Low Oxygen Tension is Critical for the Culture of Human Mesenchymal Stem Cells with Strong Osteogenic Potential from Haemarthrosis Fluid

Abstract

Satisfactory osseous tissue integration of the soft tissue graft with bone is the mainstay of healing following surgical reconstruction of the anterior cruciate ligament (ACL). However, tissue remodelling is slow and significantly impacts on quality of life by delaying return to work and sport and accelerating the onset of degenerative diseases such as osteoarthritis. Delivery of multipotent human mesenchymal stem cells (hMSCs) at surgery could enhance osseous tissue integration. We aim to use hMSCs derived from haemarthrosis fluid (HF) (the intra-articular bleed accrued post-trauma) which is aspirated and discarded as clinical waste. With the aim of improving our bioprocessing methodologies for clinical translation we have investigated the effect of low oxygen tension on the derivation and osteogenic potential of this novel HF-hMSC population. Mononuclear cells were isolated from HF aspirated samples and divided for derivation and culture under normal or low oxygen tension. HF-hMSCs were derived from 100 % of cultures under low oxygen tension compared to 71 % for normal oxygen tension; this was coupled with increased CFU-Fs. We investigated the osteogenic potential and cellular health of HF-hMSC populations following ex vivo expansion. HF-hMSC populations showed enhanced matrix mineralisation and cellular health when differentiated under low oxygen tension. This positive effect of low oxygen on osteogenesis and cellular health was reduced with prolonged culture. These data demonstrate that derivation and culture of HF-hMSC populations under low oxygen tension will enable the translation of a cellular therapy for the treatment of broad patient numbers with optimal osteogenic potency and cellular vitality.

Updates in biological therapies for knee injuries: anterior cruciate ligament

“…An MSC-based tissue-engineering strategy applied at the time of surgery can be used to accelerate and enhance osseous integration of the graft tissue by providing a cellular component involved at ligament healing. A novel human MSC population derived from the hemarthrosis fluid within the joint at injury was identified [33,34]. The osteogenic response of this hemarthrosis-derived MSC was later proved to be enhanced when cultured with PLGA, as compared with control cultures, which can lead to an enhanced osseous integration of soft tissue grafts during ligament reconstruction [35].…”

Investigating the biological response of human mesenchymal stem cells to titanium surfaces

“…Human mesenchymal stem cells (HF-hMSCs) were derived as previously described [17][18][19]. Mononuclear cells were isolated by Ficoll® gradient centrifugation (700 × g, 20 min), collected by centrifugation (90,000 × g, 3 min) and seeded into T-75 culture flasks in MSC medium (alpha minimum essential medium (αMEM), 10% (v/v) foetal bovine serum (FBS), 5 ng/ml fibroblast growth factor-2 (FGF2)).…”

“…Cell lysate was prepared using 200-μl volumes of lysis buffer (0.5% Triton-X100 in DPBS) which was repeatedly rinsed over the surfaces using a pipette before overnight incubation at 4°C to allow complete penetration of the detergent and solubilisation of the biological material. The total amount of DNA in cultures was measured using the Quant-iT™ PicoGreen® dsDNA kit calibrated with known concentrations of λ double-stranded DNA as previously described [18]. Fluorescence from samples was measured at excitation: 484 nm/emission: 538 nm using a Fluoroskan I plate reader (MTX Lab Systems, Inc., Vienna, Virginia, USA).…”

“…Ti discs and Ti screws were transferred to empty wells of the 24-well cell culture cluster plate before a 4-h culture in a 500-μl volume of alamarBlue® (stock alamarBlue® reagent diluted by 1:10 in osteogenic medium; as previously described [18]. Equivalent reactions of Ti discs and Ti screws without cells were assembled in parallel to serve as a negative control.…”

“…In previous work we have identified a novel population of osteogenic human mesenchymal stem cells derived from haemarthrosis fluid, the intra-articular bleed that is aspirated from the joint space during the acute phase of injury [17,18]. An investigation of the performance of these cells (termed heamarthrosis fluid-derived human mesenchymal stem cell (HF-hMSCs)) with a biomaterial capable of facilitating cell delivery and retention at the site of surgery has demonstrated the attachment, spreading and osteogenic differentiation on microparticles manufactured from PLGA [19].…”