The role of energy dissipation of polymeric scaffolds in the mechanobiological modulation of chondrogenic expression

Mechanical stimulation has been proposed to induce chondrogenesis in cell-seeded scaffold. However, the effects of mechanical stimuli on engineered cartilage may vary substantially between different scaffolds. This advocates for the need to identify an overarching mechanobiological variable. We hypothesize that energy dissipation of scaffolds subjected to dynamic loading may be used as a mechanobiology variable. The energy dissipation would furnish a general criterion to adjust the mechanical stimulation favoring chondrogenesis in scaffold. Epiphyseal chondro-progenitor cells were then subject to unconfined compression two hours per day during four days in different scaffolds, which differ only by the level of dissipation they generated while keeping the same loading conditions. Scaffolds with higher dissipation levels upregulated the mRNA of chondrogenic markers. In contrast lower dissipation of scaffolds was associated with downregulation of chondrogenic markers. These results showed that energy dissipation could be considered as a mechanobiology variable in cartilage. This study also indicated that scaffold with energy dissipation level close to the one of cartilage favors chondrogenic expression when dynamical loading is present

Epiphyseal chondro-progenitors provide a stable cell source for cartilage cell therapy

Articular cartilage regeneration poses particularly tough challenges for implementing cell-­‐based therapies. Many cell types have been investigated looking for a balanced combination of responsiveness and stability, yet techniques are still far from defining a gold standard. The work presented focuses on the reliable expansion and characterization of a clinical-­‐grade human epiphyseal chondro-­‐progenitor (ECP) cell bank from a single tissue donation. A parental human ECP cell bank was established which provides the seed material for master and working cell banks. ECPs were investigated at both low and high cumulative population doublings looking at morphology, monolayer expansion kinetics, resistance to cryogenic shock, colony forming efficiency and cell surface markers. Three dimensional micro-­‐pellet assays were used to determine spontaneous extracellular matrix deposition at varying population doublings and monolayer 2D differentiation studies were undertaken to assess the propensity for commitment into other lineages and their stability. ECPs exhibited remarkable homogeneity in expansion with a steady proliferative potential averaging 3 population doublings over eight days. Surface marker analysis revealed no detectable contaminating subpopulations or population enrichment during prolonged culture periods. Despite a slight reduction in Sox9 expression levels at higher population doublings in monolayer, nuclear localization was equivalent both in monolayer and in micro-­‐pellet format. Equally, ECPs were capable of depositing glycosaminoglycans, producing aggrecan, collagen I and collagen II in 3D pellets both at after low and high population doublings indicating a stable spontaneous chondrogenic potential. Osteogenic induction was differentially restricted in low and high population doublings as observed by Von Kossa staining of calcified matrix, with a notable collagen X, MMP13 and ADAMTS5 down-­‐regulation. Rare adipogenic induction was seen as evidenced by cytoplasmic lipid accumulation detectable by Oil Red O staining. These findings highlight the reliability, stability and responsiveness of ECPs over prolonged culture, making them ideal candidates in defining novel strategies for cartilage regeneration

Mechanotransduction of epiphyseal chondro-progenitors in 3D hydrogel system.

Of the many external factors that affect cell behavior, mechanical cues are fundamental in modulating a cell’s phenotype. Biomimetic mechanosensing requires the three-dimensional encapsulation of cells within a controlled microenvironment. As musculoskeletal tissues are both routinely subjected and highly sensitive to mechanical cues, studying the effect of substrate properties on cellular behavior takes on a more complex angle when looked at in 3D, as is the case in vivo. This project will therefore focus on evaluating the phenotypic modulation of epiphyseal chondro-progenitors in hyaluronic acid based hydrogels both in static, free swelling culture conditions as well as the effect of dynamic unconfined compression, looking at differentially regulated cell markers which may prove fundamental for cartilage tissue regeneration

Epiphyseal Chondro-Progenitor Cell Therapy For Articular Cartilage Regeneration

Articular cartilage regeneration poses particularly tough challenges for implementing cell-based therapies. Many cell types have been investigated looking for a balanced combination of responsiveness and stability, yet techniques are still far from defining a gold standard. The work presented here focuses on the reliable expansion and characterization of a clinical-grade human epiphyseal chondro-progenitor [ECP] cell bank from a single tissue donation. ECPs were investigated at both low and high cumulative population doublings, tracking morphology, monolayer expansion kinetics and resistance to cryogenic shock. Three dimensional micro-pellet assays were used to determine spontaneous cartilage-like extracellular matrix deposition. Differentiation studies were undertaken to assess the propensity for commitment into other lineages and their stability. ECPs exhibited remarkable homogeneity in expansion with a steady proliferative potential and a stable population surface marker profile (CD14-, CD34-, CD45-, HLA-DP, DQ, DR-, and CD26+, CD44+, CD73+, CD90+, CD105+, CD166+, HLA-A,B,C+). ECPs also exhibit a stable spontaneous chondrogenic potential, depositing glycosaminoglycan rich matrix as well as Collagen I, Collagen II and display an inherent resistance to multilineage differentiation. ECPs were photoencapsulated in methacrylated hyaluronic acid hydrogels and subjected to dynamic compression. In response to 3D mechanostimulation, ECPs modulated the presentation of surface receptors for TGF|3, a potent chondrogenic morphogen. When co-encapsulated with bone marrow derived MSCs, the trend was reversed, pointing to potential crosstalk regulation between ECPs prone to shifting their baseline expression and the modulating MSCs. As a first step in defining a novel strategy for cartilage regeneration, we have conducted a GLP-grade pre-clinical safety study in goats to assess the effect of implanted ECPs in a full thickness cartilage defect. ECPs were delivered within a collagen-based matrix to optimize therapeutic cellular localization. The cell-laden construct is delivered in combination with microfracture to direct new tissue repair and remodeling. The results from the 3-months pre-clinical study highlight the safety of ECPs, the feasibility of the proposed treatment protocol as well as early indications as to their regenerative role and potential. The findings presented herein demonstrate the reliability, stability and responsiveness of Epiphyseal Chondro-Progenitor cells, granting them clear advantages for their use in defining novel strategies for cartilage regeneration

Temperature effect on connective tissue cells

Intervertebral discs (IVD) are soft tissues that have viscoelastic behavior. Under cyclic loading, viscoelastic tissues dissipate the mechanical loading through the production of heat. In IVD, it is hypothesized that heat cannot be convected from the nucleus pulposus (NP), since it is not perfused by the cardio-vascular system. This may result in local increase of temperature in the NP, and therefore may affect the metabolism of cells present in NP or its mechanical behavior. The overall goal of the project is to study the only thermal contribution on NP cells metabolism, without any mechanical effect. A screening of potential effects, due to different temperatures, will be performed on fetal cells and on cells extracted from bovine NP

Fetal Epiphyseal Chondrocytes Behavior within 3D Hydrogels

Of the many external factors that affect cell behavior, mechanical cues have been found to be fundamental in modulating a cell’s phenotype. Indeed, merely changing substrate stiffness in monolayer culture systems generates drastically different phenotypes in adult stem cells. As musculoskeletal tissues are both routinely subjected and highly sensitive to mechanical cues, studying the effect of substrate properties on cellular behavior takes on a more complex angle when looked at in 3D, as is the case in vivo. This project will therefore focus on (1) Defining the mechanical properties of a range of biocompatible 3D gels and (2) attempt to correlate that information with cellular mechanotransduction, precisely looking at matrix deposition and gene expression modulation in gel-encapsulated cells

Biomechanical Evaluation of Human Skin on Various Anatomical Sites (Semester Project)

Measures of skin elasticity, immediate retraction and distention as well as pigmentation and erythema will be conducted on various anatomical sites to compile a profile of skin properties