Manufacturing of 3D-Printed Hybrid Scaffolds with Polyelectrolyte Multilayer Coating in Static and Dynamic Culture Conditions.

Three-dimensional printing (3DP) has emerged as a promising method for creating intricate scaffold designs. This study assessed three 3DP scaffold designs fabricated using biodegradable poly(lactic) acid (PLA) through fused deposition modelling (FDM): mesh, two channels (2C), and four channels (4C). To address the limitations of PLA, such as hydrophobic properties and poor cell attachment, a post-fabrication modification technique employing Polyelectrolyte Multilayers (PEMs) coating was implemented. The scaffolds underwent aminolysis followed by coating with SiCHA nanopowders dispersed in hyaluronic acid and collagen type I, and finally crosslinked the outermost coated layers with EDC/NHS solution to complete the hybrid scaffold production. The study employed rotating wall vessels (RWVs) to investigate how simulating microgravity affects cell proliferation and differentiation. Human mesenchymal stem cells (hMSCs) cultured on these scaffolds using proliferation medium (PM) and osteogenic media (OM), subjected to static (TCP) and dynamic (RWVs) conditions for 21 days, revealed superior performance of 4C hybrid scaffolds, particularly in OM. Compared to commercial hydroxyapatite scaffolds, these hybrid scaffolds demonstrated enhanced cell activity and survival. The pre-vascularisation concept on 4C hybrid scaffolds showed the proliferation of both HUVECs and hMSCs throughout the scaffolds, with a positive expression of osteogenic and angiogenic markers at the early stages.

Modeling the neuroimmune system in Alzheimer's and Parkinson's diseases.

Parkinson's disease (PD) and Alzheimer's disease (AD) are neurodegenerative disorders caused by the interaction of genetic, environmental, and familial factors. These diseases have distinct pathologies and symptoms that are linked to specific cell populations in the brain. Notably, the immune system has been implicated in both diseases, with a particular focus on the dysfunction of microglia, the brain's resident immune cells, contributing to neuronal loss and exacerbating symptoms. Researchers use models of the neuroimmune system to gain a deeper understanding of the physiological and biological aspects of these neurodegenerative diseases and how they progress. Several in vitro and in vivo models, including 2D cultures and animal models, have been utilized. Recently, advancements have been made in optimizing these existing models and developing 3D models and organ-on-a-chip systems, holding tremendous promise in accurately mimicking the intricate intracellular environment. As a result, these models represent a crucial breakthrough in the transformation of current treatments for PD and AD by offering potential for conducting long-term disease-based modeling for therapeutic testing, reducing reliance on animal models, and significantly improving cell viability compared to conventional 2D models. The application of 3D and organ-on-a-chip models in neurodegenerative disease research marks a prosperous step forward, providing a more realistic representation of the complex interactions within the neuroimmune system. Ultimately, these refined models of the neuroimmune system aim to aid in the quest to combat and mitigate the impact of debilitating neuroimmune diseases on patients and their families.

Debiased ambient vibrations optical coherence elastography to profile cell, organoid and tissue mechanical properties.

The role of the mechanical environment in defining tissue function, development and growth has been shown to be fundamental. Assessment of the changes in stiffness of tissue matrices at multiple scales has relied mostly on invasive and often specialist equipment such as AFM or mechanical testing devices poorly suited to the cell culture workflow.In this paper, we have developed a unbiased passive optical coherence elastography method, exploiting ambient vibrations in the sample that enables real-time noninvasive quantitative profiling of cells and tissues. We demonstrate a robust method that decouples optical scattering and mechanical properties by actively compensating for scattering associated noise bias and reducing variance. The efficiency for the method to retrieve ground truth is validated in silico and in vitro, and exemplified for key applications such as time course mechanical profiling of bone and cartilage spheroids, tissue engineering cancer models, tissue repair models and single cell. Our method is readily implementable with any commercial optical coherence tomography system without any hardware modifications, and thus offers a breakthrough in on-line tissue mechanical assessment of spatial mechanical properties for organoids, soft tissues and tissue engineering.

Donor variability of ovine bone marrow derived mesenchymal stem cell - implications for cell therapy.

It is assumed that all species, including sheep, demonstrate significant variation between individuals including the characteristics of their bone marrow-derived mesenchymal stem cells (BM-MSCs). These differences may account for limited success in pre-clinical animal studies and may also impact on treatment strategies that are used within regenerative medicine. This study investigates variations between ovine MSCs (oMSCs) isolated from 13 English Mule sheep donors by studying cell viability, expansion, the cells' trilineage differentiation potential and the expression of cell surface markers. In addition to the primary objective, this article also compares various differentiation media used for the trilineage differentiation of oMSCs. In this study, a clear individual variation between the sheep donors regarding oMSCs characterization, tri-lineage differentiation potential and marker expression was effectively demonstrated. The results set out to systematically explore the ovine mesenchymal stem cell population derived from multiple donors. With this information, it is possible to start addressing the issues of personalized approaches to regenerative therapies.

Triphasic 3D In Vitro Model of Bone-Tendon-Muscle Interfaces to Study Their Regeneration.

The transition areas between different tissues, known as tissue interfaces, have limited ability to regenerate after damage, which can lead to incomplete healing. Previous studies focussed on single interfaces, most commonly bone-tendon and bone-cartilage interfaces. Herein, we develop a 3D in vitro model to study the regeneration of the bone-tendon-muscle interface. The 3D model was prepared from collagen and agarose, with different concentrations of hydroxyapatite to graduate the tissues from bones to muscles, resulting in a stiffness gradient. This graduated structure was fabricated using indirect 3D printing to provide biologically relevant surface topographies. MG-63, human dermal fibroblasts, and Sket.4U cells were found suitable cell models for bones, tendons, and muscles, respectively. The biphasic and triphasic hydrogels composing the 3D model were shown to be suitable for cell growth. Cells were co-cultured on the 3D model for over 21 days before assessing cell proliferation, metabolic activity, viability, cytotoxicity, tissue-specific markers, and matrix deposition to determine interface formations. The studies were conducted in a newly developed growth chamber that allowed cell communication while the cell culture media was compartmentalised. The 3D model promoted cell viability, tissue-specific marker expression, and new matrix deposition over 21 days, thereby showing promise for the development of new interfaces.

Current Advances on the Regeneration of Musculoskeletal Interfaces.

The regeneration of the musculoskeletal system has been widely investigated. There is now detailed knowledge about the organs composing this system. Research has also investigated the zones between individual tissues where physical, mechanical, and biochemical properties transition. However, the understanding of the regeneration of musculoskeletal interfaces is still lacking behind. Numerous disorders and injuries can degrade or damage tissue interfaces. Their inability to regenerate can delay the tissue repair and regeneration process, leading to graft instability, high morbidity, and pain. Moreover, the knowledge of the mechanism of tissue interface development is not complete. This review presents an overview of the most recent approaches of the regeneration of musculoskeletal interfaces, including the latest in vitro, preclinical, and clinical studies. Impact statement Interfaces between soft and hard tissues are ubiquitous within the body. These transition zones are crucial for joint motion, stabilisation and load transfer between tissues, but do not seem to regenerate well after injury or deterioration. The knowledge about their biology is vast, but little is known about their development. Various musculoskeletal disorders in combination with risk factors including aging and unhealthy lifestyle, can lead to local imbalances, misalignments, inflammation, pain and restricted mobility. Our manuscript reviews the current approaches taken to promote the regeneration of musculoskeletal interfaces through in vitro, pre-clinical and clinical studies.

Hydrostatic pressure in combination with topographical cues affects the fate of bone marrow-derived human mesenchymal stem cells for bone tissue regeneration.

Topographical and mechanical cues are vital for cell fate, tissue development in vivo, and to mimic the native cell growth environment in vitro. To date, the combinatory effect of mechanical and topographical cues as not been thoroughly investigated. This study investigates the effect of PCL nanofiber alignment and hydrostatic pressure on stem cell differentiation for bone tissue regeneration. Bone marrow-derived human mesenchymal stem cells were seeded onto standard tissue culture plastic and electrospun random and aligned nanofibers. These substrates were either cultured statically or subjected to intermittent hydrostatic pressure at 270 kPa, 1 Hz for 60 min daily over 21 days in osteogenic medium. Data revealed higher cell metabolic activities for all mechanically stimulated cell culture formats compared with non-stimulated controls; and random fibers compared with aligned fibers. Fiber orientation influenced cell morphology and patterns of calcium deposition. Significant up-regulation of Collagen-I, ALP, and Runx-2 were observed for random and aligned fibers following mechanical stimulation; highest levels of osteogenic markers were expressed when hydrostatic pressure was applied to random fibers. These results indicate that fiber alignment and hydrostatic pressure direct stem cell fate and are important stimulus for tissue regeneration. © 2017 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: A: 629-640, 2018.

Comparability: manufacturing, characterization and controls, report of a UK Regenerative Medicine Platform Pluripotent Stem Cell Platform Workshop, Trinity Hall, Cambridge, 14-15 September 2015.

This paper summarizes the proceedings of a workshop held at Trinity Hall, Cambridge to discuss comparability and includes additional information and references to related information added subsequently to the workshop. Comparability is the need to demonstrate equivalence of product after a process change; a recent publication states that this 'may be difficult for cell-based medicinal products'. Therefore a well-managed change process is required which needs access to good science and regulatory advice and developers are encouraged to seek help early. The workshop shared current thinking and best practice and allowed the definition of key research questions. The intent of this report is to summarize the key issues and the consensus reached on each of these by the expert delegates.

Dynamic 3D culture: models of chondrogenesis and endochondral ossification.

The formation of cartilage from stem cells during development is a complex process which is regulated by both local growth factors and biomechanical cues, and results in the differentiation of chondrocytes into a range of subtypes in specific regions of the tissue. In fetal development cartilage also acts as a precursor scaffold for many bones, and mineralization of this cartilaginous bone precursor occurs through the process of endochondral ossification. In the endochondral formation of bones during fetal development the interplay between cell signalling, growth factors, and biomechanics regulates the formation of load bearing bone, in addition to the joint capsule containing articular cartilage and synovium, generating complex, functional joints from a single precursor anlagen. These joint tissues are subsequently prone to degeneration in adult life and have poor regenerative capabilities, and so understanding how they are created during development may provide useful insights into therapies for diseases, such as osteoarthritis, and restoring bone and cartilage lost in adulthood. Of particular interest is how these tissues regenerate in the mechanically dynamic environment of a living joint, and so experiments performed using 3D models of cartilage development and endochondral ossification are proving insightful. In this review, we discuss some of the interesting models of cartilage development, such as the chick femur which can be observed in ovo, or isolated at a specific developmental stage and cultured organotypically in vitro. Biomaterial and hydrogel-based strategies which have emerged from regenerative medicine are also covered, allowing researchers to make informed choices on the characteristics of the materials used for both original research and clinical translation. In all of these models, we illustrate the essential importance of mechanical forces and mechanotransduction as a regulator of cell behavior and ultimate structural function in cartilage.

Evaluation of the growth environment of a hydrostatic force bioreactor for preconditioning of tissue-engineered constructs.

Bioreactors have been widely acknowledged as valuable tools to provide a growth environment for engineering tissues and to investigate the effect of physical forces on cells and cell-scaffold constructs. However, evaluation of the bioreactor environment during culture is critical to defining outcomes. In this study, the performance of a hydrostatic force bioreactor was examined by experimental measurements of changes in dissolved oxygen (O2), carbon dioxide (CO2), and pH after mechanical stimulation and the determination of physical forces (pressure and stress) in the bioreactor through mathematical modeling and numerical simulation. To determine the effect of hydrostatic pressure on bone formation, chick femur skeletal cell-seeded hydrogels were subjected to cyclic hydrostatic pressure at 0-270 kPa and 1 Hz for 1 h daily (5 days per week) over a period of 14 days. At the start of mechanical stimulation, dissolved O2 and CO2 in the medium increased and the pH of the medium decreased, but remained within human physiological ranges. Changes in physiological parameters (O2, CO2, and pH) were reversible when medium samples were placed in a standard cell culture incubator. In addition, computational modeling showed that the distribution and magnitude of physical forces depends on the shape and position of the cell-hydrogel constructs in the tissue culture format. Finally, hydrostatic pressure was seen to enhance mineralization of chick femur skeletal cell-seeded hydrogels.

Interconnectivity analysis of supercritical CO2-foamed scaffolds.

This paper describes a computer algorithm for the determination of the interconnectivity of the pore space inside scaffolds used for tissue engineering. To validate the algorithm and its computer implementation, the algorithm was applied to a computer-generated scaffold consisting of a set of overlapping spherical pores, for which the interconnectivity was calculated exactly. The algorithm was then applied to micro-computed X-ray tomography images of supercritical CO(2)-foamed scaffolds made from poly(lactic-co-glycolic acid) (PLGA), whereby the effect of using different weight average molecular weight polymer on the interconnectivity was investigated.

In vivo bone formation by human marrow stromal cells in biodegradable scaffolds that release dexamethasone and ascorbate-2-phosphate.

An unsolved problem with stem cell-based engineering of bone tissue is how to provide a microenvironment that promotes the osteogenic differentiation of multipotent stem cells. Previously, we fabricated porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds that released biologically active dexamethasone (Dex) and ascorbate-2-phosphate (AsP), and that acted as osteogenic scaffolds. To determine whether these osteogenic scaffolds can be used for bone formation in vivo, we seeded multipotent human marrow stromal cells (hMSCs) onto the scaffolds and implanted them subcutaneously into athymic mice. Higher alkaline phosphatase expression was observed in hMSCs in the osteogenic scaffolds compared with that of hMSCs in control scaffolds. Furthermore, there was more calcium deposition and stronger von Kossa staining in the osteogenic scaffolds, which suggested that there was enhanced mineralized bone formation. We failed to detect cartilage in the osteogenic scaffolds (negative Safranin O staining), which implied that there was intramembranous ossification. This is the first study to demonstrate the successful formation of mineralized bone tissue in vivo by hMSCs in PLGA scaffolds that release Dex and AsP.