Abstract
Summary Statement
Mesenchymal stem cells from minced umbilical cord fragments may represent a valuable cell population for cartilage and bone tissue engineering
Introduction
A promising approach for cartilage and bone repair is the use of umbilical cord mesenchymal stem cell (UC-MSC)-based tissue engineering. Through a simple and efficient protocol based on mincing the umbilical cord, a consistent number of multipotent UC-MSCs can be obtained. The aim of this in-vitro study is to investigate the pluripotency of UC-MSCs and, in particular, the chondrogenic and osteogenic potential of UC-MSCs grown in tridimensional scaffold, in order to identify a potential clinical relevance for patients who might benefit from MSCs-therapy.
Materials/Methods
Fresh UC samples from women with healthy pregnancies were retrieved at the end of caesarean deliveries. The UC samples were manually minced without any enzymatic digestion into very small fragments (less than 4 mm length) and cultivated in an MSC expansion medium. At day 14, the UC tissue was removed and adherent cells were allowed to expand for 2 additional weeks. At day 28, the adherent cells were collected and replated until confluence was reached (Passage 1 or P1). Immunophenotypic characterization, Fluorescence In Situ Hybridization (FISH), telomere length analysis, Immunosuppression of T lymphocyte cultures, and multilineage differentiation was performed in UC-MSCs at P1 or P2. For chondrogenic differentiation on scaffold, UC-MSCs at P2 were loaded either on a hyaluronic-acid (HA)-felt(Hyaff-11) or on a collagen-I/III membrane(Chondro-gide), with a coating of fibrin glue(Tisseel), and grown in chondrogenic medium both in normoxic and hypoxic(10%O2) conditions. After 1 month, sections were stained with haematoxylin/eosin and Safranin-O. Expression of chondrocyte markers(sox-9, type II collagen) and hypoxic markers(HIFs) was assessed using immunofluorescence(IF). For osteogenic differentiation on scaffold, UC-MSCs at P2 were loaded into a bone-graft-substitute(Orthoss) and grown in osteogenic medium. After 10, 20,30 days, sections were stained with haematoxylin/eosin and expression of osteocalcin and RunX2 was assessed using immunofluorescence.
Results
At P1, we obtained a mean value of 22.8 × 106 cells(SD 1.7) from each UC, corresponding to 0.66 (SD 0.14) × 106 cells/gram of UC. Cells were positive for CD73, CD90, CD105, CD44, CD29 and HLA-I, and negative for CD34 and HLA-class II, with a subpopulation that was negative for both HLA-I and HLA-II. Results from FISH demonstrated that 95–100% of UC-MSCs were of fetal origin. Telomere length of UC-MSCs was similar to that of Bone Marrow (BM) MSC from young donors (aged 20–30 years). At 5 days, the supernatant of UC-MSC cultures had immunosuppressive activity upon T-Lymphocyte cultures. The mixed UC-MSC population was able to differentiate towards osteogenic (monolayer), adipogenic, miogenic and chondrogenic (pellet culture). Chondrogenic differentiation on scaffolds was also confirmed; hypoxic condition improved the expression of chondrogenic markers. Osteogenic differentiation on scaffold was also confirmed after 20 days of culture.
Discussion/Conclusions
These results suggest that the straightforward procedure of collecting UC-MCS at P1 from minced umbilical cord fragments can achieve a valuable cell population that seems to have a potential for orthopaedics tissue engineering such as the on-demand cell delivery using chondrogenic or osteogenic scaffold. The concept of this study may have a clinical relevance as a future hypothetical option for single-stage cartilage repair and bone regeneration.
