Faculty Bibliography

Cell-based cartilage repair procedures are becoming more widely available and have shown promising potential to treat a wide range of cartilage lesion types and sizes, particularly in the knee joint. More recently, techniques have evolved from 2-step techniques that use autologous chondrocyte expansion to 1-step techniques that make use of mesenchymal stem cells (MSCs) embedded onto biocompatible scaffolding. Our 1-step technique has been further developed to provide cell-based cartilage repair using MSCs that have the potential to be used in an off-the-shelf manner, without the need for autologous tissue harvest. Precursor MSCs can be isolated in abundance from the Wharton's jelly of umbilical cord tissue. These cells have been shown to have the desired capacity for proliferation, differentiation, and release of trophic factors that make them an excellent candidate for use in the clinical setting to provide cell-based restoration of hyaline-like cartilage. Although allogeneic in nature, these cells stimulate little or no host immune response and can be stored for long periods while maintaining viability. We present a technique of cartilage repair in the knee using Wharton's jelly-derived MSCs embedded onto scaffolding and implanted in a minimally invasive fashion using dry arthroscopy.

There is mounting evidence that the nuclear envelope, and particularly the lamina, plays a critical role in the mechanical and regulation properties of the cell and changes to the lamina can have implications for the physical properties of the whole cell. In this study we demonstrate that the optical stretcher can measure changes in the time-dependent mechanical properties of living cells with different levels of A-type lamin expression. Results from the optical stretcher shows a decrease in the deformability of cells as the levels of lamin A increases, for cells which grow both adherently and in suspension. Further detail can be probed by combining the optical stretcher with fluorescence microscopy to investigate the nuclear mechanical properties which show a larger decrease in deformability than for the whole cell.

Stem cell products, including manufactured red blood cells, require efficient sorting and purification methods to remove components potentially harmful for clinical application. However, standard approaches for cellular downstream processing rely on the use of specific and expensive labels (e.g. FACS or MACS). Techniques relying on inherent mechanical and physical properties of cells offer high-throughput scalable alternatives but knowledge of the mechanical phenotype is required. Here, we characterized for the first time deformability and size changes in CD34+ cells, and expelled nuclei, during their differentiation process into red blood cells at days 11, 14, 18 and 21, using Real-Time Deformability Cytometry (RT-DC) and Atomic Force Microscopy (AFM). We found significant differences (p < 0.0001; standardised mixed model) between the deformability of nucleated and enucleated cells, while they remain within the same size range. Expelled nuclei are smaller thus could be removed by size-based separation. An average Young's elastic modulus was measured for nucleated cells, enucleated cells and nuclei (day 14) of 1.04 ± 0.47 kPa, 0.53 ± 0.12 kPa and 7.06 ± 4.07 kPa respectively. Our identification and quantification of significant differences (p < 0.0001; ANOVA) in CD34+ cells mechanical properties throughout the differentiation process could enable development of new routes for purification of manufactured red blood cells.

We describe a quantitative, high-precision, high-throughput method for measuring the mechanical properties of cells in suspension with a microfluidic device, and for relating cell mechanical responses to protein expression levels. Using a high-speed (750 fps) charge-coupled device camera, we measure the driving pressure Δp, maximum cell deformation ε_max, and entry time t_entry of cells in an array of microconstrictions. From these measurements, we estimate population averages of elastic modulus E and fluidity β (the power-law exponent of the cell deformation in response to a step change in pressure). We find that cell elasticity increases with increasing strain ε_max according to E ∼ ε_max, and with increasing pressure according to E ∼ Δp. Variable cell stress due to driving pressure fluctuations and variable cell strain due to cell size fluctuations therefore cause significant variability between measurements. To reduce measurement variability, we use a histogram matching method that selects and analyzes only those cells from different measurements that have experienced the same pressure and strain. With this method, we investigate the influence of measurement parameters on the resulting cell elastic modulus and fluidity. We find a small but significant softening of cells with increasing time after cell harvesting. Cells harvested from confluent cultures are softer compared to cells harvested from subconfluent cultures. Moreover, cell elastic modulus increases with decreasing concentration of the adhesion-reducing surfactant pluronic. Lastly, we simultaneously measure cell mechanics and fluorescence signals of cells that overexpress the GFP-tagged nuclear envelope protein lamin A. We find a dose-dependent increase in cell elastic modulus and decrease in cell fluidity with increasing lamin A levels. Together, our findings demonstrate that histogram matching of pressure, strain, and protein expression levels greatly reduces the variability between measurements and enables us to reproducibly detect small differences in cell mechanics.

Cartilage injury of the knee that is associated with significant subchondral bone loss can result in great morbidity, and treatment options that provide durable repair are limited. Osteochondral autograft and allograft reconstruction of these lesions has been used extensively; however, these techniques often require a more invasive surgical exposure, and restoring the natural articular surface radius of curvature can be challenging, particularly in larger lesions. Cell-based repair of these lesions, using autologous chondrocytes in conjunction with bone grafting, has been used with success, although this procedure requires the patient to undergo 2 operations, and access is often restricted due to the high associated costs. Comparable medium-term clinical outcomes have been shown with scaffold-associated mesenchymal stem cell grafting, and this cell-based procedure may also be performed arthroscopically to minimize patient morbidity. In cases of cartilage injury associated with bone loss, this procedure has great potential to repair osteochondral injury when used in conjunction with bone grafting. We present the one-step arthroscopic technique of biologic inlay osteochondral reconstruction in the knee, using an autologous bone graft and a hyaluronic acid-based scaffold embedded with bone marrow aspirate concentrate, to treat full-thickness cartilage lesions associated with significant subchondral bone loss.

BACKGROUND:Articular cartilage injury is frequently encountered, yet treatment options capable of providing durable cartilage repair are limited. PURPOSE:To investigate the medium-term clinical outcomes of cartilage repair using a 1-stage technique of a hyaluronic acid-based scaffold with activated bone marrow aspirate concentrate (HA-BMAC) and compare results with those of microfracture. A secondary aim of this study was to identify specific patient demographic factors and cartilage lesion characteristics that are associated with superior outcomes. STUDY DESIGN:Cohort study; Level of evidence, 2. METHODS:) were treated with HA-BMAC or microfracture and were observed prospectively for 5 years. Patients were placed into the HA-BMAC group if the health insurance policy of the treating institution supported this option; otherwise, they were placed into the microfracture group. Objective and subjective clinical assessment tools were used preoperatively and at 2 and 5 years postoperatively to compare treatment outcomes. RESULTS:and nonsolitary lesions. Age greater than 45 years, large size of lesion, and treatment of multiple lesions were not associated with poorer outcome in patients treated with HA-BMAC. CONCLUSION:Repair of chondral injury using a hyaluronic acid-based scaffold with activated bone marrow aspirate concentrate provides better clinical outcomes and more durable cartilage repair at medium-term follow-up compared with microfracture. Positive short-term clinical outcomes can be achieved with either microfracture or HA-BMAC. Cartilage repair using HA-BMAC leads to successful medium-term outcomes independent of age or lesion size.

Cartilage lesions of the knee are a frequent finding; however, treatment options that are capable of restoring hyaline-like tissue are not routinely used. Cell-based technology such as autologous chondrocyte implantation may in some cases provide durable cartilage repair, but availability of this procedure is often restricted due to cost constraints. There have been promising outcomes reported with the use of scaffolds seeded with activated bone marrow aspirate concentrate in cases of chondral injury. There are clear advantages to cell-based cartilage repair techniques that are performed as a single-stage procedure, particularly when the repair technology can be used in a minimally invasive manner. We present an arthroscopic technique of cartilage repair using a hyaluronic acid-based scaffold associated with activated bone marrow aspirate concentrate. This technique is a cost-effective, minimally invasive, single-stage procedure that has the potential for routine use in a wide range of cartilage lesion types and locations.

Anterior shoulder dislocation in the athlete may result in an assortment of injuries that often benefit from surgical stabilization procedures. These injury patterns can be complex, requiring a multimodal approach to treatment. We present a rare case of a traumatic anterior shoulder dislocation in a teenage athlete that resulted in humeral avulsion of the glenohumeral ligament, rotator cuff tear, and axillary nerve palsy. Surgical treatment enabled return to football within 1 year of injury, and full function was restored.

PURPOSE: To evaluate the fixation integrity at time zero of a type I/III collagen patch secured to a chondral defect in the porcine knee using methods typically employed in autologous chondrocyte implantation (ACI) and matrix-assisted chondrocyte implantation. METHODS: Twenty-four porcine knee specimens underwent a medial parapatellar arthrotomy. A prefabricated template was used to create cartilage defects of 2 cm2 in the medial femoral condyle. A size-matched collagen patch was fashioned. Four methods of fixation to the chondral defect were analyzed: group 1-saline, group 2-fibrin glue around the periphery of the patch, group 3-fibrin glue applied to the base of the defect and around the periphery of the patch, group 4-6-0 vicryl suture and fibrin glue around the periphery of the patch. Collagen patch fixation was assessed at intervals of 60, 300, 600, 900, and 1,200 cycles from full extension to 90 degrees of flexion, performed manually without application of axial force. Patch fixation was evaluated by 2 independent observers using a customized scoring scale. RESULTS: Mean peripheral detachment of the patch and chondral defect uncovering remained less than 25% for all groups. Area of defect uncovering was significantly increased in group 2 compared with group 4 after 900 and 1,200 cycles (P = .0014 and P = .0025, respectively). Fibrin glue applied to the base of the defect, or suturing of the patch, reduced deformation significantly after 900 cycles. CONCLUSIONS: Suture increases the stability of fixation of a type I/III collagen patch to a chondral defect better than fibrin glue alone in the porcine knee after repetitive cycling, with respect to patch detachment and chondral defect uncovering. Application of fibrin glue to the base of the defect, or securing the patch with suture, decreases collagen patch deformation. CLINICAL RELEVANCE: In cases where minimally invasive techniques do not allow suture fixation of the collagen patch, scaffold fixation may be compromised during articular motion protocols typically used after second- and third-generation ACI procedures.

Microfluidic microdroplets have increasingly found application in biomolecular sensing as well as nanomaterials growth. More recently the synthesis of plasmonic nanostructures in microdroplets has led to surface-enhanced Raman spectroscopy (SERS)-based sensing applications. However, the study of nanoassembly in microdroplets has previously been hindered by the lack of on-chip characterization tools, particularly at early timescales. Enabled by a refractive index matching microdroplet formulation, dark-field spectroscopy is exploited to directly track the formation of nanometer-spaced gold nanoparticle assemblies in microdroplets. Measurements in flow provide millisecond time resolution through the assembly process, allowing identification of a regime where dimer formation dominates the dark-field scattering and SERS. Furthermore, it is shown that small numbers of nanoparticles can be isolated in microdroplets, paving the way for simple high-yield assembly, isolation, and sorting of few nanoparticle structures.