Ablation of the dystrophin Dp71f alternative C-terminal variant increases sarcoma tumour cell aggressiveness.

Alterations in Dp71 expression, the most ubiquitous dystrophin isoform, have been associated with patient survival across tumours. Intriguingly, in certain malignancies, Dp71 acts as a tumour suppressor, while manifesting oncogenic properties in others. This diversity could be explained by the expression of two Dp71 splice variants encoding proteins with distinct C-termini, each with specific properties. Expression of these variants has impeded the exploration of their unique roles. Using CRISPR/Cas9, we ablated the Dp71f variant with the alternative C-terminus in a sarcoma cell line not expressing the canonical C-terminal variant, and conducted molecular (RNAseq) and functional characterisation of the knockout cells. Dp71f ablation induced major transcriptomic alterations, particularly affecting the expression of genes involved in calcium signalling and ECM-receptor interaction pathways. The genome-scale metabolic analysis identified significant downregulation of glucose transport via membrane vesicle reaction (GLCter) and downregulated glycolysis/gluconeogenesis pathway. Functionally, these molecular changes corresponded with, increased calcium responses, cell adhesion, proliferation, survival under serum starvation and chemotherapeutic resistance. Knockout cells showed reduced GLUT1 protein expression, survival without attachment and their migration and invasion in vitro and in vivo were unaltered, despite increased matrix metalloproteinases release. Our findings emphasise the importance of alternative splicing of dystrophin transcripts and underscore the role of the Dp71f variant, which appears to govern distinct cellular processes frequently dysregulated in tumour cells. The loss of this regulatory mechanism promotes sarcoma cell survival and treatment resistance. Thus, Dp71f is a target for future investigations exploring the intricate functions of specific DMD transcripts in physiology and across malignancies.

Biofabrication of nanocomposite-based scaffolds containing human bone extracellular matrix for the differentiation of skeletal stem and progenitor cells.

Autograft or metal implants are routinely used in skeletal repair. However, they fail to provide long-term clinical resolution, necessitating a functional biomimetic tissue engineering alternative. The use of native human bone tissue for synthesizing a biomimetic material ink for three-dimensional (3D) bioprinting of skeletal tissue is an attractive strategy for tissue regeneration. Thus, human bone extracellular matrix (bone-ECM) offers an exciting potential for the development of an appropriate microenvironment for human bone marrow stromal cells (HBMSCs) to proliferate and differentiate along the osteogenic lineage. In this study, we engineered a novel material ink (LAB) by blending human bone-ECM (B) with nanoclay (L, Laponite®) and alginate (A) polymers using extrusion-based deposition. The inclusion of the nanofiller and polymeric material increased the rheology, printability, and drug retention properties and, critically, the preservation of HBMSCs viability upon printing. The composite of human bone-ECM-based 3D constructs containing vascular endothelial growth factor (VEGF) enhanced vascularization after implantation in an ex vivo chick chorioallantoic membrane (CAM) model. The inclusion of bone morphogenetic protein-2 (BMP-2) with the HBMSCs further enhanced vascularization and mineralization after only seven days. This study demonstrates the synergistic combination of nanoclay with biomimetic materials (alginate and bone-ECM) to support the formation of osteogenic tissue both in vitro and ex vivo and offers a promising novel 3D bioprinting approach to personalized skeletal tissue repair. Supplementary Information: The online version contains supplementary material available at 10.1007/s42242-023-00265-z.

Self-Assembly of Structured Colloidal Gels for High-Resolution 3D Micropatterning of Proteins at Scale.

Self-assembly, the spontaneous ordering of components into patterns, is widespread in nature and fundamental to generating function across length scales. Morphogen gradients in biological development are paradigmatic as both products and effectors of self-assembly and various attempts have been made to reproduce such gradients in biomaterial design. To date, approaches have typically utilized top-down fabrication techniques that, while allowing high-resolution control, are limited by scale and require chemical cross-linking steps to stabilize morphogen patterns in time. Here, a bottom-up approach to protein patterning is developed based on a novel binary reaction-diffusion process where proteins function as diffusive reactants to assemble a nanoclay-protein composite hydrogel. Using this approach, it is possible to generate scalable and highly stable 3D patterns of target proteins down to sub-cellular resolution through only physical interactions between clay nanoparticles and the proteins and ions present in blood. Patterned nanoclay gels are able to guide cell behavior to precisely template bone tissue formation in vivo. These results demonstrate the feasibility of stabilizing 3D gradients of biological signals through self-assembly processes and open up new possibilities for morphogen-based therapeutic strategies and models of biological development and repair.

Tri-Lineage Differentiation Potential of Osteosarcoma Cell Lines and Human Bone Marrow Stromal Cells from Different Anatomical Locations.

The bone cancer osteosarcoma, found mainly in adolescents, routinely forms around the growth plate/metaphysis of long bones. Bone marrow composition changes with age, shifting from a more hematopoietic to an adipocyte-rich tissue. This conversion occurs in the metaphysis during adolescence, implicating a link between bone marrow conversion and osteosarcoma initiation. To assess this, the tri-lineage differentiation potential of human bone marrow stromal cells (HBMSCs) isolated from the femoral diaphysis/metaphysis (FD) and epiphysis (FE) was characterized and compared to two osteosarcoma cell lines, Saos-2 and MG63. Compared to FE-cells, FD-cells showed an increase in tri-lineage differentiation. Additionally, differences were found between the Saos-2 cells exhibiting higher levels of osteogenic differentiation, lower adipogenic differentiation, and a more developed chondrogenic phenotype than MG63, with the Saos-2 being more comparable to FD-derived HBMSCs. The differences found between the FD and FE derived cells are consistent with the FD region containing more hematopoietic tissue compared to the FE. This may be related to the similarities between FD-derived cells and Saos-2 cells during osteogenic and chondrogenic differentiation. These studies reveal distinct differences in the tri-lineage differentiations of 'hematopoietic' and 'adipocyte rich' bone marrow, which correlate with specific characteristics of the two osteosarcoma cell lines.

Comparison of bone formation mediated by bone morphogenetic protein delivered by nanoclay gels with clinical techniques (autograft and InductOs®) in an ovine bone model.

Development of a growth factor delivery vehicle providing appropriate temporal-spatial release together with an appropriate preclinical large animal model to evaluate bone formation is critical in the development of delivery strategies for bone tissue regeneration. Smectite nanoclays such as LAPONITE™ possess unique thixotropic and protein retention properties offering promise for use in growth factor delivery in bone repair and regeneration. This study has examined bone formation mediated by a clinically approved growth factor delivery system (InductOs®) in combination with Laponite gel in an aged female ovine femoral condyle defect preclinical model (10 weeks). Two different designs, one containing a low volume of Laponite gel (LLG) in combination with the InductOs® absorbable collagen sponge (ACS), the other in which Laponite gel formed the implant (HLG), were compared against InductOs® alone and an autograft positive control. Thus, five groups: (i) empty defect, (ii) autograft, (iii) BMP2 + ACS, (iv) BMP2 + ACS + LLG and (v) BMP2 + HLG + ACS were examined in 9 mm × 12 mm defects performed bilaterally in the medial femoral condyles of 24 aged (>5 years) sheep. Bone formation within the defect was assessed using micro-computed tomography (micro-CT), digital volume correlation (DVC) for biomechanical characterisation as well as histology. The autograft and InductOs® mediated enhanced bone formation (p < 0001) compared to blank controls, while no significant differences were observed between the Laponite/Collagen/BMP delivery vehicles. However, the current study illustrated the excellent biocompatibility of Laponite and its ability to deliver localised active BMP-2, with the opportunity for improved efficacy with further optimisation. Interestingly, DVC-computed strain distributions indicated that the regenerated bone structure is mechanically adapted to bear external loads from the early remodelling stages of the bone reparation cascade. The current studies of selected nanoclay delivery platforms for BMP, assessed in a clinically relevant large animal model auger well for the development of bone fracture therapeutics for an ageing population.

Nonlinear micro finite element models based on digital volume correlation measurements predict early microdamage in newly formed bone.

Bone regeneration in critical-sized defects is a clinical challenge, with biomaterials under constant development aiming at enhancing the natural bone healing process. The delivery of bone morphogenetic proteins (BMPs) in appropriate carriers represents a promising strategy for bone defect treatment but optimisation of the spatial-temporal release is still needed for the regeneration of bone with biological, structural, and mechanical properties comparable to the native tissue. Nonlinear micro finite element (µFE) models can address some of these challenges by providing a tool able to predict the biomechanical strength and microdamage onset in newly formed bone when subjected to physiological or supraphysiological loads. Yet, these models need to be validated against experimental data. In this study, experimental local displacements in newly formed bone induced by osteoinductive biomaterials subjected to in situ X-ray computed tomography compression in the apparent elastic regime and measured using digital volume correlation (DVC) were used to validate µFE models. Displacement predictions from homogeneous linear µFE models were highly correlated to DVC-measured local displacements, while tissue heterogeneity capturing mineralisation differences showed negligible effects. Nonlinear µFE models improved the correlation and showed that tissue microdamage occurs at low apparent strains. Microdamage seemed to occur next to large cavities or in biomaterial-induced thin trabeculae, independent of the mineralisation. While localisation of plastic strain accumulation was similar, the amount of damage accumulated in these locations was slightly higher when including material heterogeneity. These results demonstrate the ability of the nonlinear µFE model to capture local microdamage in newly formed bone tissue and can be exploited to improve the current understanding of healing bone and mechanical competence. This will ultimately aid the development of BMPs delivery systems for bone defect treatment able to regenerate bone with optimal biological, mechanical, and structural properties.

The future of bone regeneration: integrating AI into tissue engineering.

Tissue engineering is a branch of regenerative medicine that harnesses biomaterial and stem cell research to utilise the body's natural healing responses to regenerate tissue and organs. There remain many unanswered questions in tissue engineering, with optimal biomaterial designs still to be developed and a lack of adequate stem cell knowledge limiting successful application. Advances in artificial intelligence (AI), and deep learning specifically, offer the potential to improve both scientific understanding and clinical outcomes in regenerative medicine. With enhanced perception of how to integrate artificial intelligence into current research and clinical practice, AI offers an invaluable tool to improve patient outcome.

Harnessing Polyhydroxyalkanoates and Pressurized Gyration for Hard and Soft Tissue Engineering.

Organ dysfunction is a major cause of morbidity and mortality. Transplantation is typically the only definitive cure, challenged by the lack of sufficient donor organs. Tissue engineering encompasses the development of biomaterial scaffolds to support cell attachment, proliferation, and differentiation, leading to tissue regeneration. For efficient clinical translation, the forming technology utilized must be suitable for mass production. Herein, uniaxial polyhydroxyalkanoate scaffolds manufactured by pressurized gyration, a hybrid scalable spinning technique, are successfully used in bone, nerve, and cardiovascular applications. Chorioallantoic membrane and in vivo studies provided evidence of vascularization, collagen deposition, and cellular invasion for bone tissue engineering. Highly efficient axonal outgrowth was observed in dorsal root ganglion-based 3D ex vivo models. Human induced pluripotent stem cell derived cardiomyocytes exhibited a mature cardiomyocyte phenotype with optimal calcium handling. This study confirms that engineered polyhydroxyalkanoate-based gyrospun fibers provide an exciting and unique toolbox for the development of scalable scaffolds for both hard and soft tissue regeneration.

Endothelial Cells: Co-culture Spheroids.

The development and maintenance of a functioning vascular system is a critical function for many aspects of tissue growth and regeneration. Vascular endothelial cell in vitro co-culture spheroids are self-organized cell composites that have the capacity to recapitulate the three-dimensional tissue microenvironment. These spheroid testing platforms aim to better understand the mechanisms of functional tissue and how new therapeutic agents can drive these 3D co-culture processes. Here we describe direct cell-cell 3D endothelial co-culture spheroid methods, to examine the physiological spatial growth and cell-cell interaction of vascular cells and surrounding native tissue cells in the formation of vascular networks within spheroids and the potential to regenerate tissue.

Evolving applications of the egg: chorioallantoic membrane assay and ex vivo organotypic culture of materials for bone tissue engineering.

The chick chorioallantoic membrane model has been around for over a century, applied in angiogenic, oncology, dental and xenograft research. Despite its often perceived archaic, redolent history, the chorioallantoic membrane assay offers new and exciting opportunities for material and growth factor evaluation in bone tissue engineering. Currently, superior/improved experimental methodology for the chorioallantoic membrane assay are difficult to identify, given an absence of scientific consensus in defining experimental approaches, including timing of inoculation with materials and the analysis of results. In addition, critically, regulatory and welfare issues impact upon experimental designs. Given such disparate points, this review details recent research using the ex vivo chorioallantoic membrane assay and the ex vivo organotypic culture to advance the field of bone tissue engineering, and highlights potential areas of improvement for their application based on recent developments within our group and the tissue engineering field.

Modeling adult skeletal stem cell response to laser-machined topographies through deep learning.

The response of adult human bone marrow stromal stem cells to surface topographies generated through femtosecond laser machining can be predicted by a deep neural network. The network is capable of predicting cell response to a statistically significant level, including positioning predictions with a probability P < 0.001, and therefore can be used as a model to determine the minimum line separation required for cell alignment, with implications for tissue structure development and tissue engineering. The application of a deep neural network, as a model, reduces the amount of experimental cell culture required to develop an enhanced understanding of cell behavior to topographical cues and, critically, provides rapid prediction of the effects of novel surface structures on tissue fabrication and cell signaling.

The Role of Pre-Clinical 3-Dimensional Models of Osteosarcoma.

Treatment for osteosarcoma (OS) has been largely unchanged for several decades, with typical therapies being a mixture of chemotherapy and surgery. Although therapeutic targets and products against cancer are being continually developed, only a limited number have proved therapeutically active in OS. Thus, the understanding of the OS microenvironment and its interactions are becoming more important in developing new therapies. Three-dimensional (3D) models are important tools in increasing our understanding of complex mechanisms and interactions, such as in OS. In this review, in vivo animal models, in vitro 3D models and in ovo chorioallantoic membrane (CAM) models, are evaluated and discussed as to their contribution in understanding the progressive nature of OS, and cancer research. We aim to provide insight and prospective future directions into the potential translation of 3D models in OS.

Nanopatterned Titanium Implants Accelerate Bone Formation In Vivo.

Accelerated de novo formation of bone is a highly desirable aim of implants targeting musculoskeletal injuries. To date, this has primarily been addressed by biologic factors. However, there is an unmet need for robust, highly reproducible yet economic alternative strategies that strongly induce an osteogenic cell response. Here, we present a surface engineering method of translating bioactive nanopatterns from polymeric in vitro studies to clinically relevant material for orthopedics: three-dimensional, large area metal. We use a titanium-based sol-gel whereby metal implants can be engineered to induce osteoinduction both in vitro and in vivo. We show that controlled disordered nanotopographies presented as pillars with 15-25 nm height and 100 nm diameter on titanium dioxide effectively induce osteogenesis when seeded with STRO-1-enriched human skeletal stem cells in vivo subcutaneous implantation in mice. After 28 days, samples were retrieved, which showed a 20-fold increase in osteogenic gene induction of nanopatterned substrates, indicating that the sol-gel nanopatterning method offers a promising route for translation to future clinical orthopedic implants.

Genetically-programmed, mesenchymal stromal cell-laden & mechanically strong 3D bioprinted scaffolds for bone repair.

Additive manufacturing processes used to create regenerative bone tissue engineered implants are not biocompatible, thereby restricting direct use with stem cells and usually require cell seeding post-fabrication. Combined delivery of stem cells with the controlled release of osteogenic factors, within a mechanically-strong biomaterial combined during manufacturing would replace injectable defect fillers (cements) and allow personalized implants to be rapidly prototyped by 3D bioprinting. Through the use of direct genetic programming via the sustained release of an exogenously delivered transcription factor RUNX2 (delivered as recombinant GET-RUNX2 protein) encapsulated in PLGA microparticles (MPs), we demonstrate that human mesenchymal stromal (stem) cells (hMSCs) can be directly fabricated into a thermo-sintered 3D bioprintable material and achieve effective osteogenic differentiation. Importantly we observed osteogenic programming of gene expression by released GET-RUNX2 (8.2-, 3.3- and 3.9-fold increases in OSX, RUNX2 and OPN expression, respectively) and calcification (von Kossa staining) in our scaffolds. The developed biodegradable PLGA/PEG paste formulation augments high-density bone development in a defect model (~2.4-fold increase in high density bone volume) and can be used to rapidly prototype clinically-sized hMSC-laden implants within minutes using mild, cytocompatible extrusion bioprinting. The ability to create mechanically strong 'cancellous bone-like' printable implants for tissue repair that contain stem cells and controlled-release of programming factors is innovative, and will facilitate the development of novel localized delivery approaches to direct cellular behaviour for many regenerative medicine applications including those for personalized bone repair.

Nanoclay-based 3D printed scaffolds promote vascular ingrowth ex vivo and generate bone mineral tissue in vitro and in vivo.

Acellular soft hydrogels are not ideal for hard tissue engineering given their poor mechanical stability, however, in combination with cellular components offer significant promise for tissue regeneration. Indeed, nanocomposite bioinks provide an attractive platform to deliver human bone marrow stromal cells (HBMSCs) in three dimensions producing cell-laden constructs that aim to facilitate bone repair and functionality. Here we present the in vitro, ex vivo and in vivo investigation of bioprinted HBMSCs encapsulated in a nanoclay-based bioink to produce viable and functional three-dimensional constructs. HBMSC-laden constructs remained viable over 21 d in vitro and immediately functional when conditioned with osteogenic media. 3D scaffolds seeded with human umbilical vein endothelial cells (HUVECs) and loaded with vascular endothelial growth factor (VEGF) implanted ex vivo into a chick chorioallantoic membrane (CAM) model showed integration and vascularisation after 7 d of incubation. In a pre-clinical in vivo application of a nanoclay-based bioink to regenerate skeletal tissue, we demonstrated bone morphogenetic protein-2 (BMP-2) absorbed scaffolds produced extensive mineralisation after 4 weeks (p < 0.0001) compared to the drug-free and alginate controls. In addition, HBMSC-laden 3D printed scaffolds were found to significantly (p < 0.0001) support bone tissue formation in vivo compared to acellular and cast scaffolds. These studies illustrate the potential of nanoclay-based bioink, to produce viable and functional constructs for clinically relevant skeletal tissue regeneration.

Disordered protein-graphene oxide co-assembly and supramolecular biofabrication of functional fluidic devices.

Supramolecular chemistry offers an exciting opportunity to assemble materials with molecular precision. However, there remains an unmet need to turn molecular self-assembly into functional materials and devices. Harnessing the inherent properties of both disordered proteins and graphene oxide (GO), we report a disordered protein-GO co-assembling system that through a diffusion-reaction process and disorder-to-order transitions generates hierarchically organized materials that exhibit high stability and access to non-equilibrium on demand. We use experimental approaches and molecular dynamics simulations to describe the underlying molecular mechanism of formation and establish key rules for its design and regulation. Through rapid prototyping techniques, we demonstrate the system's capacity to be controlled with spatio-temporal precision into well-defined capillary-like fluidic microstructures with a high level of biocompatibility and, importantly, the capacity to withstand flow. Our study presents an innovative approach to transform rational supramolecular design into functional engineering with potential widespread use in microfluidic systems and organ-on-a-chip platforms.

Exploratory Full-Field Strain Analysis of Regenerated Bone Tissue from Osteoinductive Biomaterials.

Biomaterials for bone regeneration are constantly under development, and their application in critical-sized defects represents a promising alternative to bone grafting techniques. However, the ability of all these materials to produce bone mechanically comparable with the native tissue remains unclear. This study aims to explore the full-field strain evolution in newly formed bone tissue produced in vivo by different osteoinductive strategies, including delivery systems for BMP-2 release. In situ high-resolution X-ray micro-computed tomography (microCT) and digital volume correlation (DVC) were used to qualitatively assess the micromechanics of regenerated bone tissue. Local strain in the tissue was evaluated in relation to the different bone morphometry and mineralization for specimens (n = 2 p/treatment) retrieved at a single time point (10 weeks in vivo). Results indicated a variety of load-transfer ability for the different treatments, highlighting the mechanical adaptation of bone structure in the early stages of bone healing. Although exploratory due to the limited sample size, the findings and analysis reported herein suggest how the combination of microCT and DVC can provide enhanced understanding of the micromechanics of newly formed bone produced in vivo, with the potential to inform further development of novel bone regeneration approaches.

In vivo delivery of VEGF RNA and protein to increase osteogenesis and intraosseous angiogenesis.

Deficient bone vasculature is a key component in pathological conditions ranging from developmental skeletal abnormalities to impaired bone repair. Vascularisation is dependent upon vascular endothelial growth factor (VEGF), which drives both angiogenesis and osteogenesis. The aim of this study was to examine the efficacy of blood vessel and bone formation following transfection with VEGF RNA or delivery of recombinant human VEGF165 protein (rhVEGF165) across in vitro and in vivo model systems. To quantify blood vessels within bone, an innovative approach was developed using high-resolution X-ray computed tomography (XCT) to generate quantifiable three-dimensional reconstructions. Application of rhVEGF165 enhanced osteogenesis, as evidenced by increased human osteoblast-like MG-63 cell proliferation in vitro and calvarial bone thickness following in vivo administration. In contrast, transfection with VEGF RNA triggered angiogenic effects by promoting VEGF protein secretion from MG-63VEGF165 cells in vitro, which resulted in significantly increased angiogenesis in the chorioallantoic (CAM) assay in ovo. Furthermore, direct transfection of bone with VEGF RNA in vivo increased intraosseous vascular branching. This study demonstrates the importance of continuous supply as opposed to a single high dose of VEGF on angiogenesis and osteogenesis and, illustrates the potential of XCT in delineating in 3D, blood vessel connectivity in bone.

Regulation of the Bone Vascular Network is Sexually Dimorphic.

Osteoblast (OB) lineage cells are an important source of vascular endothelial growth factor (VEGF), which is critical for bone growth and repair. During bone development, pubertal differences in males and females exist, but little is known about whether VEGF signaling contributes to skeletal sexual dimorphism. We have found that in mice, conditional disruption of VEGF in osteocalcin-expressing cells (OcnVEGFKO) exerts a divergent influence on morphological, cellular, and whole bone properties between sexes. Furthermore, we describe an underlying sexual divergence in VEGF signaling in OB cultures in vitro independent of circulating sex hormones. High-resolution synchrotron computed tomography and backscattered scanning electron microscopy revealed, in males, extensive unmineralized osteoid encasing enlarged blood vessel canals and osteocyte lacunae in cortical bone after VEGF deletion, which contributed to increased porosity. VEGF was deleted in male and female long bone-derived OBs (OBVEGKO) in vitro and Raman spectroscopic analyses of mineral and matrix repertoires highlighted differences between male and female OBVEGFKO cells, with increased immature phosphate species prevalent in male OBVEGFKO cultures versus wild type (WT). Further sexual dimorphism was observed in bone marrow endothelial cell gene expression in vitro after VEGF deletion and in sclerostin protein expression, which was increased in male OcnVEGFKO bones versus WT. The impact of altered OB matrix composition after VEGF deletion on whole bone geometry was assessed between sexes, although significant differences between OcnVEGFKO and WT were identified only in females. Our results suggest that bone-derived VEGF regulates matrix mineralization and vascularization distinctly in males and females, which results in divergent physical bone traits.

Isolation, Differentiation, and Characterization of Human Bone Marrow Stem Cells In Vitro and In Vivo.

In this chapter, we describe techniques for the isolation and characterisation of skeletal stem cells from human bone marrow. The methods for enrichment of STRO-1+ and STRO-4+ cells using magnetic activated cell sorting are described and we also detail techniques for establishing and characterizing osteogenic, adipogenic, and chondrogenic cultures from these cells. Finally, we present methods for studying the ability of these cells to produce bone in vivo using diffusion chambers which have been implanted subcutaneously into mice.