Can We Build Artificial Stem Cell Compartments?

Animals carry stem cells throughout their entire life, from embryogenesis to senescence. Their function during development and adulthood consists basically of forming and sustaining functional tissues while maintaining a small self-renewing population. They reside in a complex three-dimensional environment consisting of other nearby cells extracellular matrix components, endogenous or exogenous soluble factors, and physical, structural, or mechanical properties of the tissues they inhabit. Can we artificially recreate tissue development such that stem cells can both self-renew and be instructed to mature properly? The main factors required to regulate the maintenance and differentiation of some types of stem cells are known. In addition, new bioengineered synthetic materials that mimic extracellular matrix components can be used as initial scaffolding for building stem cell microenvironments

Cartilage Tissue Engineering Using Self-Assembling Peptides Composite Scaffolds

Adult articular cartilage presents poor intrinsic capacity for regeneration, and after injury, cellular or biomaterial-based therapeutic platforms are required to assist repair promotion. Cartilage tissue engineering (CTE) aims to produce cartilage-like tissues that recreate the complex mechanical, biophysical and biological properties found in vivo. In terms of biomaterials used for CTE, three-dimensional (3D) self-assembling peptide scaffolds (SAPS) are very attractive for their unique properties, such as biocompatibility, optional possibility of rationally design cell-signaling capacity, biodegradability and modulation of its biomechanical properties. The most attractive cell types currently used for CTE are autologous chondrocytes and adult stem cells. The use of chondrocytes in cell-based therapies for cartilage lesions is limited by quantity and requires an in vitro 2D expansion, which leads to cell dedifferentiation. In the present chapter, we report the development of heparin-, chondroitin sulfate-, decorin-, and poly(ε-caprolactone)-based self-assembling peptide composite scaffolds to promote re-differentiation of expanded human articular chondrocytes and induction of adipose-derived stem cells to chondrogenic commitment

Elastomeric cardiopatch scaffold for myocardial repair and ventricular support

[EN] OBJECTIVES: Prevention of postischaemic ventricular dilatation progressing towards pathological remodelling is necessary to decrease ventricular wall deterioration. Myocardial tissue engineering may play a therapeutic role due to its capacity to replace the extracellular matrix, thereby creating niches for cell homing. In this experimental animal study, a biomimetic cardiopatch was created with elastomeric scaffolds and nanotechnologies. METHODS: In an experimental animal study in 18 sheep, a cardiopatch was created with adipose tissue-derived progenitor cells seeded into an engineered bioimplant consisting of 3-dimensional bioabsorbable polycaprolactone scaffolds filled with a peptide hydrogel (PuraMatrix (TM)). This patch was then transplanted to cover infarcted myocardium. Non-absorbable poly(ethyl) acrylate polymer scaffolds were used as controls. RESULTS: Fifteen sheep were followed with ultrasound scans at 6 months, including echocardiography scans, tissue Doppler and spectral flow analysis and speckle-tracking imaging, which showed a reduction in longitudinal left ventricular deformation in the cardiopatch-treated group. Magnetic resonance imaging (late gadolinium enhancement) showed reduction of infarct size relative to left ventricular mass in the cardiopatch group versus the controls. Histopathological analysis at 6 months showed that the cardiopatch was fully anchored and integrated to the infarct area with minimal fibrosis interface, thereby promoting angiogenesis and migration of adipose tissue-derived progenitor cells to surrounding tissues. CONCLUSIONS: This study shows the feasibility and effectiveness of a cardiopatch grafted onto myocardial infarction scars in an experimental animal model. This treatment decreased fibrosis, limited infarct scar expansion and reduced postischaemic ventricular deformity. A capillary network developed between our scaffold and the heart. The elastomeric cardiopatch seems to have a positive impact on ventricular remodelling and performance in patients with heart failure.The RECATABI Project (Regeneration of Cardiac Tissue Assisted by Bioactive Implants) was financially supported by the 7th Framework Programme (FP7) of the European Commission. Project ID: 229239. Funded under FP7-NMP and the European Regional Development Fund (FEDER Spain).Chachques, JC.; Lila, N.; Soler Botija, C.; Martínez-Ramos, C.; Vallés Lluch, A.; Autret, G.; Perier, M.... (2020). Elastomeric cardiopatch scaffold for myocardial repair and ventricular support. European Journal of Cardio-Thoracic Surgery. 57(3):545-555. https://doi.org/10.1093/ejcts/ezz252S545555573Madonna, R., Van Laake, L. W., Botker, H. E., Davidson, S. M., De Caterina, R., Engel, F. B., … Sluijter, J. P. G. (2019). ESC Working Group on Cellular Biology of the Heart: position paper for Cardiovascular Research: tissue engineering strategies combined with cell therapies for cardiac repair in ischaemic heart disease and heart failure. Cardiovascular Research, 115(3), 488-500. doi:10.1093/cvr/cvz010Nielsen, S. H., Mouton, A. J., DeLeon-Pennell, K. Y., Genovese, F., Karsdal, M., & Lindsey, M. L. (2019). Understanding cardiac extracellular matrix remodeling to develop biomarkers of myocardial infarction outcomes. Matrix Biology, 75-76, 43-57. doi:10.1016/j.matbio.2017.12.001Spinale, F. G., Frangogiannis, N. G., Hinz, B., Holmes, J. W., Kassiri, Z., & Lindsey, M. L. (2016). Crossing Into the Next Frontier of Cardiac Extracellular Matrix Research. Circulation Research, 119(10), 1040-1045. doi:10.1161/circresaha.116.309916Chachques, J. C., Pradas, M. M., Bayes-Genis, A., & Semino, C. (2013). Creating the bioartificial myocardium for cardiac repair: challenges and clinical targets. Expert Review of Cardiovascular Therapy, 11(12), 1701-1711. doi:10.1586/14779072.2013.854165Bayés-Genís, A., Gálvez-Montón, C., & Roura, S. (2016). Cardiac Tissue Engineering. Journal of the American College of Cardiology, 68(7), 724-726. doi:10.1016/j.jacc.2016.05.055Shafy, A., Fink, T., Zachar, V., Lila, N., Carpentier, A., & Chachques, J. C. (2012). Development of cardiac support bioprostheses for ventricular restoration and myocardial regeneration. European Journal of Cardio-Thoracic Surgery, 43(6), 1211-1219. doi:10.1093/ejcts/ezs480Castells-Sala, C., Recha-Sancho, L., Llucià-Valldeperas, A., Soler-Botija, C., Bayes-Genis, A., & Semino, C. E. (2016). Three-Dimensional Cultures of Human Subcutaneous Adipose Tissue-Derived Progenitor Cells Based on RAD16-I Self-Assembling Peptide. Tissue Engineering Part C: Methods, 22(2), 113-124. doi:10.1089/ten.tec.2015.0270Martínez-Ramos, C., Rodríguez-Pérez, E., Garnes, M. P., Chachques, J. C., Moratal, D., Vallés-Lluch, A., & Monleón Pradas, M. (2014). Design and Assembly Procedures for Large-Sized Biohybrid Scaffolds as Patches for Myocardial Infarct. Tissue Engineering Part C: Methods, 20(10), 817-827. doi:10.1089/ten.tec.2013.0489Biswas, M., Sudhakar, S., Nanda, N. C., Buckberg, G., Pradhan, M., Roomi, A. U., … Houle, H. (2013). Two- and Three-Dimensional Speckle Tracking Echocardiography: Clinical Applications and Future Directions. Echocardiography, 30(1), 88-105. doi:10.1111/echo.12079Dorsey, S. M., McGarvey, J. R., Wang, H., Nikou, A., Arama, L., Koomalsingh, K. J., … Burdick, J. A. (2015). MRI evaluation of injectable hyaluronic acid-based hydrogel therapy to limit ventricular remodeling after myocardial infarction. Biomaterials, 69, 65-75. doi:10.1016/j.biomaterials.2015.08.011Chachques, J. C. (2009). Cellular cardiac regenerative therapy in which patients? Expert Review of Cardiovascular Therapy, 7(8), 911-919. doi:10.1586/erc.09.84Chachques, J. (1997). Dynamic cardiomyoplasty: clinical follow-up at 12 years. European Journal of Cardio-Thoracic Surgery, 12(4), 560-568. doi:10.1016/s1010-7940(97)00214-5Varela, C. E., Fan, Y., & Roche, E. T. (2019). Optimizing Epicardial Restraint and Reinforcement Following Myocardial Infarction: Moving Towards Localized, Biomimetic, and Multitherapeutic Options. Biomimetics, 4(1), 7. doi:10.3390/biomimetics4010007Van den Borne, S. W. M., Cleutjens, J. P. M., Hanemaaijer, R., Creemers, E. E., Smits, J. F. M., Daemen, M. J. A. P., & Blankesteijn, W. M. (2009). Increased matrix metalloproteinase-8 and -9 activity in patients with infarct rupture after myocardial infarction. Cardiovascular Pathology, 18(1), 37-43. doi:10.1016/j.carpath.2007.12.012Ducharme, A., Frantz, S., Aikawa, M., Rabkin, E., Lindsey, M., Rohde, L. E., … Lee, R. T. (2000). Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. Journal of Clinical Investigation, 106(1), 55-62. doi:10.1172/jci8768Sieminski, A. L., Semino, C. E., Gong, H., & Kamm, R. D. (2008). Primary sequence of ionic self-assembling peptide gels affects endothelial cell adhesion and capillary morphogenesis. Journal of Biomedical Materials Research Part A, 87A(2), 494-504. doi:10.1002/jbm.a.31785Bagó, J. R., Soler-Botija, C., Casaní, L., Aguilar, E., Alieva, M., Rubio, N., … Blanco, J. (2013). Bioluminescence imaging of cardiomyogenic and vascular differentiation of cardiac and subcutaneous adipose tissue-derived progenitor cells in fibrin patches in a myocardium infarct model. International Journal of Cardiology, 169(4), 288-295. doi:10.1016/j.ijcard.2013.09.013Chachques, J. C., Trainini, J. C., Lago, N., Cortes-Morichetti, M., Schussler, O., & Carpentier, A. (2008). Myocardial Assistance by Grafting a New Bioartificial Upgraded Myocardium (MAGNUM Trial): Clinical Feasibility Study. The Annals of Thoracic Surgery, 85(3), 901-908. doi:10.1016/j.athoracsur.2007.10.052Lee, H., Ahn, S., Bonassar, L. J., & Kim, G. (2012). Cell(MC3T3-E1)-Printed Poly(ϵ-caprolactone)/Alginate Hybrid Scaffolds for Tissue Regeneration. Macromolecular Rapid Communications, 34(2), 142-149. doi:10.1002/marc.201200524Strub, M., Van Bellinghen, X., Fioretti, F., Bornert, F., Benkirane-Jessel, N., Idoux-Gillet, Y., … Clauss, F. (2018). Maxillary Bone Regeneration Based on Nanoreservoirs Functionalizedε-Polycaprolactone Biomembranes in a Mouse Model of Jaw Bone Lesion. BioMed Research International, 2018, 1-12. doi:10.1155/2018/7380389Rohman, G., Huot, S., Vilas-Boas, M., Radu-Bostan, G., Castner, D. G., & Migonney, V. (2015). The grafting of a thin layer of poly(sodium styrene sulfonate) onto poly(ε-caprolactone) surface can enhance fibroblast behavior. Journal of Materials Science: Materials in Medicine, 26(7). doi:10.1007/s10856-015-5539-7Spadaccio, C., Nappi, F., De Marco, F., Sedati, P., Taffon, C., Nenna, A., … Rainer, A. (2017). Implantation of a Poly-l-Lactide GCSF-Functionalized Scaffold in a Model of Chronic Myocardial Infarction. Journal of Cardiovascular Translational Research, 10(1), 47-65. doi:10.1007/s12265-016-9718-9Monnet, E., & Chachques, J. C. (2005). Animal Models of Heart Failure: What Is New? The Annals of Thoracic Surgery, 79(4), 1445-1453. doi:10.1016/j.athoracsur.2004.04.002Bellin, G., Gardin, C., Ferroni, L., Chachques, J., Rogante, M., Mitrečić, D., … Zavan, B. (2019). Exosome in Cardiovascular Diseases: A Complex World Full of Hope. Cells, 8(2), 166. doi:10.3390/cells802016

Engineered 3D bioimplants using elastomeric scaffold, self-assembling peptide hydrogel, and adipose tissue-derived progenitor cells for cardiac regeneration

[EN] Contractile restoration of myocardial scars remains a challenge with important clinical implications. Here, a combination of porous elastomeric membrane, peptide hydrogel, and subcutaneous adipose tissue-derived progenitor cells (subATDPCs) was designed and evaluated as a bioimplant for cardiac regeneration in a mouse model of myocardial infarction. SubATDPCs were doubly transduced with lentiviral vectors to express bioluminescent-fluorescent reporters driven by constitutively active, cardiac tissue-specific promoters. Cells were seeded into an engineered bioimplant consisting of a scaffold (polycaprolactone methacryloyloxyethyl ester) filled with a peptide hydrogel (PuraMatrix(TM)), and transplanted to cover injured myocardium. Bioluminescence and fluorescence quantifications showed de novo and progressive increases in promoter expression in bioactive implant-treated animals. The bioactive implant was well adapted to the heart, and fully functional vessels traversed the myocardium-bioactive implant interface. Treatment translated into a detectable positive effect on cardiac function, as revealed by echocardiography. Thus, this novel implant is a promising construct for supporting myocardial regeneration.The research leading to these results received funding from the European Union Seventh Framework Programme (Project RECATABI, 7FP/2007-2013) under grant agreement number 229239. This work was also supported by Ministerio de Ciencia e Innovación (SAF2011- 30067-C02-01), Fundació La Marató de TV3 (080330), Red de Terapia Celular-TerCel (RD12/0019/0029), Red Cardio-vascular (RD12/0042/0047), Sociedad Española de Cardiología, and Fundació Privada Daniel Bravo AndreuSoler-Botija, C.; Bago, JR.; Llucia-Valldeperas, A.; Vallés Lluch, A.; Castells-Sala, C.; Martinez-Ramos, C.; Fernandez-Muinos, T.... (2014). Engineered 3D bioimplants using elastomeric scaffold, self-assembling peptide hydrogel, and adipose tissue-derived progenitor cells for cardiac regeneration. American Journal of Translational Research. 6:291-301. http://hdl.handle.net/10251/63949S291301

North African Influences and Potential Bias in Case-Control Association Studies in the Spanish Population

BACKGROUND: Despite the limited genetic heterogeneity of Spanish populations, substantial evidences support that historical African influences have not affected them uniformly. Accounting for such population differences might be essential to reduce spurious results in association studies of genetic factors with disease. Using ancestry informative markers (AIMs), we aimed to measure the African influences in Spanish populations and to explore whether these might introduce statistical bias in population-based association studies. METHODOLOGY/PRINCIPAL FINDINGS: We genotyped 93 AIMs in Spanish (from the Canary Islands and the Iberian Peninsula) and Northwest Africans, and conducted population and individual-based clustering analyses along with reference data from the HapMap, HGDP-CEPH, and other sources. We found significant differences for the Northwest African influence among Spanish populations from as low as ≈ 5% in Spanish from the Iberian Peninsula to as much as ≈ 17% in Canary Islanders, whereas the sub-Saharan African influence was negligible. Strikingly, the Northwest African ancestry showed a wide inter-individual variation in Canary Islanders ranging from 0% to 96%, reflecting the violent way the Islands were conquered and colonized by the Spanish in the XV century. As a consequence, a comparison of allele frequencies between Spanish samples from the Iberian Peninsula and the Canary Islands evidenced an excess of markers with significant differences. However, the inflation of p-values for the differences was adequately controlled by correcting for genetic ancestry estimates derived from a reduced number of AIMs. CONCLUSIONS/SIGNIFICANCE: Although the African influences estimated might be biased due to marker ascertainment, these results confirm that Northwest African genetic footprints are recognizable nowadays in the Spanish populations, particularly in Canary Islanders, and that the uneven African influences existing in these populations might increase the risk for false positives in association studies. Adjusting for population stratification assessed with a few dozen AIMs would be sufficient to control this effect

Development of a 3D Co-Culture System as a Cancer Model Using a Self-Assembling Peptide Scaffold

Cancer research has traditionally relied on two-dimensional (2D) cell culture, focusing mainly on cancer cells and their abnormal genetics. However, over the past decade, tumors have been accepted as complex tissues rather than a homogenous mass of proliferating cells. Consequently, cancer cells’ behavior can only be deciphered considering the contribution of the cells existing in the tumor stroma as well as its complex microenvironment. Since the tumor microenvironment plays a critical role in tumorigenesis, it is widely accepted that culturing cells in three-dimensional (3D) scaffolds, which mimic the extracellular matrix, represents a more realistic scenario. In the present work, an in vitro 3D co-culture system based on the self-assembling peptide scaffold RAD16-I (SAPS RAD16-I) was developed as a cancer model. For that, PANC-1 cells were injected into a RAD16-I peptide scaffold containing fibroblasts, resulting in a 3D system where cancer cells were localized in a defined area within a stromal cells matrix. With this system, we were able to study the effect of three well-known pharmaceutical drugs (Gemcitabine, 5-Fluorouracil (5-FU), and 4-Methylumbelliferone (4-MU)) in a 3D context in terms of cell proliferation and survival. Moreover, we have demonstrated that the anti-cancer effect of the tested compounds can be qualitatively and quantitatively evaluated on the developed 3D co-culture system. Experimental results showed that Gemcitabine and 5-FU prevented PANC-1 cell proliferation but had a high cytotoxic effect on fibroblasts as well. 4-MU had a subtle effect on PANC-1 cells but caused high cell death on fibroblasts

Chondroitin Sulfate- and Decorin-Based Self-Assembling Scaffolds for Cartilage Tissue Engineering.

Cartilage injury and degenerative tissue progression remain poorly understood by the medical community. Therefore, various tissue engineering strategies aim to recover areas of damaged cartilage by using non-traditional approaches. To this end, the use of biomimetic scaffolds for recreating the complex in vivo cartilage microenvironment has become of increasing interest in the field. In the present study, we report the development of two novel biomaterials for cartilage tissue engineering (CTE) with bioactive motifs, aiming to emulate the native cartilage extracellular matrix (ECM). We employed a simple mixture of the self-assembling peptide RAD16-I with either Chondroitin Sulfate (CS) or Decorin molecules, taking advantage of the versatility of RAD16-I. After evaluating the structural stability of the bi-component scaffolds at a physiological pH, we characterized these materials using two different in vitro assessments: re-differentiation of human articular chondrocytes (AC) and induction of human adipose derived stem cells (ADSC) to a chondrogenic commitment. Interestingly, differences in cellular morphology and viability were observed between cell types and culture conditions (control and chondrogenic). In addition, both cell types underwent a chondrogenic commitment under inductive media conditions, and this did not occur under control conditions. Remarkably, the synthesis of important ECM constituents of mature cartilage, such as type II collagen and proteoglycans, was confirmed by gene and protein expression analyses and toluidine blue staining. Furthermore, the viscoelastic behavior of ADSC constructs after 4 weeks of culture was more similar to that of native articular cartilage than to that of AC constructs. Altogether, this comparative study between two cell types demonstrates the versatility of our novel biomaterials and suggests a potential 3D culture system suitable for promoting chondrogenic differentiation

β-Sheet to Random Coil Transition in Self-Assembling Peptide Scaffolds Promotes Proteolytic Degradation

One of the most desirable properties that biomaterials designed for tissue engineering or drug delivery applications should fulfill is biodegradation and resorption without toxicity. Therefore, there is an increasing interest in the development of biomaterials able to be enzymatically degraded once implanted at the injury site or once delivered to the target organ. In this paper, we demonstrate the protease sensitivity of self-assembling amphiphilic peptides, in particular, RAD16-I (AcN-RADARADARADARADA-CONH2), which contains four potential cleavage sites for trypsin. We detected that when subjected to thermal denaturation, the peptide secondary structure suffers a transition from β-sheet to random coil. We also used Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight (MALDI-TOF) to detect the proteolytic breakdown products of samples subjected to incubation with trypsin as well as atomic force microscopy (AFM) to visualize the effect of the degradation on the nanofiber scaffold. Interestingly, thermally treated samples had a higher extent of degradation than non-denatured samples, suggesting that the transition from β-sheet to random coil leaves the cleavage sites accessible and susceptible to protease degradation. These results indicate that the self-assembling peptide can be reduced to short peptide sequences and, subsequently, degraded to single amino acids, constituting a group of naturally biodegradable materials optimal for their application in tissue engineering and regenerative medicine

Characterization of the bi-component scaffolds.

<p>(A) Toluidine blue and congo red staining of RAD16-I and composites with increasing quantities of CS. Ratios of mg RAD16-I/mg Chondroitin Sulfate ranging from 950/1 to 9.5/1. (B) Toluidine blue and congo red staining of RAD16-I and composites with increasing quantities of Decorin. Ratios of mg RAD16-I/mg Decorin ranging from 950/1 to 9.5/1. (C) Quantification of TGFβ1 released by RAD16-I and the composite RAD/CS (ratio 47.5/1) after 12, 24, 36, 60 and 84 hours of delivery (mean ± SD, n = 3). (D) Quantification of TGFβ1 released by RAD16-I and the composite RAD/Decorin (ratio 47.5/1) after 12, 24, 36, 60 and 84 hours of delivery (mean ± SD, n = 3). (E) Quantification of TGFβ1 released by RAD16-I and composite RAD/Heparin (ratio 47.5/1) after 12, 24, 36, 60 and 84 hours of delivery (mean ± SD, n = 3).</p