Osteogenic potential of reamer irrigator aspirator (RIA) aspirate collected from patients undergoing hip arthroplasty

Abstract

Intramedullary nailing preceded by canal reaming is the current standard of treatment for long-bone fractures requiring stabilization. However, conventional reaming methods can elevate intramedullary temperature and pressure, potentially resulting in necrotic bone, systemic embolism, and pulmonary complications. To address this problem, a reamer irrigator aspirator (RIA) has been developed that combines irrigation and suction for reduced-pressure reaming with temperature modulation. Osseous particles aspirated by the RIA can be recovered by filtration for use as an autograft, but the flow-through is typically discarded. The purpose of this study was to assess whether this discarded filtrate has osteogenic properties that could be used to enhance the total repair potential of aspirate. RIA aspirate was collected from five patients (ages 71-78) undergoing hip hemiarthroplasty. Osseous particles were removed using an open-pore filter, and the resulting filtrate (230 +/- 200 mL) was processed by Ficoll-gradient centrifugation to isolate mononuclear cells (6.2 +/- 5.2 x 10(6) cells/mL). The aqueous supernatant contained FGF-2, IGF-I, and latent TGF-beta1, but BMP-2 was below the limit of detection. The cell fraction included culture plastic-adherent, fibroblastic cells that displayed a surface marker profile indicative of mesenchymal stem cells and that could be induced along the osteogenic, adipogenic, and chondrogenic lineages in vitro. When compared to outgrowth cells from the culture of osseous particles, filtrate cells were more sensitive to seeding density during osteogenic culture but had similar capacity for chondrogenesis. These results suggest using RIA aspirate to develop improved, clinically expeditious, cost-effective technologies for accelerating the healing of bone and other musculoskeletal tissues.

Figure 1

RIA system. (A) System consists of reamer head, drive shaft, and tube assembly. (B) Closeup of tube assembly manifold, with irrigation and aspiration ports. (C) Aspirate is typically passed through a filter of optional mesh to collect osseous particles. Examples shown here are left: open pore, right: 750-μm mesh. Filtrate is collected in a sterile vessel (D). Reproduced by permission of Synthes, Inc. © Synthes, Inc. or its affiliates.

Figure 2

Derivation of filtrate and osseous particle-outgrowth cell populations. (A) Filtrate (left) and filtered osseous particles (right) collected with the RIA. (B) Left: filtrate was fractionated using a Ficoll density gradient. The fatty layer (1) was discarded; the aqueous supernatant (2) was collected for cytokine analysis; mononuclear cells were concentrated at the Ficoll/supernatant interface (3); additional plastic-adherent cells were found within the erythrocyte-rich pellet (4). Right: the osseous particles (boxed area) were rinsed five times with PBS to remove marrow components (image shows first rinse). (C) Left: fibroblastic colonies were derived from a plastic-adherent subpopulation of the mononuclear fraction. Right: cells with similar morphology were found to grow out of osseous particles (arrow). Scale bar = 200 μm.

Figure 3

Molecular and functional characterization of filtrate cells. (A) Flow cytometric analysis. Passage-2 cells were stained with PE- or APC-conjugated monoclonal antibodies specific for the cell surface markers CD44 (PE), CD45 (APC), CD90 (APC), and CD105 (PE). Histograms of the stains (black) are shown next to corresponding isotype controls (gray) for each surface marker. Numbers denote percentage positive cells within the univariate gates. (B) Multilineage differentiation. Left column: osteogenic cultures show Alizarin Red (top) and osteocalcin (bottom) staining at the site of mineralized nodules (scale bars = 200 μm). Center column: adipogenic conditions produced cells containing lipid vesicles, which are identified by positively Oil Red O staining (top) and negative FABP-4 immunofluorescence (bottom) relative to surrounding organelles (scale bars = 200 μm). Right column: chondrogenic cultures exhibit positive staining for Toluidine Blue (top; scale bar = 500 μm) and aggrecan core protein (bottom; scale bar = 100 μm) throughout the aggregate.

Figure 4

Functional comparison of filtrate versus osseous particle-outgrowth populations. (A) Cells were plated at graded seeding densities (10²–10⁴ cells/cm²) and cultured with or without osteogenic stimuli (OST) for 28 days. Left: Alizarin Red staining indicates density-dependent mineralization of filtrate cultures, whereas staining directly correlated with seeding density for particle outgrowth cells. Right: measurement of calcium extracted from parallel cultures (n = 3 wells/treatment) supports Alizarin Red trends. An * (filtrate) or @ (outgrowth) denotes a significant increase (p <0.05) compared to no-OST control, & (filtrate) or # (outgrowth) denotes a significant difference from 10² cells/cm², and % (outgrowth) denotes significant difference from 10³ cells/cm². (B) Cells were cultured as aggregates with or without TGF-β3 for 6 weeks. Toluidine Blue staining indicates proteoglycan synthesis within TGF-stimulated aggregates (middle column) compared to no-TGF controls (left; scale bar = 500 μm). Right column: increased magnification (scale bar = 100 μm) of aggregates from middle column.

Figure 5

Differentiation potential of Ficoll interface versus erythrocyte-rich pellet subpopulations. (A) Left: 30-mL portions of filtrate were loaded onto 15-mL Ficoll-Pacque and fractionated by centrifugation. Right: cells within the Ficoll interface (FI) and the erythrocyte-rich (ERP) pellet fractions were cultured at 5 × 10⁷ nucleated cells/flask. Spindle-shaped cells with rapid proliferation capacity were observed in both cultures (scale bar = 200 μm). (B) FI and ERP cells were expanded to passage 2 and either cultured at various seeding densities for ostegenesis (28 days) or as cell aggregates for chondrogenesis (42 days). Alizarin Red staining indicates density-dependent mineralization for both populations. Toluidine Blue staining demonstrates similar responsiveness to TGF-β3 (scale bar = 500 μm).

Figure 6

Proposed utilization of filtrate osteogenic potential. Rather than collecting only coarse osseous particles, reaming aspirate can be concentrated intraoperatively by either centrifugation or fine filtration and applied to a defect site in order to accelerate healing. Prior to reimplantation, osteoprogenitor cells within the concentrate can be treated with osteogenic stimuli.