Image-Based Feedback of Multi-Component Microdroplets for Ultra-Monodispersed Library Preparation.

Using devices with microfluidic channels can allow for precise control over liquids flowing through them. Merging flows of immiscible liquids can create emulsions with highly monodispersed microdroplets within a carrier liquid, which are ideal for miniaturised reaction vessels which can be generated with a high throughput of tens of thousands of droplets per second. Control of the size and composition of these droplets is generally performed by controlling the pumping system pushing the liquids into the device; however, this is an indirect manipulation and inadequate if absolute precision is required in the size or composition of the droplets. In this work, we extend the previous development of image-based closed-loop feedback control over microdroplet generation to allow for the control of not only the size of droplets but also the composition by merging two aqueous flows. The feedback allows direct control over the desired parameters of volume and ratio of the two components over a wide range of ratios and outperforms current techniques in terms of monodispersity in volume and composition. This technique is ideal for situations where precise control over droplets is critical, or where a library of droplets of different concentrations but the same volume is required.

Particulate and drug-induced toxicity assessed in novel quadruple cell human primary hepatic disease models of steatosis and pre-fibrotic NASH.

In an effort to replace, reduce and refine animal experimentation, there is an unmet need to advance current in vitro models that offer features with physiological relevance and enhanced predictivity of in vivo toxicological output. Hepatic toxicology is key following chemical, drug and nanomaterials (NMs) exposure, as the liver is vital in metabolic detoxification of chemicals as well as being a major site of xenobiotic accumulation (i.e., low solubility particulates). With the ever-increasing production of NMs, there is a necessity to evaluate the probability of consequential adverse effects, not only in health but also in clinically asymptomatic liver, as part of risk stratification strategies. In this study, two unique disease initiation and maintenance protocols were developed and utilised to mimic steatosis and pre-fibrotic NASH in scaffold-free 3D liver microtissues (MT) composed of primary human hepatocytes, hepatic stellate cells, Kupffer cells and sinusoidal endothelial cells. The characterized diseased MT were utilized for the toxicological assessment of a panel of xenobiotics. Highlights from the study included: 1. Clear experimental evidence for the pre-existing liver disease is important in the augmentation of xenobiotic-induced hepatotoxicity and 2. NMs are able to activate stellate cells. The data demonstrated that pre-existing disease is vital in the intensification of xenobiotic-induced liver damage. Therefore, it is imperative that all stages of the wide spectrum of liver disease are incorporated in risk assessment strategies. This is of significant consequence, as a substantial number of the general population suffer from sub-clinical liver injury without any apparent or diagnosed manifestations.

Computational optical imaging with a photonic lantern.

The thin and flexible nature of optical fibres often makes them the ideal technology to view biological processes in-vivo, but current microendoscopic approaches are limited in spatial resolution. Here, we demonstrate a route to high resolution microendoscopy using a multicore fibre (MCF) with an adiabatic multimode-to-single-mode "photonic lantern" transition formed at the distal end by tapering. We show that distinct multimode patterns of light can be projected from the output of the lantern by individually exciting the single-mode MCF cores, and that these patterns are highly stable to fibre movement. This capability is then exploited to demonstrate a form of single-pixel imaging, where a single pixel detector is used to detect the fraction of light transmitted through the object for each multimode pattern. A custom computational imaging algorithm we call SARA-COIL is used to reconstruct the object using only the pre-measured multimode patterns themselves and the detector signals.

Image-Based Single Cell Sorting Automation in Droplet Microfluidics.

The recent boom in single-cell omics has brought researchers one step closer to understanding the biological mechanisms associated with cell heterogeneity. Rare cells that have historically been obscured by bulk measurement techniques are being studied by single cell analysis and providing valuable insight into cell function. To support this progress, novel upstream capabilities are required for single cell preparation for analysis. Presented here is a droplet microfluidic, image-based single-cell sorting technique that is flexible and programmable. The automated system performs real-time dual-camera imaging (brightfield & fluorescent), processing, decision making and sorting verification. To demonstrate capabilities, the system was used to overcome the Poisson loading problem by sorting for droplets containing a single red blood cell with 85% purity. Furthermore, fluorescent imaging and machine learning was used to load single K562 cells amongst clusters based on their instantaneous size and circularity. The presented system aspires to replace manual cell handling techniques by translating expert knowledge into cell sorting automation via machine learning algorithms. This powerful technique finds application in the enrichment of single cells based on their micrographs for further downstream processing and analysis.

Complete Repair of Massive, Retracted, and "Non-Repairable" Tears of the Rotator Cuff: The Anatomic Vector Repair.

Massive and retracted tears of the supraspinatus and infraspinatus tendons of the rotator cuff are associated with great pain and disability and may be considered "non-repairable," depending on the extent of injury and the experience of the treating clinician. The technique of anatomic vector repair of the rotator cuff is a surgical treatment method that enables the surgeon to accurately characterize the injury pattern and successfully repair many of these debilitating injuries anatomically in a stepwise manner, often in cases that would have otherwise been treated with a less preferable surgical procedure that does not restore native anatomy.

Correction: Deformability-induced lift force in spiral microchannels for cell separation.

Correction for 'Deformability-induced lift force in spiral microchannels for cell separation' by Ewa Guzniczak et al., Lab Chip, 2020, 20, 614-625.

Purifying stem cell-derived red blood cells: a high-throughput label-free downstream processing strategy based on microfluidic spiral inertial separation and membrane filtration.

Cell-based therapeutics, such as in vitro manufactured red blood cells (mRBCs), are different to traditional biopharmaceutical products (the final product being the cells themselves as opposed to biological molecules such as proteins) and that presents a challenge of developing new robust and economically feasible manufacturing processes, especially for sample purification. Current purification technologies have limited throughput, rely on expensive fluorescent or magnetic immunolabeling with a significant (up to 70%) cell loss and quality impairment. To address this challenge, previously characterized mechanical properties of umbilical cord blood CD34+ cells undergoing in vitro erythropoiesis were used to develop an mRBC purification strategy. The approach consists of two main stages: (a) a microfluidic separation using inertial focusing for deformability-based sorting of enucleated cells (mRBC) from nuclei and nucleated cells resulting in 70% purity and (b) membrane filtration to enhance the purity to 99%. Herein, we propose a new route for high-throughput (processing millions of cells/min and mls of medium/min) purification process for mRBC, leading to high mRBC purity while maintaining cell integrity and no alterations in their global gene expression profile. Further adaption of this separation approach offers a potential route for processing of a wide range of cellular products.

Deformability-induced lift force in spiral microchannels for cell separation.

Cell sorting and isolation from a heterogeneous mixture is a crucial task in many aspects of cell biology, biotechnology and medicine. Recently, there has been an interest in methods allowing cell separation upon their intrinsic properties such as cell size and deformability, without the need for use of biochemical labels. Inertial focusing in spiral microchannels has been recognised as an attractive approach for high-throughput cell sorting for myriad point of care and clinical diagnostics. Particles of different sizes interact to a different degree with the fluid flow pattern generated within the spiral microchannel and that leads to particles ordering and separation based on size. However, the deformable nature of cells adds complexity to their ordering within the spiral channels. Herein, an additional force, deformability-induced lift force (FD), involved in the cell focusing mechanism within spiral microchannels has been identified, investigated and reported for the first time, using a cellular deformability model (where the deformability of cells is gradually altered using chemical treatments). Using this model, we demonstrated that spiral microchannels are capable of separating cells of the same size but different deformability properties, extending the capability of the previous method. We have developed a unique label-free approach for deformability-based purification through coupling the effect of FD with inertial focusing in spiral microchannels. This microfluidic-based purification strategy, free of the modifying immuno-labels, allowing cell processing at a large scale (millions of cells per min and mls of medium per minute), up to high purities and separation efficiency and without compromising cell quality.

Assessment of nanomaterial-induced hepatotoxicity using a 3D human primary multi-cellular microtissue exposed repeatedly over 21 days - the suitability of the in vitro system as an in vivo surrogate.

BACKGROUND: With ever-increasing exposure to engineered nanomaterials (NMs), there is an urgent need to evaluate the probability of consequential adverse effects. The potential for NM translocation to distal organs is a realistic prospect, with the liver being one of the most important target organs. Traditional in vitro or ex vivo hepatic toxicology models are often limiting (i.e. short life-span, reduced metabolic activity, lacking important cell populations, etc.). In this study, we scrutinize a 3D human liver microtissue (MT) model (composed of primary hepatocytes and non-parenchymal cells). This unique experiment benefits from long-term (3 weeks) repeated very low exposure concentrations, as well as incorporation of recovery periods (up to 2 weeks), in an attempt to account for the liver's recovery capacity in vivo. As a means of assessing the toxicological potential of NMs, cell cytotoxicity (cell membrane integrity and aspartate aminotransferase (AST) activity), pro/anti-inflammatory response and hepatic function were investigated. RESULTS: The data showed that 2 weeks of cell culture might be close to limits before subtle ageing effects start to overshadow low sub-lethal NM-induced cellular responses in this test system (adenylate kinase (AK) cytotoxicity assay). We showed that in vitro AST measurement are not suitable in a nanotoxicological context. Moreover, the cytokine analysis (IL6, IL8, IL10 and TNF-α) proved useful in highlighting recovery periods as being sufficient for allowing a reduction in the pro-inflammatory response. Next, low soluble NM-treated MT showed a concentration-dependent penetration of materials deep into the tissue. CONCLUSION: In this study the advantages and pitfalls of the multi-cellular primary liver MT are discussed. Furthermore, we explore a number of important considerations for allowing more meaningful in vitro vs. in vivo comparisons in the field of hepatic nanotoxicology.

Long-term Clinical Outcomes of One-Stage Cartilage Repair in the Knee With Hyaluronic Acid-Based Scaffold Embedded With Mesenchymal Stem Cells Sourced From Bone Marrow Aspirate Concentrate.

BACKGROUND: Cell-based cartilage repair performed as a single-stage procedure is an important advancement in the treatment of full-thickness cartilage injury and has potential for widespread clinical use. PURPOSE: To investigate the long-term clinical outcomes of cartilage repair in the knee with a hyaluronic acid-based scaffold embedded with bone marrow aspirate concentrate (HA-BMAC) for the treatment of full-thickness cartilage injury. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Patients underwent treatment of full-thickness chondral injury in the knee with HA-BMAC and were followed prospectively for a minimum of 6 years. Clinical outcomes were examined with patient-reported scoring instruments that consisted of the Tegner Activity Scale, International Knee Documentation Committee (IKDC) subjective score, visual analog scale, and Knee injury and Osteoarthritis Outcome Score (KOOS). Comparative analysis of pre- and postoperative scores was performed, and the effects of patient age, body mass index, lesion size, number of treated lesions, and concurrent treatment with associated procedures were examined. RESULTS: Twenty-three patients (mean age, 48.5 years) were followed prospectively for a mean 8 years (range, 6-10 years). Median cartilage lesion size was 6.5 cm2 (range, 2-27 cm2). At final follow-up, median Tegner, visual analog scale, and IKDC subjective scores were 4, 0.3, and 85, respectively. Final median KOOS subset scores were as follows: Pain, 94; Symptoms, 89; Activities of Daily Living, 99; Sports/Recreation, 85; and Quality of Life, 85. All scores were significantly increased at final follow-up ( P < .001). Comparable median outcome scores were demonstrated after categorization of patients by age, lesion size, treatment of multiple lesions, treatment of multiple knee compartments, and treatment by associated procedures. Rank correlation analysis demonstrated a negative correlation between patient age and final outcome scores of the IKDC, Tegner, and KOOS subsets of Pain, Activities of Daily Living, and Sports/Recreation. No associations were identified between body mass index or lesion size and outcome scores. CONCLUSION: Repair of full-thickness cartilage injury in the knee with a HA-BMAC provides good to excellent clinical outcomes at long-term follow-up in the treatment of small to large lesions. Cartilage repair with HA-BMAC leads to comparatively successful long-term outcomes in the treatment of small or large lesions, single or multiple lesions, and lesions in 1 or 2 compartments, as well as in cases of associated lesion treatment. While good outcomes can be expected among treated patients >45 years of age, outcomes may be comparatively more successful in younger patients.

Surgical repair of osteochondral lesions of the talus using biologic inlay osteochondral reconstruction: Clinical outcomes after treatment using a medial malleolar osteotomy approach compared to an arthroscopically-assisted approach.

BACKGROUND: Surgical treatment of osteochondral lesions of the talus affecting the medial aspect of the talar dome is typically performed using medial malleolar osteotomy to optimize access. This study compares clinical outcomes of lesions repaired using biologic inlay osteochondral reconstruction in patients who did or did not undergo medial malleolar osteotomy, depending on defect dimensions. METHODS: Patients treated for osteochonral lesions of the talus through a medial mallolar approach or arthroscopically-assisted approach were prospectively followed. Assessment tools consisted of the visual analogue scale (VAS) and the American Orthopaedic Foot and Ankle Society Ankle-Hindfoot score (AOFAS). The magnetic resonance observation of cartilage repair tissue (MOCART) score was used postoperatively. RESULTS: Data for 24 patients (mean age 34years, mean follow-up 22 months) was analyzed. Mean preoperative/final AOFAS and VAS in those who underwent osteotomy were 57.7/81.2 and 5.7/1.9 (p<0.001), respectively. In those who underwent arthroscopically-assisted reconstruction, mean preoperative/final AOFAS and VAS were 54.4/84.0 and 7.6/2.0 (p<0.001), respectively. There was no difference in mean MOCART score (p=0.662) for those treated with osteotomy (67.3) compared to those without (70.8). CONCLUSIONS: Osteochondral lesions of the talar dome can be treated successfully by biological inlay osteochondral reconstruction technique without medial malleolar osteotomy, with good to excellent clinical outcomes expected. MRI demonstrates good integration of the graft into surrounding tissue.

Long-term Outcomes of Primary Repair of the Anterior Cruciate Ligament Combined With Biologic Healing Augmentation to Treat Incomplete Tears.

BACKGROUND: Surgical treatment to repair partial anterior cruciate ligament (ACL) injury without reconstruction has demonstrated inconsistent clinical success. PURPOSE: To examine the long-term clinical outcomes of primary ACL repair combined with biologic healing augmentation in patients with symptomatic partial ACL tears. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: 50 patients (mean age, 29.5 years) with a partial ACL tear and symptomatic knee instability were treated with primary ligament repair in conjunction with marrow stimulation and followed prospectively for a mean duration of 10.2 years (range, 5.3-14.3 years). Comparative analysis of preinjury, preoperative, and postoperative scores using patient-reported assessment instruments was performed to examine clinical outcomes. Correlation of final outcome scores with patient age, type of ACL tear, side-to-side difference in ligamentous laxity, and body mass index (BMI) was performed through use of Spearman rank analysis. RESULTS: 44 patients were available for assessment at final follow-up. The median Tegner Activity Scale score of 7 at final follow-up was the same as the preinjury median score of 7 ( P = .128). The mean Marx Activity Scale, International Knee Documentation Committee (IKDC) Subjective, and Lysholm Knee Questionnaire scores were 10.8, 90.4, and 96.2, respectively, at final follow-up. Mean final Knee injury and Osteoarthritis Outcome Score (KOOS) subset assessments of Pain, Symptoms, Activities of Daily Living, Sports, and Quality of Life were 98.6, 97.5, 99.7, 94.3, and 95.6, respectively. Secondary ACL insufficiency occurred in 27% of patients. Clinical outcome scores were similar for all scoring instruments between patients treated for an associated diagnosis of meniscal or articular cartilage injury. No significant correlations of age, BMI, ACL tear type, or laxity and final IKDC Subjective, Lysholm, or KOOS scores were found. Analysis revealed a negative correlation of patient age and Tegner score at preinjury ( rs = -0.333, P = .022) and at final follow-up ( rs = -0.376, P = .013). The mean side-to-side difference in ligamentous laxity of 3.4 mm at short-term follow-up in those patients who developed secondary ACL insufficiency over the duration of follow-up was significantly greater than the mean of 0.9 mm in those who did not ( P = .010). CONCLUSION: Primary ACL repair combined with biologic healing augmentation to treat select cases of knee instability secondary to incomplete ACL rupture demonstrated good to excellent long-term outcomes in this cohort for those patients who did not experience secondary ACL insufficiency, with high rates of restoration of knee stability and return to preinjury athletic activities. The rate of secondary treatment for recurrent ACL insufficiency over the course of long-term follow-up was greater than would be expected for primary ACL reconstruction. Greater side-to-side differences in objective findings of ligamentous laxity were identified at shorter term follow-up in the patients who later went on to experience symptomatic secondary ACL insufficiency, compared with those who maintained stability long term.

Editorial Commentary: Acetabular Cartilage Repair: A Critically Important Frontier in Hip Preservation.

Articular cartilage damage to the acetabulum is frequently associated with femoroacetabular impingement, and there are considerable long-term implications for such injury with regard to maintenance of a healthy hip joint and quality of life. Developing treatments capable of restoring articular cartilage to acetabular cartilage defects is of great importance if hip preservation treatments are to be successful. Ideally, such methods should be performed in a minimally invasive manner and be capable of restoring durable repair tissue that reconstitutes a healthy osteochondral unit and that continues to function effectively over the long term.

A comparison of methods to assess cell mechanical properties.

The mechanical properties of cells influence their cellular and subcellular functions, including cell adhesion, migration, polarization, and differentiation, as well as organelle organization and trafficking inside the cytoplasm. Yet reported values of cell stiffness and viscosity vary substantially, which suggests differences in how the results of different methods are obtained or analyzed by different groups. To address this issue and illustrate the complementarity of certain approaches, here we present, analyze, and critically compare measurements obtained by means of some of the most widely used methods for cell mechanics: atomic force microscopy, magnetic twisting cytometry, particle-tracking microrheology, parallel-plate rheometry, cell monolayer rheology, and optical stretching. These measurements highlight how elastic and viscous moduli of MCF-7 breast cancer cells can vary 1,000-fold and 100-fold, respectively. We discuss the sources of these variations, including the level of applied mechanical stress, the rate of deformation, the geometry of the probe, the location probed in the cell, and the extracellular microenvironment.

Arthroscopic Cartilage Lesion Preparation in the Human Cadaveric Knee Using a Curette Technique Demonstrates Clinically Relevant Histologic Variation.

PURPOSE: To examine the quality of arthroscopic cartilage debridement using a curette technique by comparing regional and morphologic variations within cartilage lesions prepared in human cadaveric knee specimens for the purpose of cartilage repair procedures. A secondary aim was to compare the histologic properties of cartilage lesions prepared by surgeons of varying experience. METHODS: Standardized cartilage lesions (8 mm × 15 mm), located to the medial/lateral condyle and medial/lateral trochlea were created within 12 human cadaver knees by 40 orthopaedic surgeons. Participants were instructed to create full-thickness cartilage defects within the marked area, shouldered by uninjured vertical walls of cartilage, and to remove the calcified cartilage layer, without violating the subchondral plate. Histologic specimens were prepared to examine the verticality of surrounding cartilage walls at the front and rear aspects of the lesions, and to characterize the properties of the surrounding cartilage, the cartilage wall profile, the debrided lesion depth, bone sinusoid access, and the bone surface profile. Comparative analysis of cartilage wall verticality measured as deviation from perpendicular was performed, and Spearman's rank correlation analysis was used to examine associations between debrided wall verticality and surgeon experience. RESULTS: Mean cartilage wall verticality relative to the base of the lesion was superior at the rear aspect of the lesion compared to the front aspect (12.9° vs 29.2°, P < .001). Variability was identified in the morphology of the surrounding cartilage (P < .001), cartilage wall profile (P = .016), debrided lesion depth (P = .028), bone surface profile (P = .040), and bone sinusoid access (P = .009), with sinusoid access identified in 42% of cases. There was no significant association of cartilage lesion wall verticality and surgeon years in practice (rs = 0.161, P = .065) or arthroscopic caseload (rs = -0.071, P = .419). CONCLUSIONS: Arthroscopic cartilage lesion preparation using standard curette technique in a human cadaveric knee model results in inferior perpendicularity of the surrounding cartilage walls at the front aspect of the defect, compared to the rear aspect. This technique has shown significant variability in the depth of debridement, with debridement depths identified as either too superficial or too deep to the calcified cartilage layer in more than 60% of cases in this study. Surgeon experience does not appear to impact the morphologic properties of cartilage lesions prepared arthroscopically using ring curettes. CLINICAL RELEVANCE: To optimize restoration of hyaline-like cartilage tissue, careful attention to prepared cartilage lesion morphology is advised when arthroscopically performing cartilage repair, given the tendency for standard curette technique to create inferior verticality of cartilage walls at the front of the lesion, and the variable depth of debridement achieved.

Cartilage Repair in the Knee Using Umbilical Cord Wharton's Jelly-Derived Mesenchymal Stem Cells Embedded Onto Collagen Scaffolding and Implanted Under Dry Arthroscopy.

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.

Morphologic Properties of Cartilage Lesions in the Knee Arthroscopically Prepared by the Standard Curette Technique Are Inferior to Lesions Prepared by Specialized Chondrectomy Instruments.

BACKGROUND: Cartilage lesion preparation is an important component to cartilage repair procedures, given the effect of prepared lesion morphology on the formation of durable and well-integrated repair tissue. PURPOSE: To compare the quality of arthroscopic cartilage lesion debridement performed by (1) the standard curette (SC) technique and (2) specialized chondrectomy (CM) instruments, to provide technical guidance for optimization of cartilage lesion preparation in the setting of arthroscopic cartilage repair. STUDY DESIGN: Controlled laboratory study. METHODS: Articular cartilage lesions of standardized size (8 × 15 mm) were demarcated within the trochlea and femoral condyles of 20 human cadaver knee specimens. Orthopaedic surgeons performed arthroscopic lesion preparation using 2 techniques that consisted of SC preparation and preparation by CM instruments. A histologic comparative analysis was performed within each treatment group and between treatment groups to evaluate the morphology of prepared cartilage defects. RESULTS: The mean angle deviation from perpendicular of the cartilage wall at the front of the prepared cartilage lesions was significantly greater in the SC group versus the CM group (29.8° ± 21.4° vs 7.7° ± 7.6°, P < .001). In lesions prepared via the SC technique, the cartilage walls at the front of the prepared lesions were significantly less perpendicular than the cartilage walls at the rear of the lesions (29.8° ± 21.4° vs 11.0° ± 10.3°, P < .001), whereas lesions prepared by the CM technique demonstrated comparable verticality of surrounding cartilage walls at the front and rear aspects of the lesions (7.7° ± 7.6° vs 9.4° ± 12.3°, P = .827). Depth of lesion debridement was accomplished to the target level by the CM technique in 86% of prepared lesions, compared with 34% of lesions in the SC group. The prepared cartilage wall profile was characterized as the most ideal morphology in 55% of prepared lesions in the CM group, as opposed to 10% in the SC group. CONCLUSION: Arthroscopic cartilage lesion preparation with SC instruments results in superior perpendicularity of surrounding cartilage walls to subchondral bone and greater consistency of debrided lesion depth, as compared with the standard debridement technique with curettes. CLINICAL RELEVANCE: Arthroscopic preparation using standard curette technique leads to suboptimal morphologic characteristics of prepared lesions that likely affect the quality of repair tissue, compared to preparation using specialized chondrectomy instruments.

High-throughput assessment of mechanical properties of stem cell derived red blood cells, toward cellular downstream processing.

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.

Biologic Inlay Osteochondral Reconstruction: Arthroscopic One-Step Osteochondral Lesion Repair in the Knee Using Morselized Bone Grafting and Hyaluronic Acid-Based Scaffold Embedded With Bone Marrow Aspirate Concentrate.

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.