Chondrogenesis of infrapatellar fat pad derived adipose stem cells in 3D printed chitosan scaffold.

Ken Ye,Kathy Traianedes,Anita Quigley,Damian E Myers,Gordon G Wallace,Peter F M Choong,Simon E Moulton,Johnson Chung,Raed Felimban,Gwendolen Reilly

doi:10.1371/journal.pone.0099410

Abstract

Infrapatellar fat pad adipose stem cells (IPFP-ASCs) have been shown to harbor chondrogenic potential. When combined with 3D polymeric structures, the stem cells provide a source of stem cells to engineer 3D tissues for cartilage repair. In this study, we have shown human IPFP-ASCs seeded onto 3D printed chitosan scaffolds can undergo chondrogenesis using TGFβ3 and BMP6. By week 4, a pearlescent, cartilage-like matrix had formed that penetrated the top layers of the chitosan scaffold forming a ‘cap’ on the scaffold. Chondrocytic morphology showed typical cells encased in extracellular matrix which stained positively with toluidine blue. Immunohistochemistry demonstrated positive staining for collagen type II and cartilage proteoglycans, as well as collagen type I. Real time PCR analysis showed up-regulation of collagen type II, aggrecan and SOX9 genes when IPFP-ASCs were stimulated by TGFβ3 and BMP6. Thus, IPFP-ASCs can successfully undergo chondrogenesis using TGFβ3 and BMP6 and the cartilage-like tissue that forms on the surface of 3D-printed chitosan scaffold may prove useful as an osteochondral graft.

Highlights

Articular cartilage defects have limited capacity for selfregeneration and healing
Materials Materials used for infrapatellar fat pad (IPFP)-adipose stem cells (ASC) isolation, culture and differentiation are listed as the following: Dulbecco’s phosphate-buffered saline (D-PBS), fetal bovine serum (FBS), antibiotic/antimycotic solution (Amphotericin B, Penicillin, Streptomycin 1006), glutamax, L-ascorbic acid 2-phosphate, transforming growth factor beta-3 (TGFb3), and HEPES buffer were purchased from GIBCO, Life Technologies Corporation (Carlsbad, CA, USA); Red cell lysis buffer, Dulbecco’s modified eagle medium (DMEM), insulin-transferring-selenium (ITS), dexamethasone, and 0.1% EDTA/0.25% trypsin were from Sigma-Aldrich
Histology and Immunohistochemistry haematoxylin and eosin (H&E) staining of chondrogenic pellets showed a change in cellular morphology towards a chondrocytic phenotype showing larger cells encapsulated in lacunae when compared to control pellets

Summary

Introduction

Articular cartilage defects have limited capacity for selfregeneration and healing. Cartilage damage often results in pain and loss of function for the patient and often accelerates the development of osteoarthritis in the joint. Tissue engineering may offer treatment options that could overcome the limitations of current management options. The use of autologous chondrocytes is limited by major factors, including donor site morbidity, and chondrocytes are limited in number comprising of only 5–10% of cartilage tissue, require expansion which may lead to dedifferentiation [7,17,18,19,20]. Due to these limitations, mature chondrocytes are not ideal candidate cells to use in tissue engineering constructs

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