RESUMO
The remarkable deformability of red blood cells (RBCs) depends on the viscoelasticity of the plasma membrane and cell contents and the surface area to volume (SA:V) ratio; however, it remains unclear which of these factors is the key determinant for passage through small capillaries. We used a microfluidic device to examine the traversal of normal, stiffened, swollen, parasitised and immature RBCs. We show that dramatic stiffening of RBCs had no measurable effect on their ability to traverse small channels. By contrast, a moderate decrease in the SA:V ratio had a marked effect on the equivalent cylinder diameter that is traversable by RBCs of similar cellular viscoelasticity. We developed a finite element model that provides a coherent rationale for the experimental observations, based on the nonlinear mechanical behaviour of the RBC membrane skeleton. We conclude that the SA:V ratio should be given more prominence in studies of RBC pathologies.
Assuntos
Forma Celular , Tamanho Celular , Deformação Eritrocítica , Eritrócitos/citologia , Eritrócitos/fisiologia , Capilares/fisiologia , Movimento Celular , Humanos , Dispositivos Lab-On-A-Chip , Modelos BiológicosRESUMO
The simian parasite Plasmodium knowlesi causes severe and fatal malaria infections in humans, but the process of host cell remodelling that underpins the pathology of this zoonotic parasite is only poorly understood. We have used serial block-face scanning electron microscopy to explore the topography of P. knowlesi-infected red blood cells (RBCs) at different stages of asexual development. The parasite elaborates large flattened cisternae (Sinton Mulligan's clefts) and tubular vesicles in the host cell cytoplasm, as well as parasitophorous vacuole membrane bulges and blebs, and caveolar structures at the RBC membrane. Large invaginations of host RBC cytoplasm are formed early in development, both from classical cytostomal structures and from larger stabilised pores. Although degradation of haemoglobin is observed in multiple disconnected digestive vacuoles, the persistence of large invaginations during development suggests inefficient consumption of the host cell cytoplasm. The parasite eventually occupies ~40% of the host RBC volume, inducing a 20% increase in volume of the host RBC and an 11% decrease in the surface area to volume ratio, which collectively decreases the ability of the P. knowlesi-infected RBCs to enter small capillaries of a human erythrocyte microchannel analyser. Ektacytometry reveals a markedly decreased deformability, whereas correlative light microscopy/scanning electron microscopy and python-based skeleton analysis (Skan) reveal modifications to the surface of infected RBCs that underpin these physical changes. We show that P. knowlesi-infected RBCs are refractory to treatment with sorbitol lysis but are hypersensitive to hypotonic lysis. The observed physical changes in the host RBCs may underpin the pathology observed in patients infected with P. knowlesi.
Assuntos
Membrana Eritrocítica/metabolismo , Eritrócitos/parasitologia , Plasmodium knowlesi/ultraestrutura , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Membrana Eritrocítica/ultraestrutura , Eritrócitos/citologia , Eritrócitos/ultraestrutura , Hemoglobinas/metabolismo , Interações Hospedeiro-Parasita , Humanos , Merozoítos/ultraestrutura , Microscopia Eletrônica de Varredura , Pressão Osmótica , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/patogenicidade , Plasmodium knowlesi/crescimento & desenvolvimento , Plasmodium knowlesi/patogenicidade , Esquizontes/ultraestrutura , Trofozoítos/ultraestrutura , Vacúolos/metabolismo , Vacúolos/ultraestruturaRESUMO
Introduction: Bone tumors, characterized by diverse locations and shapes, often necessitate surgical excision followed by custom implant placement to facilitate targeted bone reconstruction. Leveraging additive manufacturing, patient-specific implants can be precisely tailored with complex geometries and desired stiffness, enhancing their suitability for bone ingrowth. Methods: In this work, a finite element model is employed to assess patient-specific lattice implants in femur bones. Our model is validated using experimental data obtained from an animal study (n = 9). Results: The results demonstrate the accuracy of the proposed finite element model in predicting the implant mechanical behavior. The model was used to investigate the influence of reducing the elastic modulus of a solid Ti6Al4V implant by tenfold, revealing that such a reduction had no significant impact on bone behavior under maximum compression and torsion loading. This finding suggests a potential avenue for reducing the endoprosthesis modulus without compromising bone integrity. Discussion: Our research suggests that employing fully lattice implants not only facilitates bone ingrowth but also has the potential to reduce overall implant stiffness. This reduction is crucial in preventing significant bone remodeling associated with stress shielding, a challenge often associated with the high stiffness of fully solid implants. The study highlights the mechanical benefits of utilizing lattice structures in implant design for enhanced patient outcomes.
RESUMO
PURPOSE: Bone tumours must be surgically excised in one piece with a margin of healthy tissue. The unique nature of each bone tumour case is well suited to the use of patient-specific implants, with additive manufacturing allowing production of highly complex geometries. This work represents the first assessment of the combination of surgical robotics and patient-specific additively manufactured implants. METHODS: The development and evaluation of a robotic system for bone tumour excision, capable of milling complex osteotomy paths, is described. The developed system was evaluated as part of an animal trial on 24 adult male sheep, in which robotic bone excision of the distal femur was followed by placement of patient-specific implants with operative time evaluated. Assessment of implant placement accuracy was completed based on post-operative CT scans. RESULTS: A mean overall implant position error of 1.05 ± 0.53 mm was achieved, in combination with a mean orientation error of 2.38 ± 0.98°. A mean procedure time (from access to implantation, excluding opening and closing) of 89.3 ± 25.25 min was observed, with recorded surgical time between 58 and 133 min, with this approximately evenly divided between robotic (43.9 ± 15.32) and implant-based (45.4 ± 18.97) tasks. CONCLUSIONS: This work demonstrates the ability for robotics to achieve repeatable and precise removal of complex bone volumes of the type that would allow en bloc removal of a bone tumour. These robotically created volumes can be precisely filled with additively manufactured patient-specific implants, with minimal gap between cut surface and implant interface.
Assuntos
Implantes Dentários , Ortopedia , Robótica , Cirurgia Assistida por Computador , Masculino , Animais , Ovinos , Cirurgia Assistida por Computador/métodos , Tomografia Computadorizada por Raios X , Fêmur/diagnóstico por imagem , Fêmur/cirurgiaRESUMO
The red blood cell (RBC) is remarkable in its ability to deform as it passages through the vasculature. Its deformability derives from a spectrin-actin protein network that supports the cell membrane and provides strength and flexibility, however questions remain regarding the assembly and maintenance of the skeletal network. Using scanning electron microscopy (SEM) and atomic force microscopy (AFM) we have examined the nanoscale architecture of the cytoplasmic side of membrane discs prepared from reticulocytes and mature RBCs. Immunofluorescence microscopy was used to probe the distribution of spectrin and other membrane skeleton proteins. We found that the cell surface area decreases by up to 30% and the spectrin-actin network increases in density by approximately 20% as the reticulocyte matures. By contrast, the inter-junctional distance and junctional density increase only by 3-4% and 5-9%, respectively. This suggests that the maturation-associated reduction in surface area is accompanied by an increase in spectrin self-association to form higher order oligomers. We also examined the mature RBC membrane in the edge (rim) and face (dimple) regions of mature RBCs and found the rim contains about 1.5% more junctional complexes compared to the dimple region. A 2% increase in band 4.1 density in the rim supports these structural measurements.
RESUMO
The malaria parasite hijacks host erythrocytes to shield itself from the immune system and proliferate. Red blood cell abnormalities can provide protection from malaria by impeding parasite invasion and growth within the cell or by compromising the ability of parasites to avoid host clearance. Here, we describe 2 N-ethyl-N-nitrosourea-induced mouse lines, SptbMRI26194 and SptbMRI53426 , containing single-point mutations in the erythrocyte membrane skeleton gene, ß spectrin (Sptb), which exhibit microcytosis but retain a relatively normal ratio of erythrocyte surface area to volume and are highly resistant to rodent malaria. We propose the major factor responsible for malaria protection is the specific clearance of mutant erythrocytes, although an enhanced clearance of uninfected mutant erythrocytes was also observed (ie, the bystander effect). Using an in vivo erythrocyte tracking assay, we established that this phenomenon occurs irrespective of host environment, precluding the involvement of nonerythrocytic cells in the resistance mechanism. Furthermore, we recapitulated this phenotype by disrupting the interaction between ankyrin-1 and ß spectrin in vivo using CRISPR/Cas9 genome editing technology, thereby genetically validating a potential antimalarial target. This study sheds new light on the role of ß spectrin during Plasmodium infection and highlights how changes in the erythrocyte cytoskeleton can substantially influence malaria susceptibility with minimal adverse consequences for the host.