Enhancing the osteogenic efficacy of human bone marrow aspirate: concentrating osteoprogenitors using wave-assisted filtration.

BACKGROUND: Recent approaches have sought to harness the potential of stem cells to regenerate bone that is lost as a consequence of trauma or disease. Bone marrow aspirate (BMA) provides an autologous source of osteoprogenitors for such applications. However, previous studies indicated that the concentration of osteoprogenitors present in BMA is less than required for robust bone regeneration. We provide further evidence for the importance of BMA enrichment for skeletal tissue engineering strategies using a novel acoustic wave-facilitated filtration strategy to concentrate BMA for osteoprogenitors, clinically applicable for intraoperative orthopedic use. METHODS: Femoral BMA from 15 patients of an elderly cohort was concentrated for the nucleated cell fraction against erythrocytes and excess plasma volume via size exclusion filtration facilitated by acoustic agitation. The effect of aspirate concentration was assessed by assays for colony formation, flow cytometry, multilineage differentiation and scaffold seeding efficiency. RESULTS: BMA was filtered to achieve a mean 4.2-fold reduction in volume with a corresponding enrichment of viable and functional osteoprogenitors, indicated by flow cytometry and assays for colony formation. Enhanced osteogenic and chondrogenic differentiation was observed using concentrated aspirate and enhanced cell-seeding efficiency onto allogeneic bone graft as an effect of osteoprogenitor concentration relative specifically to the concentration of erythrocytes in the aspirate. CONCLUSIONS: These studies provide evidence for the importance of BMA nucleated cell concentration for both cell differentiation and cell seeding efficiency and demonstrate the potential of this approach for intraoperative application to enhance bone healing.

Novel technology to provide an enriched therapeutic cell concentrate from bone marrow aspirate.

Current strategies to repair fractures rely on orthopaedic surgeons harvesting bone from one area of the body, typically pelvis and transferring it to the fracture site. The amount of tissue available is therefore limited, requiring a second surgical procedure and often causing the patient long term pain. An alternative approach is utilise therapeutic cells contained within bone marrow aspirate during the primary procedure. The number of therapeutic cells within a fresh aspirate is insufficient to provide clinically acceptable bone healing in a timescale that is satisfactory to the surgeon and the patient. Therefore methods to efficiently concentrate bone marrow in the clinical setting are required. Centrifugation is the current method of choice but has limitations in that it requires large capital equipment, servicing and there are potential issues of tissue contamination. We have developed a novel, acoustically-assisted filtration device that addresses these limitations, delivering a concentrated bone marrow in a point of care, single use, fully disposable, compact device. An additional advantage is that the level of concentration required can be specified by the end user. The resulting bone marrow concentrate has been characterised in terms of cell number, viability and osteogenic potential using flow cytometry and alkaline phosphatase assay. When compared to recent clinical studies using bone marrow to repair non-union fractures, the findings from our work suggest that the bone marrow concentrate is likely to be highly therapeutic and clinically efficacious as a bone fracture repair strategy. A product concept for use in the clinical setting is presented.

Pulsed low intensity ultrasound enhances mineralisation in preosteoblast cells.

Pulsed low intensity ultrasound has been shown to be highly efficacious in the treatment of nonunion fractures and in the acceleration of fresh fracture healing. MC3T3-E1 subclone 14 cells were cultured for up to 25 days either with or without a daily treatment with low intensity pulsed ultrasound. It was determined that, on day 10 there was a dramatic increase in alkaline phosphatase and MMP-13 mRNA levels detected in ultrasound-treated cultures compared with untreated controls. The activity of alkaline phosphatase was significantly increased at days 6, 8 and 10. On day 10, the amount of mineralisation within cultures, assessed using alizarin red staining, was significantly increased in ultrasound-treated cultures compared with untreated controls. These results suggest that one of the mechanisms that low intensity pulsed ultrasound has on fracture repair is to enhance the process of endochondral ossification where the soft callus is converted to mineralised hard callus.