Ex vivo culture of adult CD34+ stem cells using functional highly porous polymer scaffolds to establish biomimicry of the bone marrow niche

Haematopoiesis, the process of blood production, occurs from a tiny contingent of haematopoietic stem cells (HSC) in highly specialised three-dimensional niches located within the bone marrow. When haematopoiesis is replicated using in vitro two-dimensional culture, HSCs rapidly differentiate, limiting self-renewal. Emulsion-templated highly porous polyHIPE foam scaffolds were chosen to mimic the honeycomb architecture of human bone. The unmodified polyHIPE material supports haematopoietic stem and progenitor cell (HSPC) culture, with successful culture of erythroid progenitors and neutrophils within the scaffolds. Using erythroid culture methodology, the CD34+ population was maintained for 28 days with continual release of erythroid progenitors. These cells are shown to spontaneously repopulate the scaffolds, and the accumulated egress can be expanded and grown at large scale to reticulocytes. We next show that the polyHIPE scaffolds can be successfully functionalised using activated BM(PEG)2 (1,8-bismaleimido-diethyleneglycol) and then a Jagged-1 peptide attached in an attempt to facilitate notch signalling. Although Jagged-1 peptide had no detectable effect, the BM(PEG)2 alone significantly increased cell egress when compared to controls, without depleting the scaffold population. This work highlights polyHIPE as a novel functionalisable material for mimicking the bone marrow, and also that PEG can influence HSPC behaviour within scaffolds

Computer-assisted and patient-specific 3-D planning and evaluation of a single-cut rotational osteotomy for complex long-bone deformities

Malunion after long bone fracture results in an incorrect position of the distal bone segment. This misalignment may lead to reduced function of the limb, early osteoarthritis and chronic pain. An established treatment option is a corrective osteotomy. For complex malunions, a single-cut rotational osteotomy is sometimes preferred in cases of angular deformity in three dimensions. However, planning and performing this type of osteotomy is relatively complex. This report describes a computer-assisted method for 3-D planning and realizing a single-cut rotational osteotomy with a patient-specific cutting guide for orienting the osteotomy and an angled jig for adjusting the rotation angle. The accuracy and reproducibility of the method is evaluated experimentally using plastic bones. In addition, complex rotational deformities are simulated by a computer to investigate the relation between deformity and correction parameters. The computed relation between deformity and correction parameters enables the surgeon to judge the feasibility of a single-cut rotational osteotomy. This appears possible for deformities combining axial misalignment with sufficient axial rotation. The proposed 3-D method of preoperative planning and transfer with a patient-specific cutting guide and angled jig renders the osteotomy procedure easily applicable, accurate, reproducible, and is a good alternative for complex and expensive navigation systems

Rapid automatic assessment of microvascular density in sidestream dark field images

The purpose of this study was to develop a rapid and fully automatic method for the assessment of microvascular density and perfusion in sidestream dark field (SDF) images. We modified algorithms previously developed by our group for microvascular density assessment and introduced a new method for microvascular perfusion assessment. To validate the new algorithm for microvascular density assessment, we reanalyzed a selection of SDF video clips (n = 325) from a study in intensive care patients and compared the results to (semi-)manually found microvascular densities. The method for microvascular perfusion assessment (temporal SDF image contrast analysis, tSICA) was tested in several video simulations and in one high quality SDF video clip where the microcirculation was imaged before and during circulatory arrest in a cardiac surgery patient. We found that the new method for microvascular density assessment was very rapid (<30 s/clip) and correlated excellently with (semi-)manually measured microvascular density. The new method for microvascular perfusion assessment (tSICA) was shown to be limited by high cell densities and velocities, which severely impedes the applicability of this method in real SDF images. Hence, here we present a validated method for rapid and fully automatic assessment of microvascular density in SDF images. The new method was shown to be much faster than the conventional (semi-)manual method. Due to current SDF imaging hardware limitations, we were not able to automatically detect microvascular perfusion

Measurement of functional microcirculatory geometry and velocity distributions using automated image analysis

This study describes a new method for analyzing microcirculatory videos. It introduces algorithms for quantitative assessment of vessel length, diameter, the functional microcirculatory density distribution and red blood-cell (RBC) velocity in individual vessels as well as its distribution. The technique was validated and compared to commercial software. The method was applied to the sublingual microcirculation in a healthy volunteer and in a patient during cardiac surgery. Analysis time was reduced from hours to minutes compared to previous methods requiring manual vessel identification. Vessel diameter was detected with high accuracy (>80%, d > 3 pixels). Capillary length was estimated within 5 pixels accuracy. Velocity estimation was very accurate (>95%) in the range [2.5, 1,000] pixels/s. RBC velocity was reduced by 70% during the first 10 s of cardiac luxation. The present method has been shown to be fast and accurate and provides increased insight into the functional properties of the microcirculation

Image-Guided Surgical Robotic System for Percutaneous Reduction of Joint Fractures

Complex joint fractures often require an open surgical procedure, which is associated with extensive soft tissue damages and longer hospitalization and rehabilitation time. Percutaneous techniques can potentially mitigate these risks but their application to joint fractures is limited by the current sub-optimal 2D intra-operative imaging (fluoroscopy) and by the high forces involved in the fragment manipulation (due to the presence of soft tissue, e.g., muscles) which might result in fracture malreduction. Integration of robotic assistance and 3D image guidance can potentially overcome these issues. The authors propose an image-guided surgical robotic system for the percutaneous treatment of knee joint fractures, i.e., the robot-assisted fracture surgery (RAFS) system. It allows simultaneous manipulation of two bone fragments, safer robot-bone fixation system, and a traction performing robotic manipulator. This system has led to a novel clinical workflow and has been tested both in laboratory and in clinically relevant cadaveric trials. The RAFS system was tested on 9 cadaver specimens and was able to reduce 7 out of 9 distal femur fractures (T- and Y-shape 33-C1) with acceptable accuracy (≈1 mm, ≈5°), demonstrating its applicability to fix knee joint fractures. This study paved the way to develop novel technologies for percutaneous treatment of complex fractures including hip, ankle, and shoulder, thus representing a step toward minimally-invasive fracture surgeries

Intra-operative fiducial-based CT/fluoroscope image registration framework for image-guided robot-assisted joint fracture surgery

Purpose Joint fractures must be accurately reduced minimising soft tissue damages to avoid negative surgical outcomes. To this regard, we have developed the RAFS surgical system, which allows the percutaneous reduction of intra-articular fractures and provides intra-operative real-time 3D image guidance to the surgeon. Earlier experiments showed the effectiveness of the RAFS system on phantoms, but also key issues which precluded its use in a clinical application. This work proposes a redesign of the RAFS’s navigation system overcoming the earlier version’s issues, aiming to move the RAFS system into a surgical environment. Methods The navigation system is improved through an image registration framework allowing the intra-operative registration between pre-operative CT images and intra-operative fluoroscopic images of a fractured bone using a custom-made fiducial marker. The objective of the registration is to estimate the relative pose between a bone fragment and an orthopaedic manipulation pin inserted into it intra-operatively. The actual pose of the bone fragment can be updated in real time using an optical tracker, enabling the image guidance. Results Experiments on phantom and cadavers demonstrated the accuracy and reliability of the registration framework, showing a reduction accuracy (sTRE) of about 0.88 ±0.2mm (phantom) and 1.15±0.8mm (cadavers). Four distal femur fractures were successfully reduced in cadaveric specimens using the improved navigation system and the RAFS system following the new clinical workflow (reduction error 1.2±0.3mm, 2±1∘). Conclusion Experiments showed the feasibility of the image registration framework. It was successfully integrated into the navigation system, allowing the use of the RAFS system in a realistic surgical application

Enhancement of red blood cell transfusion compatibility using CRISPR‐mediated erythroblast gene editing

Regular blood transfusion is the cornerstone of care for patients with red blood cell (RBC) disorders such as thalassaemia or sickle-cell disease. With repeated transfusion, alloimmunisation often occurs due to incompatibility at the level of minor blood group antigens. We use CRISPR-mediated genome editing of an immortalised human erythroblast cell line (BEL-A) to generate multiple enucleation competent cell lines deficient in individual blood groups. Edits are combined to generate a single cell line deficient in multiple antigens responsible for the most common transfusion incompatibilities: ABO (Bombay phenotype), Rh (Rhnull), Kell (K0), Duffy (Fynull), GPB (S−s−U−). These cells can be differentiated to generate deformable reticulocytes, illustrating the capacity for coexistence of multiple rare blood group antigen null phenotypes. This study provides the first proof-of-principle demonstration of combinatorial CRISPR-mediated blood group gene editing to generate customisable or multi-compatible RBCs for diagnostic reagents or recipients with complicated matching requirements