Development of biomaterial self-assembling based platforms to obtain human cartilage tissue in vitro

El cartílag articular té una capacitat limitada de creixement i regeneració i, els tractaments per restaurar la funció del teixit, després d’una lesió, són limitats i poc entesos per la comunitat mèdica. Existeix, per tant, un gran interès en trobar una solució pràctica i agradable pel pacient que aconsegueixi la reparació del cartílag. La enginyeria de teixits va sorgir per restablir teixits danyats usant noves plataformes terapèutiques basades en cèl·lules i/o biomaterials. Aquestes noves teràpies pretenen crear estructures similars al cartílag que imiten les propietats mecàniques i biològiques que trobem in vivo. En aquest context, l’ús de matrius biomimètiques que reprodueixin estructural i funcionalment el microambient natiu han despertat gran interès en aquest camp. Els pèptids auto-ensamblants representen candidats ideals per crear nínxols cel·lulars, ja que les seves nanofibres i propietats biomecàniques son similars a les de la matriu extracel·lular. En aquesta tesi, s’ha desenvolupat nous biomaterials sintètics amb gran potencial per la reparació de cartílag. Aquests estan basats en el pèptid auto-ensamblant RAD16-I decorat amb motius bioactius, amb l’objectiu de reproduir la matriu del cartílag. Donada la versatilitat del hidrogel RAD16-I, les noves matrius es van formar per simple mescla del pèptid RAD16-I amb molècules d’heparina, condroitin sulfat i decorina. Aquestes matrius bi-composades presenten bona estabilitat química i estructural a pH fisiològic i son capaces d’unir i alliberar, gradualment, factors de creixement. L’avaluació d’aquestes matrius es va dur a terme mitjançant dues estratègies in vitro diferents: la rediferenciació de condròcits articulars humans i la inducció del llinatge condrogènic en cèl·lules mare derivades de teixit adipós. Ambdós tipus cel·lulars son considerats una bona font cel·lular per obtenir constructes que reparin defectes al cartílag. Els resultats presentats en aquest treball mostren diferencies a nivell de comportament cel·lular, patrons d’expressió i propietats mecàniques entre els dos tipus cel·lulars i les diferents condicions de cultiu (matrius i medis). Cal destacar que els dos tipus cel·lulars es diferencien a un llinatge condrogènic en medi d’inducció i que els constructes presenten propietats mecàniques compatibles amb un sistema condrogènic. A més s’ha determinat que la presencia de molècules d’heparina a la matriu promou la supervivència de les cèl·lules mare derivades de teixit adipós. En conjunt, les noves matrius bi-composades representen un material fàcil de preparar i prometedor per promoure la diferenciació condrogènica. Finalment, part d’aquesta tesi s’ha centrat en el desenvolupament d’una nova matriu composta mitjançant la infiltració del pèptid RAD16-I amb cèl·lules en microfibres de policaprolactona (PCL). S’ha demostrat que aquesta nova combinació ofereix una estructura funcional i biomimètica, ja que proporciona suport mecànic per les fibres de PCL i a la vegada, facilita l’adhesió i el creixement cel·lular per l’hidrogel RAD16-I. El cultiu in vitro de condròcits humans desdiferenciats demostra que la nova matriu composada promou la supervivència cel·lular i el restabliment del llinatge condrogènic. En general, les propietats sinèrgiques de la nova matriu composada proporcionen una plataforma terapèutica ideal per ajudar a la reparació del cartílag.El cartílago articular tiene una capacidad limitada de crecimiento y regeneración y, los tratamientos para restaurar la función del tejido, después de una lesión, son limitados y poco entendidos por la comunidad médica. Existe, por tanto, un gran interés en encontrar una solución práctica y agradable para el paciente que consiga la reparación del cartílago. La ingeniería de tejidos surgió para restaurar tejidos dañados usando nuevas plataformas terapéuticas basadas en células y/o biomateriales. Estas nuevas terapias pretenden crear estructuras similares al cartílago que imiten las propiedades mecánicas y biológicas que se dan in vivo. En este sentido, el uso de matrices biomiméticas que reproduzcan estructural y funcionalmente el microambiente nativo ha generado gran interés en este campo. Los péptidos auto-ensamblantes representan candidatos ideales para crear nichos celulares dado que, sus nanofibras y propiedades biomecánicas son similares a las de la matriz extracelular. En esta tesis, se han desarrollado nuevos biomateriales sintéticos con gran potencial para la reparación de cartílago. Éstos, están basados en el péptido auto-ensamblante RAD16-I decorado con motivos bioactivos, tratando de reproducir la matriz del cartílago. Dada la versatilidad del hidrogel RAD16-I, las nuevas matrices se formaron por simple mezcla del péptido RAD16-I con moléculas de heparina, condroitin sulfato y decorina. Estas matrices bi-compuestas presentan buena estabilidad química y estructural a pH fisiológico y son capaces de unir y liberar, gradualmente, factores de crecimiento. La evaluación de estas matrices se llevó a cabo mediante dos estrategias in vitro diferentes: la rediferenciación de condrocitos articulares humanos y, la inducción del linaje condrogénico en células madre derivadas de tejido adiposo. Ambos tipos celulares son considerados una buena fuente de células para obtener constructos que reparen defectos en el cartílago. Los resultados presentados en este trabajo muestran diferencias a nivel de comportamiento celular, patrones de expresión y propiedades mecánicas entre los dos tipos celulares y las diferentes condiciones de cultivo (matrices y medios). Cabe destacar que, ambos tipos celulares se diferencian a un linaje condrogénico en medio de inducción y que los constructos presentan propiedades mecánicas compatibles con un sistema condrogénico. Además, se ha determinado que la presencia de moléculas de heparina en la matriz promueve la supervivencia de las células madre derivadas de tejido adiposo. En conjunto, las nuevas matrices bi-compuestas representan un material fácil de preparar y prometedor para promover la diferenciación condrogénica. Por último, parte de esta tesis se ha centrado en el desarrollo de una nueva matriz compuesta mediante la infiltración del péptido RAD16-I con células en microfibras de policaprolactona (PCL). Se ha demostrado que esta nueva combinación ofrece una estructura funcional y biomimética, dado que, proporciona soporte mecánico por las fibras PCL y a su vez, facilita la adhesión y el crecimiento celular debido al hidrogel RAD16-I. El cultivo in vitro de condrocitos humanos desdiferenciados demuestra que la nueva matriz compuesta promueve la supervivencia celular y el restablecimiento del linaje condrogénico. En general, las propiedades sinérgicas de la nueva matriz compuesta proporcionan una plataforma terapéutica ideal para ayudar a la reparación del cartílago.Adult articular cartilage has a limited capacity for growth and regeneration and, after injury, treatments to restore tissue function remain poorly understood by the medical community. Therefore, there is currently great interest in finding practical and patient-friendly strategies for cartilage repair. Tissue engineering has emerged to restore damaged tissue by using new cellular or biomaterial-based therapeutic platforms. These approaches aim to produce cartilage-like structures that reproduce the complex mechanical and biological properties found in vivo. To this end, the use of biomimetic scaffolds that recreate structurally and functionally the native cell microenvironment has become of increasing interest in the field. Self-assembling peptides are attractive candidates to create artificial cellular niches, because their nanoscale network and biomechanical properties are similar to those of the natural extracellular matrix (ECM). In the present thesis, new composite synthetic biomaterials were developed for cartilage tissue engineering (CTE). They were based on the non-instructive self-assembling peptide RAD16-I and decorated with bioactive motifs, aiming to emulate the native cartilage ECM. We employed a simple mixture of the self-assembling peptide RAD16-I with either heparin, chondroitin sulfate or decorin molecules, taking advantage of the versatility of RAD16-I. The bi-component scaffolds presented good structural and chemical stability at a physiological pH and the capacity to bind and gradually release growth factors. Then, these composite scaffolds were characterized using two different in vitro assessments: re-differentiation of human articular chondrocytes (ACs) and induction of human adipose derived stem cells (ADSCs) to a chondrogenic commitment. Both native chondrocytes and adult mesenchymal stem cells (MSCs), either bone marrow or adipose-tissue derived, are considered good cell sources for CTE applications. The results presented in this work revealed differences in cellular behavior, expression patterns and mechanical properties between cell types and culture conditions (scaffolds and media). Remarkably, both cell types underwent into chondrogenic commitment under inductive media conditions and 3D constructs presented mechanical properties compatible to a system undergoing chondrogenesis. Interestingly, as a consequence of the presence of heparin moieties in the scaffold cell survival of ADSCs was enhanced. Altogether, the new bi-component scaffolds represent a promising "easy to prepare" material for promoting chondrogenic differentiation. Finally, part of this thesis was focus on developing a composite scaffold by infiltrating a three-dimensional (3D) woven microfiber poly (ε-caprolactone) (PCL) scaffold with the RAD16-I self-assembling peptide and cells. This new combination resulted into a multi-scale functional and biomimetic tissue-engineered structure providing mechanical support by PCL scaffold and facilitating cell attachment and growth by RAD16-I hydrogel. The in vitro 3D culture of dedifferentiated human ACs evidenced that the new composite supports cell survival and promotes the reestablishment of the chondrogenic lineage commitment. Overall, the synergistic properties of the novel composite scaffold may provide an ideal therapeutic platform to assist cartilage repair

Development of a three-dimensional bioengineered platform for articular cartilage regeneration

Degenerative cartilage pathologies are nowadays a major problem for the world population. Factors such as age, genetics or obesity can predispose people to suffer from articular cartilage degeneration, which involves severe pain, loss of mobility and consequently, a loss of quality of life. Current strategies in medicine are focused on the partial or total replacement of affected joints, physiotherapy and analgesics that do not address the underlying pathology. In an attempt to find an alternative therapy to restore or repair articular cartilage functions, the use of bioengineered tissues is proposed. In this study we present a three-dimensional (3D) bioengineered platform combining a 3D printed polycaprolactone (PCL) macrostructure with RAD16-I, a soft nanofibrous self-assembling peptide, as a suitable microenvironment for human mesenchymal stem cells’ (hMSC) proliferation and differentiation into chondrocytes. This 3D bioengineered platform allows for long-term hMSC culture resulting in chondrogenic differentiation and has mechanical properties resembling native articular cartilage. These promising results suggest that this approach could be potentially used in articular cartilage repair and regenerationPeer ReviewedPostprint (published version

Multi-component hybrid hydrogels – understanding the extent of orthogonal assembly and its impact on controlled release

This paper reports self-assembled multi-component hybrid hydrogels including a range of nanoscale systems and characterizes the extent to which each component maintains its own unique functionality, demonstrating that multi-functionality can be achieved by simply mixing carefully-chosen constituents. Specifically, the individual components are: (i) pH-activated low-molecular-weight gelator (LMWG) 1,3;2,4-dibenzylidenesorbitol-4′,4′′-dicarboxylic acid (DBS–COOH), (ii) thermally-activated polymer gelator (PG) agarose, (iii) anionic biopolymer heparin, and (iv) cationic self-assembled multivalent (SAMul) micelles capable of binding heparin. The LMWG still self-assembles in the presence of PG agarose, is slightly modified on the nanoscale by heparin, but is totally disrupted by the micelles. However, if the SAMul micelles are bound to heparin, DBS–COOH self-assembly is largely unaffected. The LMWG endows hybrid materials with pH-responsive behavior, while the PG provides mechanical robustness. The rate of heparin release can be controlled through network density and composition, with the LMWG and PG behaving differently in this regard, while the presence of the heparin binder completely inhibits heparin release through complexation. This study demonstrates that a multi-component approach can yield exquisite control over self-assembled materials. We reason that controlling orthogonality in such systems will underpin further development of controlled release systems with biomedical applications

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

SEM images of ADSCs and ACs cultured in 3D scaffolds after 4 weeks.

<p>Cells were seeded into RAD16-I, RAD/CS or RAD/Decorin scaffolds. ADSCs were cultured with control or chondrogenic media; ACs were cultured with expansion, control or chondrogenic media. Two images per condition are shown.</p

Human ADSCs and ACs cultured under different media conditions with the self-assembling RAD16-I peptide scaffold and bi-component composites.

<p>ADSCs and ACs were encapsulated in the control scaffold (RAD16-I) and in the composites (RAD/CS and RAD/Decorin), maintained for 4 weeks in the different media compositions and evaluated throughout the culture period for cell and construct morphology by phase contrast images. Images of 3D constructs show a contracted structure under chondrogenic culture conditions. Fluorescent images of DAPI and phalloidin staining of the three scaffolds after 4 weeks of culture in different culture media (Scale bars = 100 μm).</p

Viability of human ADSCs cultured with control and chondrogenic media in the self-assembling RAD16-I peptide scaffold and in RAD/CS, RAD/Decorin and RAD/Heparin composites.

<p>(A) Fluorescent images of live/dead staining at week 4 of culture. Live cells are stained in green and dead cells in red (Scale bars = 200 μm). (B) MTT absorbance values of 3D constructs in both control and chondrogenic culture media in the four scaffold types at different weeks of culture (Significant differences are indicated as * for p<0.05, ** for p<0.01, and *** for p<0.001, One-way ANOVA, N = 2 n = 3). (C) Construct appearance after MTT incubation at week 4 of culture with the different culture media (Con, control medium; ch, chondrogenic medium). Constructs under chondrogenic medium were completely purple after MTT incubation, and constructs under control medium were faintly stained. In the case of RAD/Heparin constructs, live cells were detected in the inner part of the construct (fine arrows).</p

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

Gene expression levels of chondrogenic and hypertrophic markers of ADSCs and ACs cultured in 3D scaffolds for 4 weeks.

<p>ADSCs cultured with RAD16-I, RAD/CS and RAD/Decorin scaffolds in chondrogenic medium were analyzed by qRT-PCR for collagen type I (<i>COL1</i>, A), collagen type II (<i>COL2</i>, B), <i>SOX9</i> (C), aggrecan (<i>ACAN</i>, D), collagen type X (<i>COL10</i>, E) and <i>RUNX2</i> (F). ACs cultured with RAD16-I, RAD/CS and RAD/Decorin scaffolds in expansion (exp) and chondrogenic (ch) medium were analyzed by qRT-PCR for <i>COL1</i> (G), <i>COL2</i> (H), <i>SOX9</i> (I), <i>ACAN</i> (J), <i>COL10</i> (K) and <i>RUNX2</i> (L). Ct values relative to ribosomal protein L22 (RPL22) were obtained and reported as the fold increase (ΔΔCt) relative to 2D cultures (Significant differences are indicated as * for p<0.05, ** for p<0.01, and *** for p<0.001, One-way ANOVA, N = 2 n = 3).</p

Characterization of chondrogenic phenotypes of ADSCs and ACs cultured with RAD16-I, RAD/CS, or RAD/Decorin composite scaffolds for 4 weeks.

<p>(A) Toluidine blue staining (sulfated GAGs) of 3D ADSC constructs cultured in chondrogenic medium. (B) Toluidine blue staining of 3D AC constructs cultured in expansion, control and chondrogenic media. (C) Von Kossa staining (indicating calcium mineralization) of 3D ADSC constructs cultured in chondrogenic medium. (D) Von Kossa staining of 3D AC constructs cultured in chondrogenic medium.</p