) microspheres for stem cell culture. Biomaterials 28: 5518–5525. doi: 10.1016/j.biomaterials.2007.08.038
[19]
Ng KW, Kugler LE, Doty SB, Ateshian GA, Hung CT (2009) Scaffold degradation elevates the collagen content and dynamic compressive modulus in engineered articular cartilage. Osteoarthritis Cartilage 17: 220–227. doi: 10.1016/j.joca.2008.06.013
[20]
Boontheekul T, Kong H-J, Mooney DJ (2005) Controlling alginate gel degradation utilizing partial oxidation and bimodal molecular weight distribution. Biomaterials 26: 2455–2465. doi: 10.1016/j.biomaterials.2004.06.044
[21]
Melvik JE, Bjornstad J and Svendsen T (2011) Delayed Self-Gelling Alginate Systems and Uses Thereof. US Patent Application 20110053886.
[22]
Knott G, Marchman H, Wall D, Lich B (2008) Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling. J Neurosci 28: 2959–2964. doi: 10.1523/jneurosci.3189-07.2008
[23]
Bushby AJ, P’ng KMY, Young RD, Pinali C, Knupp C, et al. (2011) Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy. Nat Protoc 6: 845–858. doi: 10.1038/nprot.2011.332
[24]
Lee S, Solow-Cordero DE, Kessler E, Takahara K, Greenspan DS (1997) Transforming growth factor-beta regulation of bone morphogenetic protein-1/procollagen C-proteinase and related proteins in fibrogenic cells and keratinocytes. J Biol Chem 272: 19059–19066. doi: 10.1074/jbc.272.30.19059
[25]
Herlofsen SR, Kuechler AM, Melvik JE, Brinchmann JE (2011) Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells in Self-Gelling Alginate Discs Reveals Novel Chondrogenic Signature Gene Clusters. Tissue Eng Part A 17: 1003–1013. doi: 10.1089/ten.tea.2010.0499
[26]
Fernandes AM, Herlofsen SR, Karlsen TA, Kuechler AM, Floisand Y, et al. (2013) Similar properties of chondrocytes from osteoarthritis joints and mesenchymal stem cells from healthy donors for tissue engineering of articular cartilage. PLoS One 8: e62994. doi: 10.1371/journal.pone.0062994
[27]
M？rch YA, Donati I, Strand BL, Skj？k-Br？k G (2006) Effect of Ca2+, Ba2+, and Sr2+ on alginate microbeads. Biomacromolecules 7: 1471–1480. doi: 10.1021/bm060010d
[28]
Smith AM, Hunt NC, Shelton RM, Birdi G, Grover LM (2012) Alginate Hydrogel Has a Negative Impact on in Vitro Collagen 1 Deposition by Fibroblasts. Biomacromolecules 13: 4032–4038. doi: 10.1021/bm301321d
[29]
Leal-Egana A, Braumann U-D, Diaz-Cuenca A, Nowicki M, Bader A (2011) Determination of pore size distribution at the cell-hydrogel interface. J Nanobiotechnology 9: 24. doi: 10.1186/1477-3155-9-24
Trewenack AJ, Please CP, Landman KA (2009) A continuum model for the development of tissue-engineered cartilage around a chondrocyte. Math Med Biol 26: 241–262. doi: 10.1093/imammb/dqp013
[32]
Friedmann A, Hoess A, Cismak A, Heilmann A (2011) Investigation of cell-substrate interactions by focused ion beam preparation and scanning electron microscopy. Acta Biomater 7: 2499–2507. doi: 10.1016/j.actbio.2011.02.024
[33]
Zehbe R, Riesemeier H, Kirkpatrick CJ, Brochhausen C (2012) Imaging of articular cartilage - Data matching using X-ray tomography, SEM, FIB slicing and conventional histology. Micron 43: 1060–1067. doi: 10.1016/j.micron.2012.05.001
