Chitosan-based scaffold counteracts hypertrophic and fibrotic markers in chondrogenic differentiated mesenchymal stromal cells
Cristina Manferdini
IRCCS Istituto Ortopedico Rizzoli, SC Laboratorio di Immunoreumatologia e Rigenerazione Tissutale, Bologna, Italy
Search for more papers by this authorElena Gabusi
IRCCS Istituto Ortopedico Rizzoli, SC Laboratorio di Immunoreumatologia e Rigenerazione Tissutale, Bologna, Italy
Search for more papers by this authorLuciana Sartore
Dipartimento di Ingegneria Meccanica e Industriale, Università degli studi di Brescia, Brescia, Italy
Search for more papers by this authorKamol Dey
Dipartimento di Ingegneria Meccanica e Industriale, Università degli studi di Brescia, Brescia, Italy
Search for more papers by this authorSilvia Agnelli
Dipartimento di Ingegneria Meccanica e Industriale, Università degli studi di Brescia, Brescia, Italy
Search for more papers by this authorCamillo Almici
Laboratory for Stem Cell Manipulation and Cyopreservation, Department of Transfusion Medicine, ASST Spedali Civili, Brescia, Italy
Search for more papers by this authorAndrea Bianchetti
Laboratory for Stem Cell Manipulation and Cyopreservation, Department of Transfusion Medicine, ASST Spedali Civili, Brescia, Italy
Search for more papers by this authorNicoletta Zini
IGM, CNR–National Research Council of Italy, Bologna, Italy
IRCCS Istituto Ortopedico Rizzoli, Bologna, Italy
Search for more papers by this authorDomenico Russo
Unità di Malattie del Sangue e Trapianto Midollo Osseo, Dipartimento di Scienze Cliniche e Sperimentali, Università degli studi di Brescia, Brescia, Italy
Search for more papers by this authorFederica Re
Unità di Malattie del Sangue e Trapianto Midollo Osseo, Dipartimento di Scienze Cliniche e Sperimentali, Università degli studi di Brescia, Brescia, Italy
Search for more papers by this authorErminia Mariani
IRCCS Istituto Ortopedico Rizzoli, SC Laboratorio di Immunoreumatologia e Rigenerazione Tissutale, Bologna, Italy
DIMEC, Alma Mater Studiorum, Università di Bologna, Bologna, Italy
Search for more papers by this authorCorresponding Author
Gina Lisignoli
IRCCS Istituto Ortopedico Rizzoli, SC Laboratorio di Immunoreumatologia e Rigenerazione Tissutale, Bologna, Italy
Correspondence
Gina Lisignoli, SC Laboratorio di Immunoreumatologia e Rigenerazione Tissutale, IRCCS Istituto Ortopedico Rizzoli, via di Barbiano 1/10, Bologna 40136, Italy.
Email: [email protected]
Search for more papers by this authorCristina Manferdini
IRCCS Istituto Ortopedico Rizzoli, SC Laboratorio di Immunoreumatologia e Rigenerazione Tissutale, Bologna, Italy
Search for more papers by this authorElena Gabusi
IRCCS Istituto Ortopedico Rizzoli, SC Laboratorio di Immunoreumatologia e Rigenerazione Tissutale, Bologna, Italy
Search for more papers by this authorLuciana Sartore
Dipartimento di Ingegneria Meccanica e Industriale, Università degli studi di Brescia, Brescia, Italy
Search for more papers by this authorKamol Dey
Dipartimento di Ingegneria Meccanica e Industriale, Università degli studi di Brescia, Brescia, Italy
Search for more papers by this authorSilvia Agnelli
Dipartimento di Ingegneria Meccanica e Industriale, Università degli studi di Brescia, Brescia, Italy
Search for more papers by this authorCamillo Almici
Laboratory for Stem Cell Manipulation and Cyopreservation, Department of Transfusion Medicine, ASST Spedali Civili, Brescia, Italy
Search for more papers by this authorAndrea Bianchetti
Laboratory for Stem Cell Manipulation and Cyopreservation, Department of Transfusion Medicine, ASST Spedali Civili, Brescia, Italy
Search for more papers by this authorNicoletta Zini
IGM, CNR–National Research Council of Italy, Bologna, Italy
IRCCS Istituto Ortopedico Rizzoli, Bologna, Italy
Search for more papers by this authorDomenico Russo
Unità di Malattie del Sangue e Trapianto Midollo Osseo, Dipartimento di Scienze Cliniche e Sperimentali, Università degli studi di Brescia, Brescia, Italy
Search for more papers by this authorFederica Re
Unità di Malattie del Sangue e Trapianto Midollo Osseo, Dipartimento di Scienze Cliniche e Sperimentali, Università degli studi di Brescia, Brescia, Italy
Search for more papers by this authorErminia Mariani
IRCCS Istituto Ortopedico Rizzoli, SC Laboratorio di Immunoreumatologia e Rigenerazione Tissutale, Bologna, Italy
DIMEC, Alma Mater Studiorum, Università di Bologna, Bologna, Italy
Search for more papers by this authorCorresponding Author
Gina Lisignoli
IRCCS Istituto Ortopedico Rizzoli, SC Laboratorio di Immunoreumatologia e Rigenerazione Tissutale, Bologna, Italy
Correspondence
Gina Lisignoli, SC Laboratorio di Immunoreumatologia e Rigenerazione Tissutale, IRCCS Istituto Ortopedico Rizzoli, via di Barbiano 1/10, Bologna 40136, Italy.
Email: [email protected]
Search for more papers by this authorAbstract
Cartilage tissue engineering remains problematic because no systems are able to induce signals that contribute to native cartilage structure formation. Therefore, we tested the potentiality of gelatin-polyethylene glycol scaffolds containing three different concentrations of chitosan (CH; 0%, 8%, and 16%) on chondrogenic differentiation of human platelet lysate-expanded human bone marrow mesenchymal stromal cells (hBM-MSCs). Typical chondrogenic (SOX9, collagen type 2, and aggrecan), hypertrophic (collagen type 10), and fibrotic (collagen type 1) markers were evaluated at gene and protein level at Days 1, 28, and 48. We demonstrated that 16% CH scaffold had the highest percentage of relaxation with the fastest relaxation rate. In particular, 16% CH scaffold, combined with chondrogenic factor TGFβ3, was more efficient in inducing hBM-MSCs chondrogenic differentiation compared with 0% or 8% scaffolds. Collagen type 2, SOX9, and aggrecan showed the same expression in all scaffolds, whereas collagen types 10 and 1 markers were efficiently down-modulated only in 16% CH. We demonstrated that using human platelet lysate chronically during hBM-MSCs chondrogenic differentiation, the chondrogenic, hypertrophic, and fibrotic markers were significantly decreased. Our data demonstrate that only a high concentration of CH, combined with TGFβ3, creates an environment capable of guiding in vitro hBM-MSCs towards a phenotypically stable chondrogenesis.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
Supporting Information
| Filename | Description |
|---|---|
| term2941-supp_0001_T1.docxWord 2007 document , 36.5 KB |
Table S1: Oligonucleotide primers used for real-time PCR. |
| term2941-supp_0002_F1.docxWord 2007 document , 506.1 KB |
Figure S1. hBM-MSCs immunophenotypic and differentiative characteristics. (A) Show hBM-MSCs antigenic profile. (B) Shows the differentiation capacity of expanded hBM-MSCs; stained with Alizarine Red for osteogenic differentiation and Oil Red O for adipogenic differentation |
| term2941-supp_0003_F2.docxWord 2007 document , 6.1 MB |
Figure S2. hBM-MSCs concentration and distribution inside the scaffold. Representative picture of hematoxylin-eosin stained 5+105 (a) and 106 (b) hBM-MSCs seeded on G-PEG-CH2 scaffold and evaluated at day 14 to define the best cell concentration. Representative picture of Toluidine blue stained 106 hBM-MSCs seeded on G-PEG-CH2 scaffold and evaluated at days 28 (c) and 48 (d) to evaluate the distribution inside the scaffold. Bars =50μm |
| term2941-supp_0004_F3.docxWord 2007 document , 591 KB |
Figure S3. hBM-MSCs proliferation time-course. Proliferation of chondrogenic differentiated (TGF3) hBM-MSCs was evaluated on three different scaffolds containing no chitosan (G-PEG) or 8% (G-PEG-CH1) or 16% (G-PEG-CH2) of chitosan in presence (HPL) or absence of HPL (-HPL) at 1, 14, 28 and 48 days by Alamar blue. Data were expressed as mean of the percentage reduction of Alamar blue |
| term2941-supp_0005_F4.docxWord 2007 document , 5.7 MB |
Figure S4. hBM-MSCs concentration and distribution inside the scaffold. Representative picture of hematoxylin-eosin stained 5+105 (a) and 106 (b) hBM-MSCs seeded on G-PEG-CH2 scaffold and evaluated at day 14 to define the best cell concentration. Representative picture of Toluidine blue stained 106 chondrogenic differentiated (+TGFβ3) hBM-MSCs seeded on F-PEG-CH2 scaffold and evaluated at days 28 (c) and 48 (d) to evaluate the distribution inside the scaffold. Bars=50μm |
| term2941-supp_0006_F5.docxWord 2007 document , 1.1 MB |
Figure S5. Chondrogenic markers on hBM-MSCs untreated with TGF3 chondrogenic factor. SOX-9, ACAN, COL1A1 and COL10A1 genes were evaluated on hBM-MSCs untreated with TGFβ3 grown on three different scaffolds containing no chitosan (G-PEG) or 8% (G-PEG-CH1) or 16% (G-PEG-CH2) of chitosan in presence (+HPL) both at 28 and 48 days. Data were expressed as mean percentage of target genes respect too the RPS9 housekeeping gene ± SD. Dotted line indicates genes at day 1 |
| term2941-supp_0007_F6.docxWord 2007 document , 2.8 MB |
Figure S6. Proliferation and Aggrecan immunohistochemical evaluation on hBM-MSCs untreated with chondrogenic factor TGFβ3. A. Proliferation of hBM-MSCs was evaluated on three different scaffold containing no chitosan (G-PEG) or 8% (G-PEG-CH1) or 16% (G-PEG-CH2) of chitosan in presence of HPL both at 28 and 48 days. Data were expressed as mean percentage of MKI67 target gene respect to the RPS9 housekeeping gene ± SD. Dotted line indicates the MKI67 expression at Day 1. Significant results are indicated as *=p<0.01. B. Representative images of aggrecan evaluated on hBM-MSCs grown on three different scaffolds containing o chitosan (G-PEG) or 8% (G-PEG-CH1) or 16% (G-PEG-CH2) of chitosan in presence of HPL at 28 day. Bars= 100 μm. |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
REFERENCES
- Agrawal, P., Pramanik, K., Vishwanath, V., Biswas, A., Bissoyi, A., & Patra, P. K. (2018). Enhanced chondrogenesis of mesenchymal stem cells over silk fibroin/chitosan-chondroitin sulfate three dimensional scaffold in dynamic culture condition. Journal of biomedical materials research Part B, Applied biomaterials, 106(7), 2576–2587. https://doi.org/10.1002/jbm.b.34074
- Bernhard, J. C., & Vunjak-Novakovic, G. (2016). Should we use cells, biomaterials, or tissue engineering for cartilage regeneration? Stem Cell Research & Therapy, 7(1), 56. https://doi.org/10.1186/s13287-016-0314-3
- Boschetti, F., Pennati, G., Gervaso, F., Peretti, G. M., & Dubini, G. (2004). Biomechanical properties of human articular cartilage under compressive loads. Biorheology, 41(3-4), 159–166.
- Broom, N. D., & Poole, C. A. (1982). A functional-morphological study of the tidemark region of articular cartilage maintained in a non-viable physiological condition. Journal of Anatomy, 135(Pt 1), 65–82.
- Burnouf, T., Strunk, D., Koh, M. B., & Schallmoser, K. (2016). Human platelet lysate: Replacing fetal bovine serum as a gold standard for human cell propagation? Biomaterials, 76, 371–387. https://doi.org/10.1016/j.biomaterials.2015.10.065
- Caldwell, K. L., & Wang, J. (2015). Cell-based articular cartilage repair: The link between development and regeneration. Osteoarthritis and Cartilage, 23(3), 351–362. https://doi.org/10.1016/j.joca.2014.11.004
- Candela, M. E., Yasuhara, R., Iwamoto, M., & Enomoto-Iwamoto, M. (2014). Resident mesenchymal progenitors of articular cartilage. Matrix biology: journal of the International Society for Matrix Biology, 39, 44–49. https://doi.org/10.1016/j.matbio.2014.08.015
- Capelli, C., Domenghini, M., Borleri, G., Bellavita, P., Poma, R., Carobbio, A., … Introna, M. (2007). Human platelet lysate allows expansion and clinical grade production of mesenchymal stromal cells from small samples of bone marrow aspirates or marrow filter washouts. Bone Marrow Transplantation, 40(8), 785–791. https://doi.org/10.1038/sj.bmt.1705798
- Carballo, C. B., Nakagawa, Y., Sekiya, I., & Rodeo, S. A. (2017). Basic Science of Articular Cartilage. Clinics in Sports Medicine, 36(3), 413–425. https://doi.org/10.1016/j.csm.2017.02.001
- Chaudhuri, O., Gu, L., Klumpers, D., Darnell, M., Bencherif, S. A., Weaver, J. C., … Mooney, D. J. (2016). Hydrogels with tunable stress relaxation regulate stem cell fate and activity. Nature Materials, 15(3), 326–334. https://doi.org/10.1038/nmat4489
- Chen, H., Liu, Y., Jiang, Z., Chen, W., Yu, Y., & Hu, Q. (2014). Cell-scaffold interaction within engineered tissue. Experimental Cell Research, 323(2), 346–351. https://doi.org/10.1016/j.yexcr.2014.02.028
- Comblain, F., Rocasalbas, G., Gauthier, S., & Henrotin, Y. (2017). Chitosan: A promising polymer for cartilage repair and viscosupplementation. Bio-medical Materials and Engineering, 28(s1), S209–s215. https://doi.org/10.3233/BME-171643
- Darling, E. M., & Athanasiou, K. A. (2005). Rapid phenotypic changes in passaged articular chondrocyte subpopulations. Journal of Orthopaedic Research : Official Publication of the Orthopaedic Research Society, 23(2), 425–432. https://doi.org/10.1016/j.orthres.2004.08.008
- DeKosky, B. J., Dormer, N. H., Ingavle, G. C., Roatch, C. H., Lomakin, J., Detamore, M. S., & Gehrke, S. H. (2010). Hierarchically designed agarose and poly (ethylene glycol) interpenetrating network hydrogels for cartilage tissue engineering. Tissue engineering Part C, Methods, 16(6), 1533–1542. https://doi.org/10.1089/ten.tec.2009.0761
- Dey, K., Agnelli, S., & Sartore, L. (2019). Dynamic freedom: Substrate stress relaxation stimulates cell responses. Biomaterials Science, 7(3), 836–842. https://doi.org/10.1039/C8BM01305E
- Dey, K., Agnelli, S., Serzanti, M., Ginestra, P., Scarì, G., Dell'Era, P., & Sartore, L. (2019). Preparation and properties of high performance gelatin-based hydrogels with chitosan or hydroxyethyl cellulose for tissue engineering applications. International Journal of Polymeric Materials and Polymeric Biomaterials, 68(4), 183–192. https://doi.org/10.1080/00914037.2018.1429439
- Diani, J., Fayolle, B., & Gilormini, P. (2009). A review on the Mullins effect. European Polymer Journal, 45(3), 601–612. https://doi.org/10.1016/j.eurpolymj.2008.11.017
- Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., Krause, D., … Horwitz, E. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 8(4), 315–317. https://doi.org/10.1080/14653240600855905
- Doucet, C., Ernou, I., Zhang, Y., Llense, J. R., Begot, L., Holy, X., & Lataillade, J. J. (2005). Platelet lysates promote mesenchymal stem cell expansion: A safety substitute for animal serum in cell-based therapy applications. Journal of Cellular Physiology, 205(2), 228–236. https://doi.org/10.1002/jcp.20391
- Duarte Campos, D. F., Drescher, W., Rath, B., Tingart, M., & Fischer, H. (2012). Supporting biomaterials for articular cartilage repair. Cartilage, 3(3), 205–221. https://doi.org/10.1177/1947603512444722
- Gadjanski, I., Spiller, K., & Vunjak-Novakovic, G. (2012). Time-dependent processes in stem cell-based tissue engineering of articular cartilage. Stem Cell Reviews, 8(3), 863–881. https://doi.org/10.1007/s12015-011-9328-5
- Gullotta, F., Izzo, D., Scalera, F., Palazzo, B., Martin, I., Sannino, A., & Gervaso, F. (2018). Biomechanical evaluation of hMSCs-based engineered cartilage for chondral tissue regeneration. Journal of the Mechanical Behavior of Biomedical Materials, 86, 294–304. https://doi.org/10.1016/j.jmbbm.2018.06.040
- Hildner, F., Eder, M. J., Hofer, K., Aberl, J., Redl, H., van Griensven, M., … Peterbauer-Scherb, A. (2015). Human platelet lysate successfully promotes proliferation and subsequent chondrogenic differentiation of adipose-derived stem cells: A comparison with articular chondrocytes. Journal of Tissue Engineering and Regenerative Medicine, 9(7), 808–818. https://doi.org/10.1002/term.1649
- Hunziker, E. B. (2009). The elusive path to cartilage regeneration. Advanced materials (Deerfield Beach, Fla), 21(32-33), 3419–3424. https://doi.org/10.1002/adma.200801957
- Iwamoto, M., Ohta, Y., Larmour, C., & Enomoto-Iwamoto, M. (2013). Toward regeneration of articular cartilage. Birth defects research Part C, Embryo today: reviews, 99(3), 192–202. https://doi.org/10.1002/bdrc.21042
- Jayasuriya, C. T., Chen, Y., Liu, W., & Chen, Q. (2016). The influence of tissue microenvironment on stem cell-based cartilage repair. Annals of the New York Academy of Sciences, 1383(1), 21–33. https://doi.org/10.1111/nyas.13170
- Johnstone, B., Alini, M., Cucchiarini, M., Dodge, G. R., Eglin, D., Guilak, F., … Stoddart, M. J. (2013). Tissue engineering for articular cartilage repair—The state of the art. European Cells & Materials, 25, 248–267. https://doi.org/10.22203/eCM.v025a18
- Kalkan, R., Nwekwo, C. W., & Adali, T. (2018). The use of scaffolds in cartilage regeneration. Critical Reviews in Eukaryotic Gene Expression, 28(4), 343–348. https://doi.org/10.1615/CritRevEukaryotGeneExpr.2018024574
- Kothapalli, C. R., Shaw, M. T., & Wei, M. (2005). Biodegradable HA-PLA 3-D porous scaffolds: Effect of nano-sized filler content on scaffold properties. Acta Biomaterialia, 1(6), 653–662. https://doi.org/10.1016/j.actbio.2005.06.005
- Levengood, S. L., & Zhang, M. (2014). Chitosan-based scaffolds for bone tissue engineering. Journal of Materials Chemistry B, 2(21), 3161–3184. https://doi.org/10.1039/c4tb00027g
- Liou, J. J., Rothrauff, B. B., Alexander, P. G., & Tuan, R. S. (2018). Effect of platelet-rich plasma on chondrogenic differentiation of adipose- and bone marrow-derived mesenchymal stem cells. Tissue Engineering Part A, 24(19-20), 1432–1443. https://doi.org/10.1089/ten.tea.2018.0065
- Lisignoli, G., Manferdini, C., Lambertini, E., Zini, N., Angelozzi, M., Gabusi, E., … Piva, R. (2014). Chondrogenic potential of slug-depleted human mesenchymal stem cells. Tissue Engineering Part A, 20(19-20), 2795–2805. https://doi.org/10.1089/ten.tea.2013.0343
- Lu, T. J., Chiu, F. Y., Chiu, H. Y., Chang, M. C., & Hung, S. C. (2017). Chondrogenic differentiation of mesenchymal stem cells in three-dimensional chitosan film culture. Cell Transplantation, 26(3), 417–427. https://doi.org/10.3727/096368916X693464
- Manferdini, C., Maumus, M., Gabusi, E., Piacentini, A., Filardo, G., Peyrafitte, J. A., … Lisignoli, G. (2013). Adipose-derived mesenchymal stem cells exert antiinflammatory effects on chondrocytes and synoviocytes from osteoarthritis patients through prostaglandin E2. Arthritis and Rheumatism, 65(5), 1271–1281. https://doi.org/10.1002/art.37908
- Maumus, M., Manferdini, C., Toupet, K., Peyrafitte, J. A., Ferreira, R., Facchini, A., … Noel, D. (2013). Adipose mesenchymal stem cells protect chondrocytes from degeneration associated with osteoarthritis. Stem Cell Research, 11(2), 834–844. https://doi.org/10.1016/j.scr.2013.05.008
- Merlin Rajesh Lal, L. P., Suraishkumar, G. K., & Nair, P. D. (2017). Chitosan-agarose scaffolds supports chondrogenesis of human Wharton's jelly mesenchymal stem cells. Journal of biomedical materials research Part A, 105(7), 1845–1855. https://doi.org/10.1002/jbm.a.36054
- Moreira Teixeira, L. S., Leijten, J. C., Wennink, J. W., Chatterjea, A. G., Feijen, J., van Blitterswijk, C. A., … Karperien, M. (2012). The effect of platelet lysate supplementation of a dextran-based hydrogel on cartilage formation. Biomaterials, 33(14), 3651–3661. https://doi.org/10.1016/j.biomaterials.2012.01.051
- Mortada, I., & Mortada, R. (2018). Epigenetic changes in mesenchymal stem cells differentiation. European Journal of Medical Genetics, 61(2), 114–118. https://doi.org/10.1016/j.ejmg.2017.10.015
- Ng, J. J., Wei, Y., Zhou, B., Bernhard, J., Robinson, S., Burapachaisri, A., … Vunjak-Novakovic, G. (2017). Recapitulation of physiological spatiotemporal signals promotes in vitro formation of phenotypically stable human articular cartilage. Proceedings of the National Academy of Sciences of the United States of America, 114(10), 2556–2561. https://doi.org/10.1073/pnas.1611771114
- Occhetta, P., Pigeot, S., Rasponi, M., Dasen, B., Mehrkens, A., Ullrich, T., … Martin, I. (2018). Developmentally inspired programming of adult human mesenchymal stromal cells toward stable chondrogenesis. Proceedings of the National Academy of Sciences of the United States of America, 115(18), 4625–4630. https://doi.org/10.1073/pnas.1720658115
- Pelttari, K., Winter, A., Steck, E., Goetzke, K., Hennig, T., Ochs, B. G., … Richter, W. (2006). Premature induction of hypertrophy during in vitro chondrogenesis of human mesenchymal stem cells correlates with calcification and vascular invasion after ectopic transplantation in SCID mice. Arthritis and Rheumatism, 54(10), 3254–3266. https://doi.org/10.1002/art.22136
- Piluso, S., Lendlein, A., & Neffe, A. T. (2017). Enzymatic action as switch of bulk to surface degradation of clicked gelatin-based networks. Polymers for Advanced Technologies, 28(10), 1318–1324. https://doi.org/10.1002/pat.3962
- Quiroga, J. M. P., Wilson, W., Ito, K., & van Donkelaar, C. C. (2017). Relative contribution of articular cartilage's constitutive components to load support depending on strain rate. Biomechanics and Modeling in Mechanobiology, 16(1), 151–158. https://doi.org/10.1007/s10237-016-0807-0
- Richter, D. L., Schenck, R. C. Jr., Wascher, D. C., & Treme, G. (2016). Knee articular cartilage repair and restoration techniques: A review of the literature. Sports health, 8(2), 153–160. https://doi.org/10.1177/1941738115611350
- Rodriguez-Vazquez, M., Vega-Ruiz, B., Ramos-Zuniga, R., Saldana-Koppel, D. A., & Quinones-Olvera, L. F. (2015). Chitosan and its potential use as a scaffold for tissue engineering in regenerative medicine. BioMed Research International, 2015, 821279.
- Roffi, A., Nakamura, N., Sanchez, M., Cucchiarini, M., & Filardo, G. (2018). Injectable systems for intra-articular delivery of mesenchymal stromal cells for cartilage treatment: A systematic review of preclinical and clinical evidence. International Journal of Molecular Sciences, 19(11). https://doi.org/10.3390/ijms19113322
- Scotti, C., Tonnarelli, B., Papadimitropoulos, A., Scherberich, A., Schaeren, S., Schauerte, A., … Martin, I. (2010). Recapitulation of endochondral bone formation using human adult mesenchymal stem cells as a paradigm for developmental engineering. Proceedings of the National Academy of Sciences of the United States of America, 107(16), 7251–7256. https://doi.org/10.1073/pnas.1000302107
- Tan, H., Chu, C. R., Payne, K. A., & Marra, K. G. (2009). Injectable in situ forming biodegradable chitosan-hyaluronic acid based hydrogels for cartilage tissue engineering. Biomaterials, 30(13), 2499–2506. https://doi.org/10.1016/j.biomaterials.2008.12.080
- Vinatier, C., & Guicheux, J. (2016). Cartilage tissue engineering: From biomaterials and stem cells to osteoarthritis treatments. Annals of Physical and Rehabilitation Medicine, 59(3), 139–144. https://doi.org/10.1016/j.rehab.2016.03.002
- Webber, M. J., Khan, O. F., Sydlik, S. A., Tang, B. C., & Langer, R. (2015). A perspective on the clinical translation of scaffolds for tissue engineering. Annals of Biomedical Engineering, 43(3), 641–656. https://doi.org/10.1007/s10439-014-1104-7
- Wells, R. G. (2008). The role of matrix stiffness in regulating cell behavior. Hepatology (Baltimore, Md), 47(4), 1394–1400. https://doi.org/10.1002/hep.22193
- Zaky, S. H., Ottonello, A., Strada, P., Cancedda, R., & Mastrogiacomo, M. (2008). Platelet lysate favours in vitro expansion of human bone marrow stromal cells for bone and cartilage engineering. Journal of Tissue Engineering and Regenerative Medicine, 2(8), 472–481. https://doi.org/10.1002/term.119
- Zhao, X., Huebsch, N., Mooney, D. J., & Suo, Z. (2010). Stress-relaxation behavior in gels with ionic and covalent crosslinks. Journal of Applied Physics, 107(6), 63509. https://doi.org/10.1063/1.3343265
