Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Ramon Llull University

The prolonged ischemia after myocardial infarction leads to a high degree of cardiomyocyte death, which leads to a reduction of normal heart function. Valuable lessons can be learnt from human myocardium and stem cell biology that would help scientists to develop new, effective, safe, and affordable regenerative therapies. In vivo models are of high interest, but their high complexity limits the possibility to analyze specific factors. In vitro models permit analyzing specific factors of tissue physiology or pathophysiology providing accurate approaches that may guide the creation of three-dimensional (3D) engineered cell aggregates. These systems provide a simplistic way to examine individual factors as compared to animal models, and better mimic the reality than 2D models. In this sense, the objective of this work is to better understand the behavior of a human mesenchymal stem cell-like cell line (subcutaneous adipose tissue-derived progenitor cells [subATDPCs], susceptible to be used in cell therapies) when they are embedded in the 3D environment provided by RAD16-I self-assembling peptide (SAP). Specifically, we study the effect in subATDPCs viability, morphology, proliferation, and protein and gene expression of matrix composition (i.e., RGD motif and heparin polysaccharide modifications) in RAD16-I matrix under different media conditions. Results demonstrated that the 3D environment provided by RAD16-I SAP is able to maintain subATDPCs in this new milieu and at the same time its cardiac commitment. Additionally, it has been observed that chemical induction can induce upregulation of cardiac markers, such as TBX5, MEF2C, ACTN1, and GJA1. Therefore, we propose this 3D model as a promising platform to analyze the effect of specific cues that can help improve cell performance for future cell therapy.

Three-Dimensional Cultures of Human Subcutaneous Adipose Tissue-Derived Progenitor Cells Based on RAD16-I Self-Assembling Peptide.

The use of chondrocytes in cell-based therapies for cartilage lesions are limited by quantity and, therefore, require an in vitro expansion. As monolayer culture leads to de-differentiation, different culture techniques are currently under development to recover chondrocyte phenotype after cell expansion. In the present work, we studied the capacity of the bimolecular heparin-based self-assembling peptide scaffold (RAD16-I) as a three-dimensional (3D) culture system to foster reestablishment of chondrogenic phenotype of de-differentiated human Articular Chondrocytes (AC). The culture was performed in a serum-free medium under control and chondrogenic induction and good viability results were observed after 4 weeks of culture in both conditions. Cells changed their morphology to a more elongated shape and established a cellular network that induced the condensation of the constructs in the case of chondrogenic medium, leading to a compacted structure with improved mechanical properties. Specific extracellular matrix (ECM) proteins of mature cartilage, such as collagen type II and aggrecan were up-regulated under chondrogenic medium and significantly enhanced with the presence of heparin in the scaffold. 3D constructs became highly stained with toluidine blue dye after 4 weeks of culture, indicating the presence of synthetized proteoglycans (PGs) by the cells. Interestingly, the full viscoelastic behavior was closely related to that found in chicken native cartilage. Altogether, the results suggest that the 3D culture model described can help de-differentiated human chondrocytes to recover its cartilage phenotype. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1694-1706, 2016.

Heparin-based self-assembling peptide scaffold reestablish chondrogenic phenotype of expanded de-differentiated human chondrocytes.

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. 

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

Adult articular cartilage has a limited capacity for growth and regeneration and, with injury, new cellular or biomaterial-based therapeutic platforms are required to promote repair. Tissue engineering aims to produce cartilage-like tissues that recreate the complex mechanical and biological properties found . In this study, a unique composite scaffold was developed by infiltrating a three-dimensional (3D) woven microfiber poly (ε-caprolactone) (PCL) scaffold with the RAD16-I self-assembling nanofibers to obtain multi-scale functional and biomimetic tissue-engineered constructs. The scaffold was seeded with expanded dedifferentiated human articular chondrocytes and cultured for four weeks in control and chondrogenic growth conditions. The composite constructs were compared to control constructs obtained by culturing cells with 3D woven PCL scaffolds or RAD16-I independently. High viability and homogeneous cell distribution were observed in all three scaffolds used during the term of the culture. Moreover, gene and protein expression profiles revealed that chondrogenic markers were favored in the presence of RAD16-I peptide (PCL/RAD composite or alone) under chondrogenic induction conditions. Further, constructs displayed positive staining for toluidine blue, indicating the presence of synthesized proteoglycans. Finally, mechanical testing showed that constructs containing the PCL scaffold maintained the initial shape and viscoelastic behavior throughout the culture period, while constructs with RAD16-I scaffold alone contracted during culture time into a stiffer and compacted structure. Altogether, these results suggest that this new composite scaffold provides important mechanical requirements for a cartilage replacement, while providing a biomimetic microenvironment to re-establish the chondrogenic phenotype of human expanded articular chondrocytes.

Dedifferentiated Human Articular Chondrocytes Redifferentiate to a Cartilage-Like Tissue Phenotype in a Poly(ε-Caprolactone)/Self-Assembling Peptide Composite Scaffold.

Lourdes Recha-Sancho

Tissue Engineering Research Laboratory,
Department of Bioengineering,
IQS-School of Engineering,
Tissue Engineering Research Laboratory, Department of Bioengineering, IQS-School of Engineering

Ramon Llull University

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

Lourdes Recha-Sancho

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Ramon Llull University

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

Tissue Engineering Research Laboratory,
Department of Bioengineering,
IQS-School of Engineering,
Tissue Engineering Research Laboratory, Department of Bioengineering, IQS-School of Engineering