Biofabrication of the osteochondral unit and its applications: Current and future directions for 3D bioprinting

Multiple prevalent diseases, such as osteoarthritis (OA), for which there is no cure or full understanding, affect the osteochondral unit; a complex interface tissue whose architecture, mechanical nature and physiological characteristics are still yet to be successfully reproduced in vitro. Although there have been multiple tissue engineering-based approaches to recapitulate the three dimensional (3D) structural complexity of the osteochondral unit, there are various aspects that still need to be improved. This review presents the different pre-requisites necessary to develop a human osteochondral unit construct and focuses on 3D bioprinting as a promising manufacturing technique. Examples of 3D bioprinted osteochondral tissues are reviewed, focusing on the most used bioinks, chosen cell types and growth factors. Further information regarding the applications of these 3D bioprinted tissues in the fields of disease modelling, drug testing and implantation is presented. Finally, special attention is given to the limitations that currently hold back these 3D bioprinted tissues from being used as models to investigate diseases such as OA. Information regarding improvements needed in bioink development, bioreactor use, vascularisation and inclusion of additional tissues to further complete an OA disease model, are presented. Overall, this review gives an overview of the evolution in 3D bioprinting of the osteochondral unit and its applications, as well as further illustrating limitations and improvements that could be performed explicitly for disease modelling

Mechanisms of Action of Human Mesenchymal Stem Cells in Tissue Repair Regeneration and their Implications

Cell replacement therapy holds a promising future in the treatment of degenerative diseases related to neuronal, cardiac and bone tissues. In such kind of diseases, there is a progressive loss of specific types of cells. Currently the most upcoming and trusted cell candidate is Mesenchymal Stem Cells (MSCs) as these cells are easy to isolate from the tissue, easy to maintain and expand and no ethical concerns are linked. MSCs can be obtained from a number of sources like bone marrow, umbilical cord blood, umbilical cord, dental pulp, adipose tissues, etc. MSCs help in tissue repair and regeneration by various mechanisms of action like cell differentiation, immunomodulation, paracrine effect, etc. The future of regenerative medicine lies in tissue engineering and exploiting various properties to yield maximum output. In the current review article, we have targeted the repair and regeneration mechanisms of MSCs in neurodegenerative diseases, cardiac diseases and those related to bones. Yet there is a lot to understand, discover and then understand again about the molecular mechanisms of MSCs and then applying this knowledge in developing the therapy to get maximum repair and regeneration of concerned tissue and in turn the recovery of the patient

Nonmulberry Silk Braids Direct Terminal Osteocytic Differentiation through Activation of Wnt-Signaling

Silk polymers can regulate osteogenesis by mimicking some features of the extracellular matrix of bone and facilitate mineralized deposition on their surface by cultured osteoprogenitors. However, terminal differentiation of these mineralizing osteoblasts into osteocytic phenotypes has not yet been demonstrated on silk. Therefore, in this study we test the hypothesis that flat braids of natively (nonregenerated) spun nonmulberry silk <i>A. mylitta</i>, possessing mechanical stiffness in the range of trabecular bone, can regulate osteocyte differentiation within their 3D microenvironment. We seeded human preosteoblasts onto these braids and cultured them under varied temperatures (33.5 and 39 °C), soluble factors (dexamethasone, ascorbic acid, and β-glycerophosphate), and cytokine (TGF-β1). After 1 week, cell dendrites were conspicuously evident, confirming osteocyte differentiation, especially, in the presence of osteogenic factors and TGF-β1 expressing all characteristic osteocyte markers (podoplanin, DMP-1, and sclerostin). <i>A. mylitta</i> silk braids alone were sufficient to induce this differentiation, albeit only transiently. Therefore, we believe that the combinatorial effect of <i>A. mylitta</i> silk (surface chemistry, braid rigidity, and topography), osteogenic differentiation factors, and TGF-β1 were critical in stabilizing the mature osteocytic phenotype. Interestingly, Wnt signaling promoted osteocytic differentiation as evidenced by the upregulated expression of β-catenin in the presence of osteogenic factors and growth factor. This study highlights the role of nonmulberry silk braids in regulating stable osteocytic differentiation. Future studies could benefit from this understanding of the signaling mechanisms associated with silk-based matrices in order to develop 3D <i>in vitro</i> bone model systems

Developmentally inspired model of endochondral ossification

Introduction: In postnatal development, chondro-osseous transitions such as endochondral ossification (EO) are regulated by rapid matrix turnover, mineralisation and chondro-osseous transdifferentiation, all disrupted in osteoarthritis (OA). We are exploring how force governs these transitions of cartilage to bone; previous studies from our group indicate cartilage matrix and chondrocyte phenotypic plasticity in the growth plate, and stability in articular cartilage, is mechanoregulated. Here, we describe exploitation of a human ‘developmental biology-inspired platform’ alongside in vivo studies, to study cartilage mineralisation.Materials and Methods: In vivo studies reveal chondro-osseous transitions are inhibited by forces. We synthesise GelMA using click chemistry to generate hydrogels with compressive moduli of 3–5 kPa. Buoyancy-driven gradients of BMP-2 within hydrogels seeded with hMSCs were cultured for 28 days. qPCR, histology and immunohistochemistry assessed, cartilage, hypertrophic and bone markers. Uniaxial cyclic compression (0.5 Hz, 10% strain) was applied using Electroforce5500. Cells from Confetti-UBCre mice are being used for lineage tracing studies.Results: MSC-laden GelMA constructs showed differential gene expression across the gradient, indicating tri-phasic osteochondral tissue formation within 28 days. Runx2/Sox9 immunostaining confirmed osteochondral differentiation, increased expression of SPP1, SP7, Runx2, Col1 indicated osteogenesis at one end, while distinct expression of Col10 and Runx2 in the central region marked cellular hypertrophy. The effects of cyclic loading on cell signalling, hyaline cartilage formation and thickness, calcification and phenotypic plasticity/stability are being assessed and compared with in vivo findings.Discussion: These data validate the formation of a humanised osteochondral gradient recapitulating developmental processes, in vitro. It is hypothesised that the generated osteochondral tissues will reflect the results of phenotypic changes in cells and ECM regulation, under physiological and pathological loads. The model will be integrated with snRNA sequencing and lineage tracing, to study trans-differentiation. This approach provides an experimentally tractable mechanobiology model and clinically conformant osteochondral tissue development model enabling fundamental biology and disease modelling across scales

Preconditioned 70S30C bioactive glass foams promote osteogenesis in vivo

AbstractBioactive glass scaffolds (70S30C; 70% SiO2 and 30% CaO) produced by a sol–gel foaming process are thought to be suitable matrices for bone tissue regeneration. Previous in vitro data showed bone matrix production and active remodelling in the presence of osteogenic cells. Here we report their ability to act as scaffolds for in vivo bone regeneration in a rat tibial defect model, but only when preconditioned. Pretreatment methods (dry, pre-wetted or preconditioned without blood) for the 70S30C scaffolds were compared against commercial synthetic bone grafts (NovaBone® and Actifuse®). Poor bone ingrowth was found for both dry and wetted sol–gel foams, associated with rapid increase in pH within the scaffolds. Bone ingrowth was quantified through histology and novel micro-CT image analysis. The percentage bone ingrowth into dry, wetted and preconditioned 70S30C scaffolds at 11weeks were 10±1%, 21±2% and 39±4%, respectively. Only the preconditioned sample showed above 60% material–bone contact, which was similar to that in NovaBone and Actifuse. Unlike the commercial products, preconditioned 70S30C scaffolds degraded and were replaced with new bone. The results suggest that bioactive glass compositions should be redesigned if sol–gel scaffolds are to be used without preconditioning to avoid excess calcium release