Rapid Chondrocyte Isolation for Tissue Engineering Applications: The Effect of Enzyme Concentration and Temporal Exposure on the Matrix Forming Capacity of Nasal Derived Chondrocytes.

Srujana Vedicherla,Conor Timothy Buckley

doi:10.1155/2017/2395138

Srujana Vedicherla, Conor Timothy Buckley

Open Access

https://doi.org/10.1155/2017/2395138

Copy DOI

Journal: BioMed research international	Publication Date: Jan 1, 2017
Citations: 24	License type: CC BY 4.0

Affiliation: Trinity College Dublin

Abstract

Laboratory based processing and expansion to yield adequate cell numbers had been the standard in Autologous Disc Chondrocyte Transplantation (ADCT), Allogeneic Juvenile Chondrocyte Implantation (NuQu®), and Matrix-Induced Autologous Chondrocyte Implantation (MACI). Optimizing cell isolation is a key challenge in terms of obtaining adequate cell numbers while maintaining a vibrant cell population capable of subsequent proliferation and matrix elaboration. However, typical cell yields from a cartilage digest are highly variable between donors and based on user competency. The overall objective of this study was to optimize chondrocyte isolation from cartilaginous nasal tissue through modulation of enzyme concentration exposure (750 and 3000 U/ml) and incubation time (1 and 12 h), combined with physical agitation cycles, and to assess subsequent cell viability and matrix forming capacity. Overall, increasing enzyme exposure time was found to be more detrimental than collagenase concentration for subsequent viability, proliferation, and matrix forming capacity (sGAG and collagen) of these cells resulting in nonuniform cartilaginous matrix deposition. Taken together, consolidating a 3000 U/ml collagenase digest of 1 h at a ratio of 10 ml/g of cartilage tissue with physical agitation cycles can improve efficiency of chondrocyte isolation, yielding robust, more uniform matrix formation.

Highlights

Degenerative defects in articular cartilage or cartilage-like tissues, such as disc nucleus pulposus, are a significant cause of morbidity and socioeconomic burden especially in the context of an active ageing population
While cellular repopulation in replenishing and regenerating the cartilaginous matrix has been established in the literature [1], there has been a paradigm shift in recent years, focusing on the role of primary cells or predifferentiated cells in the absence of growth factors that can maintain their phenotype in vivo [2, 3]
The overall objective of this study was to evaluate the effect of enzyme exposure, incubation time, and additional physical agitation cycles in optimizing chondrocyte isolation from nasal cartilage biopsies using the commonly employed collagenase enzyme

Summary

Introduction

Degenerative defects in articular cartilage or cartilage-like tissues, such as disc nucleus pulposus, are a significant cause of morbidity and socioeconomic burden especially in the context of an active ageing population. While cellular repopulation in replenishing and regenerating the cartilaginous matrix has been established in the literature [1], there has been a paradigm shift in recent years, focusing on the role of primary cells or predifferentiated cells in the absence of growth factors that can maintain their phenotype in vivo [2, 3]. A crucial step in these approaches is cell isolation, usually obtained through mechanical and enzymatic breakdown of a tissue biopsy and subsequent laboratory expansion in cell processing facilities. Cartilage is a relatively acellular tissue with only 5–10% of its volume consisting of chondrocytes [7]. In vivo, these cells reside within a BioMed Research International pericellular matrix as chondrons [8], surrounded by dense extracellular matrix (ECM) consisting of collagens and proteoglycans. A high cell density is critical for maximising chondrogenesis [10] and remains a pertinent issue in cartilage regeneration

Objectives

Methods

Results

Discussion

Conclusion