Search

Recent studies have shown spray cooling of the skin surface with millisecond cryogen spurts to be an effective method for protecting the epidermis from non-specific thermal injury during various laser mediated dermatological procedures. We have investigated the effects of ambient humidity level, spraying distance, and cryogen boiling point on the resulting radiometric surface temperature. Our findings indicate that: 1) decreasing the ambient humidity level results in less ice formation on the skin surface without altering the radiometric surface temperature during a cryogen spurt; 2) increasing the spraying distance to 85 mm lowers the radiometric surface temperature; and 3) boiling point of the cryogen does not directly affect the surface temperature in the geometries studied.

Objective: Equilibrium Partitioning of an Ionic Contrast agent with microcomputed tomography (EPIC-μCT) is a non-invasive technique to quantify and visualize the three-dimensional distribution of glycosaminoglycans (GAGs) in fresh cartilage tissue. However, it is unclear whether this technique is applicable to already fixed tissues. Therefore, this study aimed at investigating whether formalin fixation of bovine cartilage affects X-ray attenuation, and thus the interpretation of EPIC-μCT data.Design: Osteochondral samples (n=24) were incubated with ioxaglate, an ionic contrast agent, for 22h prior to μCT scanning. The samples were scanned in both formalin-fixed and fresh conditions. GAG content was measured using a biochemical assay and normalized to wet weight, dry weight, and water content to determine potential reasons for differences in X-ray attenuation.Results: The expected zonal distribution of contrast agent/GAGs was observed for both fixed and fresh cartilage specimens. However, despite no significant differences in GAG concentrations or physical properties between fixed and fresh samples, the average attenuation levels of formalin-fixed cartilage were 14.3% lower than in fresh samples.Conclusions: EPIC-μCT is useful for three-dimensional visualization of GAGs in formalin-fixed cartilage. However, a significant reduction in X-ray attenuation for fixed (compared to fresh) cartilage must be taken into account and adjusted for accordingly when quantifying GAG concentrations using EPIC-μCT. [ABSTRACT FROM AUTHOR]

This article focuses on how patterns and structures arise largely through cell-to-cell signaling, directed by signaling molecules (ligands) and their receptor targets. These signaling pathways control key developmental processes like cell proliferation and orientation (also called polarity). A relatively small cadre of molecules is enlisted over and over again to initiate an equally limited number of pathways to shape a developing embryo. Though the mechanics and effects of many of these pathways are understood, far less is known about the mechanisms that regulate which pathway is activated. One well-studied family of proteins, called Frizzled, regulates body symmetry and cell polarity, which,among other things, makes sure the bristles on a fly's wing all point in the same direction.

Extrusion-based bioprinting has gained widespread popularity in biofabrication due to its ability to assemble cells and biomaterials in precise patterns and form tissue-like constructs. To achieve this, bioinks must have rheological properties suitable for printing while maintaining cytocompatibility. However, many commonly used biomaterials do not meet the rheological requirements and therefore require modification for bioprinting applications. This study demonstrates the incorporation of Laponite-RD (LPN) into gelatin methacryloyl (GelMA) to produce highly customizable bioinks with desired rheological and mechanical properties for extrusion-based bioprinting. Bioink formulations were based on GelMA (5%-15% w/v) and LPN (0%-4% w/v), and a comprehensive rheological design was applied to evaluate key rheological properties necessary for extrusion-based bioprinting. The results showed that GelMA bioinks with LPN (1%-4% w/v) exhibited pronounced shear thinning and viscoelastic behavior, as well as improved thermal stability. Furthermore, a concentration window of 1%-2% (w/v) LPN to 5%-15% GelMA demonstrated enhanced rheological properties and printability required for extrusion-based bioprinting. Construct mechanical properties were highly tunable by varying polymer concentration and photocrosslinking parameters, with Young's moduli ranging from ∼0.2 to 75 kPa. Interestingly, at higher Laponite concentrations, GelMA cross-linking was inhibited, resulting in softer hydrogels. High viability of MCF-7 breast cancer cells was maintained in both free-swelling droplets and printed hydrogels, and metabolically active spheroids formed over 7 days of culture in all conditions. In summary, the addition of 1%-2% (w/v) LPN to gelatin-based bioinks significantly enhanced rheological properties and retained cell viability and proliferation, suggesting its suitability for extrusion-based bioprinting., Competing Interests: C.M. is a shareholder and the Chief Executive Officer of Gelomics Pty Ltd. T.J.K. is a shareholder of Gelomics Pty. Ltd and J.W.D. is an employee of Gelomics Pty Ltd., (© 2024 Author(s).)

Biofabrication approaches toward the development of tissue-engineered vascular grafts (TEVGs) have been widely investigated. However, successful translation has been limited to large diameter applications, with small diameter grafts frequently failing due to poor mechanical performance, in particular mismatched radial compliance. Herein, melt electrowriting (MEW) of poly( ϵ -caprolactone) has enabled the manufacture of highly porous, biocompatible microfibre scaffolds with physiological anisotropic mechanical properties, as substrates for the biofabrication of small diameter TEVGs. Highly reproducible scaffolds with internal diameter of 4.0 mm were designed with 500 and 250 µ m pore sizes, demonstrating minimal deviation of less than 4% from the intended architecture, with consistent fibre diameter of 15 ± 2 µ m across groups. Scaffolds were designed with straight or sinusoidal circumferential microfibre architecture respectively, to investigate the influence of biomimetic fibre straightening on radial compliance. The results demonstrate that scaffolds with wave-like circumferential microfibre laydown patterns mimicking the architectural arrangement of collagen fibres in arteries, exhibit physiological compliance (12.9 ± 0.6% per 100 mmHg), while equivalent control geometries with straight fibres exhibit significantly reduced compliance (5.5 ± 0.1% per 100 mmHg). Further mechanical characterisation revealed the sinusoidal scaffolds designed with 250 µ m pores exhibited physiologically relevant burst pressures of 1078 ± 236 mmHg, compared to 631 ± 105 mmHg for corresponding 500 µ m controls. Similar trends were observed for strength and failure, indicating enhanced mechanical performance of scaffolds with reduced pore spacing. Preliminary in vitro culture of human mesenchymal stem cells validated the MEW scaffolds as suitable substrates for cellular growth and proliferation, with high cell viability (>90%) and coverage (>85%), with subsequent seeding of vascular endothelial cells indicating successful attachment and preliminary endothelialisation of tissue-cultured constructs. These findings support further investigation into long-term tissue culture methodologies for enhanced production of vascular extracellular matrix components, toward the development of the next generation of small diameter TEVGs., (Creative Commons Attribution license.)

Hydrogels provide a promising platform for cartilage repair and regeneration. Although hydrogels have shown some efficacy, they still have shortcomings including poor mechanical properties and suboptimal integration with surrounding cartilage. Herein, hydrogels that are injectable, cytocompatible, mechanically robust, and highly adhesive to cartilage are developed. This approach uses GelMA-glycol chitosan (GelMA-GC) that is crosslinkable with visible light and photoinitiators (lithium acylphosphinate and tris (2,2'-bipyridyl) dichlororuthenium (II) hexahydrate ([RuII(bpy) 3 ] 2+ and sodium persulfate (Ru/SPS)). Ru/SPS-cross-linked hydrogels have higher compressive and tensile modulus, and most prominently higher adhesive strength with cartilage, which also depends on inclusion of GC. Tensile and push-out tests of the Ru/SPS-cross-linked GelMA-GC hydrogels demonstrate adhesive strength of ≈100 and 46 kPa, respectively. Hydrogel precursor solutions behave in a Newtonian manner and are injectable. After injection in focal bovine cartilage defects and in situ cross-linking, this hydrogel system remains intact and integrated with cartilage following joint manipulation ex vivo. Cells remain viable (>85%) in the hydrogel system and further show tissue regeneration potential after three weeks of in vitro culture. These preliminary results provide further motivation for future research on bioadhesive hydrogels for cartilage repair and regeneration., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

If it were possible to purchase tumour-spheroids as a standardised product, ready for direct use in assays, this may contribute to greater research reproducibility, potentially reducing costs and accelerating outcomes. Herein, we describe a workflow where uniformly sized cancer tumour-spheroids are mass-produced using microwell culture, cryopreserved with high viability, and then cultured in neutral buoyancy media for drug testing. C4-2B prostate cancer or MCF-7 breast cancer cells amalgamated into uniform tumour-spheroids after 48 h of culture. Tumour-spheroids formed from 100 cells each tolerated the cryopreservation process marginally better than tumour-spheroids formed from 200 or 400 cells. Post-thaw, tumour-spheroid metabolic activity was significantly reduced, suggesting mitochondrial damage. Metabolic function was rescued by thawing the tumour-spheroids into medium supplemented with 10 µM N -Acetyl-l-cysteine (NAC). Following thaw, the neutral buoyancy media, Happy Cell ASM, was used to maintain tumour-spheroids as discrete tissues during drug testing. Fresh and cryopreserved C4-2B or MCF-7 tumour-spheroids responded similarly to titrations of Docetaxel. This protocol will contribute to a future where tumour-spheroids may be available for purchase as reliable and reproducible products, allowing laboratories to efficiently replicate and build on published research, in many cases, making tumour-spheroids simply another cell culture reagent.

Untreated osteochondral defects are a leading cause of osteoarthritis, a condition that places a heavy burden on both patients and orthopedic surgeons. Although tissue engineering has shown promise for creating mechanically similar cartilage-like constructs, their integration with cartilage remains elusive. Therefore, a formulation of biodegradable, biocompatible biomaterial with sufficient mechanical and adhesive properties for cartilage repair is required. To accomplish this, we prepared biocompatible, photo-curable, mechanically robust, and highly adhesive GelMA-glycol chitosan (GelMA-GC) hydrogels. GelMA-GC hydrogels had a modulus of 283 kPa and provided a biocompatible environment (>70% viability of embedded chondrocytes) in long-term culture within a bovine cartilage ring. The adhesive strength of bovine chondrocyte-laden GelMA-GC hydrogel to bovine cartilage increased from 38 to 52 kPa over four weeks of culture. Moreover, intermittent uniaxial mechanical stimulation enhanced the adhesive strength to ∼60 kPa, indicating that the cartilage-hydrogel integration could remain secure and functional under dynamic loading conditions. Furthermore, gene expression data and immunofluorescence staining revealed the capacity of chondrocytes in GelMA-GC hydrogel to synthesize chondrogenic markers (COL2A1 and ACAN), suggesting the potential for tissue regeneration. The promising in vitro results of this work motivate further exploration of the potential of photo-curable GelMA-GC bioadhesive hydrogels for cartilage repair and regeneration., Competing Interests: T. J. Klein and C. Meinert are co-founders and shareholders of Gelomics Pty Ltd. C. Meinert is also the Chief Executive Officer of Gelomics Pty Ltd. A. Weekes and J. W. Davern are employees of Gelomics Pty Ltd., (© 2023 Author(s).)

The financial viability of a cell and tissue-engineered therapy may depend on the compatibility of the therapy with mass production and cryopreservation. Herein, we developed a method for the mass production and cryopreservation of 3D cartilage microtissues. Cartilage microtissues were assembled from either 5000 human bone marrow-derived stromal cells (BMSC) or 5000 human articular chondrocytes (ACh) each using a customized microwell platform (the Microwell-mesh). Microtissues rapidly accumulate homogenous cartilage-like extracellular matrix (ECM), making them potentially useful building blocks for cartilage defect repair. Cartilage microtissues were cultured for 5 or 10 days and then cryopreserved in 90% serum plus 10% dimethylsulfoxide (DMSO) or commercial serum-free cryopreservation media. Cell viability was maximized during thawing by incremental dilution of serum to reduce oncotic shock, followed by washing and further culture in serum-free medium. When assessed with live/dead viability dyes, thawed microtissues demonstrated high viability but reduced immediate metabolic activity relative to unfrozen control microtissues. To further assess the functionality of the freeze-thawed microtissues, their capacity to amalgamate into a continuous tissue was assess over a 14 day culture. The amalgamation of microtissues cultured for 5 days was superior to those that had been cultured for 10 days. Critically, the capacity of cryopreserved microtissues to amalgamate into a continuous tissue in a subsequent 14-day culture was not compromised, suggesting that cryopreserved microtissues could amalgamate within a cartilage defect site. The quality ECM was superior when amalgamation was performed in a 2% O 2 atmosphere than a 20% O 2 atmosphere, suggesting that this process may benefit from the limited oxygen microenvironment within a joint. In summary, cryopreservation of cartilage microtissues is a viable option, and this manipulation can be performed without compromising tissue function., Competing Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: MRD and KF founded a company that seeks to commercialize the Microwell-mesh., (© The Author(s) 2023.)

Spray nebulization is an elegant, but relatively unstudied, technique for scaffold production. Herein we fabricated mesh scaffolds of polycaprolactone (PCL) nanofibers via spray nebulization of 8% PCL in dichloromethane (DCM) using a 55.2 kPa compressed air stream and 17 ml h -1 polymer solution flow rate. Using a refined protocol, we tested the hypothesis that spray nebulization would simultaneously generate nanofibers and eliminate solvent, yielding a benign environment at the point of fiber deposition that enabled the direct deposition of nanofibers onto cell monolayers. Nanofibers were collected onto a rotating plate 20 cm from the spray nozzle, but could be collected onto any static or moving surface. Scaffolds exhibited a mean nanofiber diameter of 910 ± 190 nm, ultimate tensile strength of 2.1 ± 0.3 MPa, elastic modulus of 3.3 ± 0.4 MPa, and failure strain of 62 ± 6%. In vitro , scaffolds supported growth of human keratinocyte cell epithelial-like layers, consistent with potential utility as a dermal scaffold. Fourier-transform infrared spectroscopy demonstrated that DCM had vaporized and was undetectable in scaffolds immediately following production. Exploiting the rapid elimination of DCM during fiber production, we demonstrated that nanofibers could be directly deposited on to cell monolayers, without compromising cell viability. This is the first description of spray nebulization generating nanofibers using PCL in DCM. Using this method, it is possible to rapidly produce nanofiber scaffolds, without need for high temperatures or voltages, yielding a method that could potentially be used to deposit nanofibers onto cell cultures or wound sites., (© 2023 IOP Publishing Ltd.)

Engineered tissues provide an alternative to graft material, circumventing the use of donor tissue such as autografts or allografts and non-physiological synthetic implants. However, their lack of vasculature limits the growth of volumetric tissue more than several millimeters thick which limits their success post-implantation. Perfused bioreactors enhance nutrient mass transport inside lab-grown tissue but remain poorly customizable to support the culture of personalized implants. Here, a multiscale framework of computational fluid dynamics (CFD), additive manufacturing, and a perfusion bioreactor system are presented to engineer personalized volumetric tissue in the laboratory. First, microscale 3D printed scaffold pore geometries are designed and 3D printed to characterize media perfusion through CFD and experimental fluid testing rigs. Then, perfusion bioreactors are custom-designed to combine 3D printed scaffolds with flow-focusing inserts in patient-specific shapes as simulated using macroscale CFD. Finally, these computationally optimized bioreactor-scaffold assemblies are additively manufactured and cultured with pre-osteoblast cells for 7, 20, and 24 days to achieve tissue growth in the shape of human calcaneus bones of 13 mL volume and 1 cm thickness. This framework enables an intelligent model-based design of 3D printed scaffolds and perfusion bioreactors which enhances nutrient transport for long-term volumetric tissue growth in personalized implant shapes. The novel methods described here are readily applicable for use with different cell types, biomaterials, and scaffold microstructures to research therapeutic solutions for a wide range of tissues., (© 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

The ultimate objective of tissue engineering is to fabricate artificial living constructs with a structural organization and function that faithfully resembles their native tissue counterparts. For example, the deep zone of articular cartilage possesses a distinctive anisotropic architecture with chondrocytes organized in aligned arrays ≈1-2 cells wide, features that are oriented parallel to surrounding extracellular matrix fibers and orthogonal to the underlying subchondral bone. Although there are major advances in fabricating custom tissue architectures, it remains a significant technical challenge to precisely recreate such fine cellular features in vitro. Here, it is shown that ultrasound standing waves can be used to remotely organize living chondrocytes into high-resolution anisotropic arrays, distributed throughout the full volume of agarose hydrogels. It is demonstrated that this cytoarchitecture is maintained throughout a five-week course of in vitro tissue engineering, producing hyaline cartilage with cellular and extracellular matrix organization analogous to the deep zone of native articular cartilage. It is anticipated that this acoustic cell patterning method will provide unprecedented opportunities to interrogate in vitro the contribution of chondrocyte organization to the development of aligned extracellular matrix fibers, and ultimately, the design of new mechanically anisotropic tissue grafts for articular cartilage regeneration., (© 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Despite a 38% decrease in global maternal mortality during the last decade, rates remain unacceptably high with greater than 800 maternal deaths occurring each day. There exists significant regional variation among rates and causes of maternal mortality, and the vast majority occurs in low-income and middle-income countries. The leading causes of direct maternal mortality are hemorrhage, hypertensive disorders of pregnancy, sepsis, complications of abortion, and thromboembolism. Eliminating preventable maternal mortality hinges on improving clinical management of these life-threatening obstetric conditions, as well as addressing the complex social and economic barriers that pregnant women face to access quality care., Competing Interests: Disclosure None of the authors has any commercial or financial conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Free flap surgery is currently the only successful method used by surgeons to reconstruct critical-sized defects of the jaw, and is commonly used in patients who have had bony lesions excised due to oral cancer, trauma, infection or necrosis. However, donor site morbidity remains a significant flaw of this strategy. Various biomaterials have been under investigation in search of a suitable alternative for segmental mandibular defect reconstruction. Hydrogels are group of biomaterials that have shown their potential in various tissue engineering applications, including bone regeneration, both through in vitro and in vivo pre-clinical animal trials. This review discusses different types of hydrogels, their fabrication techniques, 3D printing, their potential for bone regeneration, outcomes, and the limitations of various hydrogels in preclinical models for bone tissue engineering. This review also proposes a modified technique utilizing the potential of hydrogels combined with scaffolds and cells for efficient reconstruction of mandibular segmental defects.

Background: The CyberKnife Xsight lung-tracking system (XLTS) provides an alternative to fiducial-based target-tracking systems (FTTS) for non-small-cell lung cancer (NSCLC) patients without invasive fiducial insertion procedures. This study provides a method for 3D independent dosimetric verification of the accuracy of the FTTS compared to the XLTS without relying on log-files generated by the CyberKnife system., Methods: A respiratory motion trace was taken from a 4D-CT of a real lung cancer patient and applied to a modified QUASAR™ respiratory motion phantom. A novel approach to 3D dosimetry was developed using Gafchromic EBT3 film, allowing the 3D dose distribution delivered to the moving phantom to be reconstructed. Treatments were planned using the recommended margins for one and three fiducial markers and XLTS 2-view, 1-view and 0-view target-tracking modalities. The dose delivery accuracy was analysed by comparing the reconstructed dose distributions to the planned dose distributions using gamma index analysis., Results: For the 3%/2 mm gamma criterion, gamma passing rates up to 99.37% were observed for the static deliveries. The 3-fiducial and 1-fiducial-based deliveries exhibited passing rates of 93.74% and 97.82%, respectively, in the absence of target rotation. When target rotation was considered, the passing rate for 1-fiducial tracking degraded to 91.24%. The passing rates observed for XLTS 2-view, 1-view and 0-view target-tracking were 92.78%, 96.22% and 76.08%, respectively., Conclusions: Except for the XLTS 0-view, the dosimetric accuracy of the XLTS was comparable to the FTTS under equivalent treatment conditions. This study gives us further confidence in the CyberKnife XLTS and FTTS systems., (© 2022. The Author(s).)

Current clinical treatment strategies for the bypassing of small diameter (<6 mm) blood vessels in the management of cardiovascular disease frequently fail due to a lack of suitable autologous grafts, as well as infection, thrombosis, and intimal hyperplasia associated with synthetic grafts. The rapid advancement of 3D printing and regenerative medicine technologies enabling the manufacture of biological, tissue-engineered vascular grafts (TEVGs) with the ability to integrate, remodel, and repair in vivo, promises a paradigm shift in cardiovascular disease management. This review comprehensively covers current state-of-the-art biofabrication technologies for the development of biomimetic TEVGs. Various scaffold based additive manufacturing methods used in vascular tissue engineering, including 3D printing, bioprinting, electrospinning and melt electrowriting, are discussed and assessed against the biomechanical and functional requirements of human vasculature, while the efficacy of decellularization protocols currently applied to engineered and native vessels are evaluated. Further, we provide interdisciplinary insight into the outlook of regenerative medicine for the development of vascular grafts, exploring key considerations and perspectives for the successful clinical integration of evolving technologies. It is expected that continued advancements in microscale additive manufacturing, biofabrication, tissue engineering and decellularization will culminate in the development of clinically viable, off-the-shelf TEVGs for small diameter applications in the near future. STATEMENT OF SIGNIFICANCE: Current clinical strategies for the management of cardiovascular disease using small diameter vessel bypassing procedures are inadequate, with up to 75% of synthetic grafts failing within 3 years of implantation. It is this critically important clinical problem that researchers in the field of vascular tissue engineering and regenerative medicine aim to alleviate using biofabrication methods combining additive manufacturing, biomaterials science and advanced cellular biology. While many approaches facilitate the development of bioengineered constructs which mimic the structure and function of native blood vessels, several challenges must still be overcome for clinical translation of the next generation of tissue-engineered vascular grafts., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Understanding how health care utilization responds to cost-sharing is of central importance for providing high quality care and limiting the growth of costs. We study whether the framing of cost-sharing incentives has an effect on health care utilization. For this we make use of a policy change in the Netherlands. Until 2007, patients received a refund if they consumed little or no health care; the refund was the lower the more care they had consumed. From 2008 onward, there was a deductible. This means that very similar economic incentives were first framed in terms of smaller gains and later as losses. We find that patients react to incentives much more strongly when they are framed in terms of losses. The effect on yearly spending is 8.6 percent. This suggests that discussions on the optimal design of cost-sharing incentives should also revolve around the question how these are presented to patients., Competing Interests: Declaration of Competing Interest There are no conflicts of interest., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Oncogenic RAS mutations are associated with tumor resistance to radiation therapy. Cell-cell interactions in the tumor microenvironment (TME) profoundly influence therapy outcomes. However, the nature of these interactions and their role in Ras tumor radioresistance remain unclear. Here we use Drosophila oncogenic Ras tissues and human Ras cancer cell radiation models to address these questions. We discover that cellular response to genotoxic stress cooperates with oncogenic Ras to activate JAK/STAT non-cell autonomously in the TME. Specifically, p53 is heterogeneously activated in Ras tumor tissues in response to irradiation. This mosaicism allows high p53-expressing Ras clones to stimulate JAK/STAT cytokines, which activate JAK/STAT in the nearby low p53-expressing surviving Ras clones, leading to robust tumor re-establishment. Blocking any part of this cell-cell communication loop re-sensitizes Ras tumor cells to irradiation. These findings suggest that coupling STAT inhibitors to radiotherapy might improve clinical outcomes for Ras cancer patients.

Bone marrow stromal cells (BMSC) show promise in cartilage repair, and sheep are the most common large animal pre-clinical model., Objective: The objective of this study was to characterise ovine BMSC (oBMSC) in vitro, and to evaluate the capacity of chondrogenic micro-pellets manufactured from oBMSC or ovine articular chondrocytes (oACh) to repair osteochondral defects in sheep., Design: oBMSC were characterised for surface marker expression using flow cytometry and evaluated for tri-lineage differentiation capacity. oBMSC micro-pellets were manufactured in a microwell platform, and chondrogenesis was compared at 2%, 5%, and 20% O 2 . The capacity of cartilage micro-pellets manufactured from oBMSC or oACh to repair osteochondral defects in adult sheep was evaluated in an 8-week pilot study., Results: Expanded oBMSC were positive for CD44 and CD146 and negative for CD45. The common adipogenic induction ingredient, 3-Isobutyl-1-methylxanthine (IBMX), was toxic to oBMSC, but adipogenesis could be restored by excluding IBMX from the medium. BMSC chondrogenesis was optimal in a 2% O 2 atmosphere. Micro-pellets formed from oBMSC or oACh appeared morphologically similar, but hypertrophic genes were elevated in oBMSC micro-pellets. While oACh micro-pellets formed cartilage-like repair tissue in sheep, oBMSC micro-pellets did not., Conclusion: The sensitivity of oBMSC, compared to human BMSC, to IBMX in standard adipogenic assays highlights species-associated differences. Micro-pellets manufactured from oACh were more effective than micro-pellets manufactured from oBMSC in the repair of osteochondral defects in sheep. While oBMSC can be driven to form cartilage-like tissue in vitro, the effective use of these cells in cartilage repair will depend on the successful mitigation of hypertrophy and tissue integration.

Virtually all bone marrow-derived stromal cell (BMSC) chondrogenic induction cultures include greater than 2 weeks exposure to transforming growth factor-β (TGF-β), but fail to generate cartilage-like tissue suitable for joint repair. Herein we used a micro-pellet model (5 × 10 3 BMSC each) to determine the duration of TGF-β1 exposure required to initiate differentiation machinery, and to characterize the role of intrinsic programming. We found that a single day of TGF-β1 exposure was sufficient to trigger BMSC chondrogenic differentiation and tissue formation, similar to 21 days of TGF-β1 exposure. Despite cessation of TGF-β1 exposure following 24 hours, intrinsic programming mediated further chondrogenic and hypertrophic BMSC differentiation. These important behaviors are obfuscated by diffusion gradients and heterogeneity in commonly used macro-pellet models (2 × 10 5 BMSC each). Use of more homogenous micro-pellet models will enable identification of the critical differentiation cues required, likely in the first 24-hours, to generate high quality cartilage-like tissue from BMSC.

Background: Bone marrow stromal cells (BMSC) have promise in cartilage tissue engineering, but for their potential to be fully realised, the propensity to undergo hypertrophy must be mitigated. The literature contains diverging reports on the effect of parathyroid hormone (PTH) on BMSC differentiation. Cartilage tissue models can be heterogeneous, confounding efforts to improve media formulations., Methods: Herein, we use a novel microwell platform (the Microwell-mesh) to manufacture hundreds of small-diameter homogeneous micro-pellets and use this high-resolution assay to quantify the influence of constant or intermittent PTH(1-34) medium supplementation on BMSC chondrogenesis and hypertrophy. Micro-pellets were manufactured from 5000 BMSC each and cultured in standard chondrogenic media supplemented with (1) no PTH, (2) intermittent PTH, or (3) constant PTH., Results: Relative to control chondrogenic cultures, BMSC micro-pellets exposed to intermittent PTH had reduced hypertrophic gene expression following 1 week of culture, but this was accompanied by a loss in chondrogenesis by the second week of culture. Constant PTH treatment was detrimental to chondrogenic culture., Conclusions: This study provides further clarity on the role of PTH on chondrogenic differentiation in vitro and suggests that while PTH may mitigate BMSC hypertrophy, it does so at the expense of chondrogenesis.

A previous study identified kartogenin (KGN) as a potent modulator of bone marrow mesenchymal stem/stromal cell (BMSC) chondrogenesis. This initial report did not contrast KGN directly against transforming growth factor-beta 1 (TGF-β1), the most common growth factor used in chondrogenic induction medium. Herein, we directly compared the in vitro chondrogenic potency of TGF-β1 and KGN using a high resolution micropellet model system. Micropellets were cultured for 7-14 days in medium supplemented with TGF-β1, KGN, or both TGF-β1 + KGN. Following 14 days of induction, micropellets exposed to TGF-β1 alone or TGF-β1 + KGN in combination were larger and produced more glycosominoglycan (GAG) than KGN-only cultures. When TGF-β1 + KGN was used, GAG quantities were similar or slightly greater than the TGF-β1-only cultures, depending on the BMSC donor. BMSC micropellet cultures supplemented with KGN alone contracted in size over the culture period and produced minimal GAG. Indicators of hypertrophy were not mitigated in TGF-β1 + KGN cultures, suggesting that KGN does not obstruct BMSC hypertrophy. KGN appears to have weak chondrogenic potency in human BMSC cultures relative to TGF-β1, does not obstruct hypertrophy, and may not be a viable alternative to growth factors in cartilage tissue engineering.

We aimed to capture the outstanding mechanical properties of meshes, manufactured using textile technologies, in thin biodegradable biphasic tissue-engineered scaffolds through encapsulation of meshes into porous structures formed from the same polymer. Our novel manufacturing process used thermally induced phase separation (TIPS), with ethylene carbonate (EC) as the solvent, to encapsulate a poly(lactic-co-glycolic acid) (PLGA) mesh into a porous PLGA network. Biphasic scaffolds (1 cm × 4 cm × 300 μm) were manufactured by immersing strips of PLGA mesh in 40 °C solutions containing 5% PLGA in EC, supercooling at 4 °C for 4 min, triggering TIPS by manually agitating the supercooled solution, and lastly eluting EC into 4 °C Milli-Q water. EC processing was rapid and did not compromise mesh tensile properties. Biphasic scaffolds exhibited a tensile strength of 40.7 ± 2.2 MPa, porosity of 94%, pore size of 16.85 ± 3.78 μm, supported HaCaT cell proliferation, and degraded in vitro linearly over the first ∼3 weeks followed by rapid degradation over the following three weeks. The successful integration of textile-type meshes yielded scaffolds with exceptional mechanical properties. This thin, porous, high-strength scaffold is potentially suitable for use in dermal wound repair or repair of tubular organs.

Volumetric imaging techniques capable of correlating structural and functional information with nanoscale resolution are necessary to broaden the insight into cellular processes within complex biological systems. The recent emergence of focused ion beam scanning electron microscopy (FIB-SEM) has provided unparalleled insight through the volumetric investigation of ultrastructure; however, it does not provide biomolecular information at equivalent resolution. Here, immunogold FIB-SEM, which combines antigen labeling with in situ FIB-SEM imaging, is developed in order to spatially map ultrastructural and biomolecular information simultaneously. This method is applied to investigate two different cell-material systems: the localization of histone epigenetic modifications in neural stem cells cultured on microstructured substrates and the distribution of nuclear pore complexes in myoblasts differentiated on a soft hydrogel surface. Immunogold FIB-SEM offers the potential for broad applicability to correlate structure and function with nanoscale resolution when addressing questions across cell biology, biomaterials, and regenerative medicine., (© 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rationally designed behavioral tests are important tools to assess the function of specific brain regions. The hippocampus is a crucial neural substrate for spatial cognition, and many studies have linked hippocampal dysfunction with defects on spatial learning and memory in neurological conditions ranging from Alzheimer's disease to autoimmune syndromes, such as neuropsychiatric lupus. While our understanding of hippocampal function, from the molecular to the system levels, has increased dramatically over the last decades, this effort has not yet translated into efficacious therapies for cognitive impairment. We think that the availability of highly validated behavioral paradigms to measure cognition in mouse models is likely to enhance the potential success of preclinical therapeutic modalities. Here, we present an extensive study of the paddling pool task (PPT), first reported by Deacon and Rawlins, in which mice learn to escape from shallow water through a peripheral exit in a circular arena dubbed the clockmaze. We show that the PPT provides highly reliable results when assaying spatial cognition in C57/BL6 mice (120 males, 40 females) and BALB/c mice (40 males, 90 females). Additionally, we develop a robust algorithm for the assessment of escape strategies with clearly quantifiable readouts, enabling fine-granular phenotyping. Notably, the use of spatial strategy increases linearly across trials in the PPT. In a separate cohort of mice, we apply muscimol injections to silence the dorsal CA1 region of the hippocampus and show that the use of the spatial strategy in the PPT relies on the integrity of the dorsal hippocampus. Additionally, we compare directly the PPT and the Morris water maze (MWM) task in C57/BL6 mice (20 males, 20 females) and BALB/c mice (20 males, 20 females) and we find that the PPT induces significantly lower anxiety, exhaustion and hypothermia than the MWM. We conclude that the PPT provides a robust assessment of spatial cognition in mice, which can be applied in conjunction with other tests, to facilitate hypothesis testing and drug development to combat cognitive impairment.

Gelatin methacryloyl (GelMA) hydrogels are a mechanically and biochemically tuneable biomaterial, facilitating chondrocyte culture for tissue engineering applications. However, a lack of characterisation and standardisation of fabrication methodologies for GelMA restricts its utilisation in surgical interventions for articular cartilage repair. The purpose of this study was to determine the effects of gelatin source and photoinitiator type on the redifferentiation capacity of monolayer-expanded human articular chondrocytes encapsulated in GelMA/hyaluronic acid methacrylate (HAMA) hydrogels. Chondrocyte-laden hydrogels reinforced with multiphasic melt-electrowritten (MEW) medical grade polycaprolactone (mPCL) microfibre scaffolds were prepared using bovine (B) or porcine-derived (P) GelMA, and photocrosslinked with either lithium acylphosphinate (LAP) and visible light (405 nm) or Irgacure 2959 (IC) and UV light (365 nm). Bulk physical properties, cell viability and biochemical features of hydrogel constructs were measured at day 1 and day 28 of chondrogenic cell culture. The compressive moduli of all groups increased after 28 days of cell culture, with B-IC displaying similar compressive strength to that of native articular cartilage (∼1.5 MPa). Compressive moduli correlated with an increase in total glycosaminoglycan (GAG) content for each group. Gene expression analysis revealed upregulation of chondrogenic marker genes in IC-crosslinked groups, whilst dedifferentiation gene markers were upregulated in LAP-crosslinked groups. mPCL reinforcement correlated with increased accumulation of collagen I and II in B-IC, B-LAP and P-IC groups compared to non-reinforced hydrogels. A reduction in cell viability was noted in all samples at day 28, potentially due to the generation of free radicals during photocrosslinking or cytotoxicity of the photoinitiators. In summary, hydrogel constructs prepared with bovine-derived GelMA and photocrosslinked with Irgacure 2959 and 365 nm light displayed properties most similar to native articular cartilage after 28 days of cell culture. The differences in biological response between investigated construct types emphasises the necessity to characterise and standardise biomaterials before translating in vitro tissue engineering research to preclinical applications for articular cartilage injuries.

Tissue engineering has offered unique opportunities for disease modeling and regenerative medicine; however, the success of these strategies is dependent on faithful reproduction of native cellular organization. Here, it is reported that ultrasound standing waves can be used to organize myoblast populations in material systems for the engineering of aligned muscle tissue constructs. Patterned muscle engineered using type I collagen hydrogels exhibits significant anisotropy in tensile strength, and under mechanical constraint, produced microscale alignment on a cell and fiber level. Moreover, acoustic patterning of myoblasts in gelatin methacryloyl hydrogels significantly enhances myofibrillogenesis and promotes the formation of muscle fibers containing aligned bundles of myotubes, with a width of 120-150 µm and a spacing of 180-220 µm. The ability to remotely pattern fibers of aligned myotubes without any material cues or complex fabrication procedures represents a significant advance in the field of muscle tissue engineering. In general, these results are the first instance of engineered cell fibers formed from the differentiation of acoustically patterned cells. It is anticipated that this versatile methodology can be applied to many complex tissue morphologies, with broader relevance for spatially organized cell cultures, organoid development, and bioelectronics., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The conventional mechanical properties of articular cartilage, such as compressive stiffness, have been shown to have limited capacity to distinguish visually normal from degraded cartilage samples. In this study, a new mechanical indentation framework for assessing functional properties of articular cartilage during loading/unloading, i.e. deformation and recovery, was established. The capacity of a ring-shaped indenter integrated with an ultrasound transducer to distinguish mechanically intact from proteoglycan-depleted tissue was investigated. To achieve this, normal and enzymatically degraded bovine osteochondral samples were subjected to loading/unloading while the response of the tissue at the middle was captured by ultrasound at the same time. The enzymatic degradation model was characterized by amount of proteoglycan content, glycosaminoglycan release and proteomic analysis. The mechanical response of a wider continuum of articular cartilage in the loaded area and its surrounding region was captured in this framework leading to investigate two parameters, L and TS, related to the surrounding tissue of the loaded area for functional assessment of cartilage. L is the distance between the ultrasound transducer and articular cartilage surface and TS is the transient strain of articular cartilage during loading and unloading. Classification Analysis based on Principal Component Analysis was used to investigate the capacity of the new parameters to assess the functionality of the tissue. Multivariate statistics based on Partial Least Squares regression was employed to identify the correlation between the response of the tissue in the indented area and its surrounding cartilage. The results of this study indicate that L during loading (deformation) can differentiate normal and mildly proteoglycan-depleted samples from severely depleted samples and L during unloading (recovery) can distinguish between normal and proteoglycan-depleted tissue. However, TS during deformation and recovery is unable to discriminate normal cartilage samples from proteoglycan-depleted tissue. The results also demonstrate a strong correlation between mechanical properties of the loaded area with the response of its surrounding cartilage during recovery. It is therefore concluded that L in this newly established framework can discriminate between normal and proteoglycan-depleted cartilage samples. However, more samples will be needed to verify the demarcation between samples degraded for varying amount of time., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Modern tissue engineering technologies have delivered tools to recreate a cell's naturally occurring niche in vitro and to investigate normal and pathological cell-cell and cell-niche interactions. Hydrogel biomaterials mimic crucial properties of native extracellular matrices, including mechanical support, cell adhesion sites and proteolytic degradability. As such, they are applied as 3D cell culture platforms to replicate tissue-like architectures observed in vivo, allowing physiologically relevant cell behaviors. Here we review bioengineered 3D approaches used for prostate and breast cancer. Furthermore, we describe the synthesis and use of gelatin methacryloyl-based hydrogels as in vitro 3D cancer model. This platform is used to engineer the microenvironments for prostate and breast cancer cells to study processes regulating spheroid formation, cell functions and responses to therapeutic compounds. Collectively, these bioengineered 3D approaches provide cell biologists with innovative pre-clinical tools that integrate the complexity of the disease seen in patients to advance our knowledge of cancer cell physiology and the contribution of a tumor's surrounding milieu.

The ex vivo engineering of autologous cartilage tissues has the potential to revolutionize the clinical management of joint disorders. Yet, high manufacturing costs and variable outcomes associated with tissue-engineered implants are still limiting their application. To improve clinical outcomes and facilitate a wider use of engineered tissues, automated bioreactor systems capable of enhancing and monitoring neotissues are required. Here, we developed an innovative system capable of applying precise uni- or biaxial mechanical stimulation to developing cartilage neotissues in a tightly controlled and automated fashion. The bioreactor allows for simple control over the loading parameters with a user-friendly graphical interface and is equipped with a load cell for monitoring tissue maturation. Applying our bioreactor, we demonstrate that human articular chondrocytes encapsulated in hydrogels composed of gelatin methacryloyl (GelMA) and hyaluronic acid methacrylate (HAMA) respond to uni- and biaxial mechanical stimulation by upregulation of hyaline cartilage-specific marker genes. We further demonstrate that intermittent biaxial mechanostimulation enhances accumulation of hyaline cartilage-specific extracellular matrix. Our study underlines the stimulatory effects of mechanical loading on the biosynthetic activity of human chondrocytes in engineered constructs and the need for easy-to-use, automated bioreactor systems in cartilage tissue engineering.

Three-dimensional (3D) bioprinting techniques can be used for the fabrication of personalized, regenerative constructs for tissue repair. The current article provides insight into the potential and opportunities of 3D bioprinting for the fabrication of cartilage regenerative constructs. Although 3D printing is already used in the orthopedic clinic, the shift toward 3D bioprinting has not yet occurred. We believe that this shift will provide an important step forward in the field of cartilage regeneration. Three-dimensional bioprinting techniques allow incorporation of cells and biological cues during the manufacturing process, to generate biologically active implants. The outer shape of the construct can be personalized based on clinical images of the patient's defect. Additionally, by printing with multiple bio-inks, osteochondral or zonally organized constructs can be generated. Relevant mechanical properties can be obtained by hybrid printing with thermoplastic polymers and hydrogels, as well as by the incorporation of electrospun meshes in hydrogels. Finally, bioprinting techniques contribute to the automation of the implant production process, reducing the infection risk. To prompt the shift from nonliving implants toward living 3D bioprinted cartilage constructs in the clinic, some challenges need to be addressed. The bio-inks and required cartilage construct architecture need to be further optimized. The bio-ink and printing process need to meet the sterility requirements for implantation. Finally, standards are essential to ensure a reproducible quality of the 3D printed constructs. Once these challenges are addressed, 3D bioprinted living articular cartilage implants may find their way into daily clinical practice.

Biochemically modified proteins have attracted significant attention due to their widespread applications as biomaterials. For instance, chemically modified gelatin derivatives have been widely explored to develop hydrogels for tissue engineering and regenerative medicine applications. Among the reported methods, modification of gelatin with methacrylic anhydride (MA) stands out as a convenient and efficient strategy to introduce functional groups and form hydrogels via photopolymerization. Combining light-activation of modified gelatin with soft lithography has enabled the materialization of microfabricated hydrogels. So far, this gelatin derivative has been referred to in the literature as gelatin methacrylate, gelatin methacrylamide, or gelatin methacryloyl, with the same abbreviation of GelMA. Considering the complex composition of gelatin and the presence of different functional groups on the amino acid residues, both hydroxyl groups and amine groups can possibly react with methacrylic anhydride during functionalization of the protein. This can also apply to the modification of other proteins, such as recombinant human tropoelastin to form MA-modified tropoelastin (MeTro). Here, we employed analytical methods to quantitatively determine the amounts of methacrylate and methacrylamide groups in MA-modified gelatin and tropoelastin to better understand the reaction mechanism. By combining two chemical assays with instrumental techniques, such as proton nuclear magnetic resonance ( 1 H NMR) and liquid chromatography tandem-mass spectrometry (LC-MS/MS), our results indicated that while amine groups had higher reactivity than hydroxyl groups and resulted in a majority of methacrylamide groups, modification of proteins by MA could lead to the formation of both methacrylamide and methacrylate groups. It is therefore suggested that the standard terms for GelMA and MeTro should be defined as gelatin methacryloyl and methacryloyl-substituted tropoelastin, respectively, to remain consistent with the widespread abbreviations used in literature., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Although end-of-life medical spending is often viewed as a major component of aggregate medical expenditure, accurate measures of this type of medical spending are scarce. We used detailed health care data for the period 2009-11 from Denmark, England, France, Germany, Japan, the Netherlands, Taiwan, the United States, and the Canadian province of Quebec to measure the composition and magnitude of medical spending in the three years before death. In all nine countries, medical spending at the end of life was high relative to spending at other ages. Spending during the last twelve months of life made up a modest share of aggregate spending, ranging from 8.5 percent in the United States to 11.2 percent in Taiwan, but spending in the last three calendar years of life reached 24.5 percent in Taiwan. This suggests that high aggregate medical spending is due not to last-ditch efforts to save lives but to spending on people with chronic conditions, which are associated with shorter life expectancies., (Project HOPE—The People-to-People Health Foundation, Inc.)

The ability to treat large tissue defects with customized, patient-specific scaffolds is one of the most exciting applications in the tissue engineering field. While an increasing number of modestly sized tissue engineering solutions are making the transition to clinical use, successfully scaling up to large scaffolds with customized geometry is proving to be a considerable challenge. Managing often conflicting requirements of cell placement, structural integrity, and a hydrodynamic environment supportive of cell culture throughout the entire thickness of the scaffold has driven the continued development of many techniques used in the production, culturing, and characterization of these scaffolds. This review explores a range of technologies and methods relevant to the design and manufacture of large, anatomically accurate tissue-engineered scaffolds with a focus on the interaction of manufactured scaffolds with the dynamic tissue culture fluid environment. Biotechnol. Bioeng. 2017;114: 1129-1139. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Articular cartilage from a material science point of view is a soft network composite that plays a critical role in load-bearing joints during dynamic loading. Its composite structure, consisting of a collagen fiber network and a hydrated proteoglycan matrix, gives rise to the complex mechanical properties of the tissue including viscoelasticity and stress relaxation. Melt electrospinning writing allows the design and fabrication of medical grade polycaprolactone (mPCL) fibrous networks for the reinforcement of soft hydrogel matrices for cartilage tissue engineering. However, these fiber-reinforced constructs underperformed under dynamic and prolonged loading conditions, suggesting that more targeted design approaches and material selection are required to fully exploit the potential of fibers as reinforcing agents for cartilage tissue engineering. In the present study, we emulated the proteoglycan matrix of articular cartilage by using highly negatively charged star-shaped poly(ethylene glycol)/heparin hydrogel (sPEG/Hep) as the soft matrix. These soft hydrogels combined with mPCL melt electrospun fibrous networks exhibited mechanical anisotropy, nonlinearity, viscoelasticity and morphology analogous to those of their native counterpart, and provided a suitable microenvironment for in vitro human chondrocyte culture and neocartilage formation. In addition, a numerical model using the p-version of the finite element method (p-FEM) was developed in order to gain further insights into the deformation mechanisms of the constructs in silico, as well as to predict compressive moduli. To our knowledge, this is the first study presenting cartilage tissue-engineered constructs that capture the overall transient, equilibrium and dynamic biomechanical properties of human articular cartilage.

Biological tissues at articulating surfaces, such as articular cartilage, typically have remarkable low-friction properties that limit tissue shear during movement. However, these frictional properties change with trauma, aging, and disease, resulting in an altered mechanical state within the tissues. Yet, it remains unclear how these surface changes affect the behaviour of embedded cells when the tissue is mechanically loaded. Here, we developed a cytocompatible, bilayered hydrogel system that permits control of surface frictional properties without affecting other bulk physicochemical characteristics such as compressive modulus, mass swelling ratio, and water content. This hydrogel system was applied to investigate the effect of variations in surface friction on the biological response of human articular chondrocytes to shear loading. Shear strain in these hydrogels during dynamic shear loading was significantly higher in high-friction hydrogels than in low-friction hydrogels. Chondrogenesis was promoted following dynamic shear stimulation in chondrocyte-encapsulated low-friction hydrogel constructs, whereas matrix synthesis was impaired in high-friction constructs, which instead exhibited increased catabolism. Our findings demonstrate that the surface friction of tissue-engineered cartilage may act as a potent regulator of cellular homeostasis by governing the magnitude of shear deformation during mechanical loading, suggesting a similar relationship may also exist for native articular cartilage., Statement of Significance: Excessive mechanical loading is believed to be a major risk factor inducing pathogenesis of articular cartilage and other load-bearing tissues. Yet, the mechanisms leading to increased transmission of mechanical stimuli to cells embedded in the tissue remain largely unexplored. Here, we demonstrate that the tribological properties of loadbearing tissues regulate cellular behaviour by governing the magnitude of mechanical deformation arising from physiological tissue function. Based on these findings, we propose that changes to articular surface friction as they occur with trauma, aging, or disease, may initiate tissue pathology by increasing the magnitude of mechanical stress on embedded cells beyond a physiological level., (Copyright © 2016 Acta Materialia Inc. All rights reserved.)

Hypoxia has been shown to be important for maintaining cartilage homeostasis as well as for inducing chondrogenic differentiation. Ensuring low oxygen levels during in vitro culture is difficult, therefore we assessed the chondro-inductive capabilities of the hypoxia-mimicking agent O-phenanthroline, which is also known as a non-specific matrix metalloproteinase (MMP) inhibitor. We found that O-phenanthroline reduced the expression of MMP3 and MMP13 mRNA levels during chondrogenic differentiation of human chondrocytes (hChs), as well as after TNFα/IL-1β exposure in an explant model. Interestingly, O-phenanthroline significantly inhibited matrix degradation in a TNFα/IL-1β-dependent model of cartilage degeneration when compared to control and natural hypoxia (2.5% O 2 ). O-Phenanthroline had limited ability to improve the chondrogenic differentiation or matrix deposition in the chondrogenic pellet model. Additionally, O-phenanthroline alleviated MMP-induced cartilage degradation without affecting chondrogenesis in the explant culture. The data presented in this study indicate that the inhibitory effect of O-phenanthroline on MMP expression is dominant over the hypoxia-mimicking effect. Copyright © 2014 John Wiley & Sons, Ltd., (Copyright © 2014 John Wiley & Sons, Ltd.)

The concept of biphasic or multi-layered compound scaffolds has been explored within numerous studies in the context of cartilage and osteochondral regeneration. To date, no system has been identified that stands out in terms of superior chondrogenesis, osteogenesis or the formation of a zone of calcified cartilage (ZCC). Herein we present a 3D plotted scaffold, comprising an alginate and hydroxyapatite paste, cast within a photocrosslinkable hydrogel made of gelatin methacrylamide (GelMA), or GelMA with hyaluronic acid methacrylate (HAMA). We hypothesized that this combination of 3D plotting and hydrogel crosslinking would form a high fidelity, cell supporting structure that would allow localization of hydroxyapatite to the deepest regions of the structure whilst taking advantage of hydrogel photocrosslinking. We assessed this preliminary design in terms of chondrogenesis in culture with human articular chondrocytes, and verified whether the inclusion of hydroxyapatite in the form presented had any influence on the formation of the ZCC. Whilst the inclusion of HAMA resulted in a better chondrogenic outcome, the effect of HAP was limited. We overall demonstrated that formation of such compound structures is possible, providing a foundation for future work. The development of cohesive biphasic systems is highly relevant for current and future cartilage tissue engineering.

Progress in advancing a system-level understanding of the complexity of human tissue development and regeneration is hampered by a lack of biological model systems that recapitulate key aspects of these processes in a physiological context. Hence, growing demand by cell biologists for organ-specific extracellular mimics has led to the development of a plethora of 3D cell culture assays based on natural and synthetic matrices. We developed a physiological microenvironment of semisynthetic origin, called gelatin methacryloyl (GelMA)-based hydrogels, which combine the biocompatibility of natural matrices with the reproducibility, stability and modularity of synthetic biomaterials. We describe here a step-by-step protocol for the preparation of the GelMA polymer, which takes 1-2 weeks to complete, and which can be used to prepare hydrogel-based 3D cell culture models for cancer and stem cell research, as well as for tissue engineering applications. We also describe quality control and validation procedures, including how to assess the degree of GelMA functionalization and mechanical properties, to ensure reproducibility in experimental and animal studies.

Photocrosslinkable hydrogels are frequently used in cartilage tissue engineering, with crosslinking systems relying on cytotoxic photoinitiators and ultraviolet (UV) light to form permanent hydrogels. These systems are rarely assessed in terms of optimization of photoinitiator or UV dosage, with non-cytotoxic concentrations from literature deemed sufficient. We hypothesized that the number of reactive functional groups present within a hydrogel polymer is highly relevant when crosslinking, affording cytoprotection to chondrocytes by preferentially interacting with the highly reactive radicals that are formed during UV-mediated activation of a photoinitiator. This was tested using two photocrosslinkable hydrogel systems: gelatin methacrylamide (GelMA) and gellan gum methacrylate (GGMA). We further assessed the effects of two different UV dosages on chondrocyte differentiation while subject to a single photoinitiator dosage in the GGMA system. Most notably, we found that a higher ratio of reactive groups to photoinitiator molecules offers cytoprotective effects, and future developments in photocrosslinkable hydrogel technology may involve assessment of such ratios. In contrast, we found there to be no effect of UV on chondrocyte differentiation at the two chosen dosages. Overall the optimization of photocrosslinkable systems is of great value in cartilage tissue engineering and these data provide a groundwork for such concepts to be developed further., Statement of Significance: Photocrosslinkable hydrogels, which use photoinitiators and predominantly ultraviolet light to form stable matrices for cell encapsulation and tissue development, are promising for cartilage tissue engineering. While both photoinitiators and ultraviolet light can damage cells, these systems have generally not been optimized. We propose that the ratio of reactive functional groups within a polymer to photoinitiator molecules is a critical parameter for optimization of photocrosslinkable hydrogels. Using photocrosslinkable gelatin and gellan gum, we found that a higher ratio of reactive groups to photoinitiator molecules protected chondrocytes, but did not affect chondrocyte differentiation. The principle of cytoprotection by functional groups developed in this work will be of great value in optimizing photocrosslinkable hydrogel systems for cartilage and other tissue engineering applications., (Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The crystal structure of 5-iodouracil, C4H3IN2O2, has been determined in the noncentrosymmetric space group P21 on a nonmerohedrally twinned crystal. Both twin components are enantiomorphically pure, but the twin element is a mirror plane perpendicular to c*. The molecular structure is discussed and stacking faults in the two-dimensional packing are proposed as a reason for the twinning.

Engineered biphasic osteochondral tissues may have utility in cartilage defect repair. As bone-marrow-derived mesenchymal stem/stromal cells (MSC) have the capacity to make both bone-like and cartilage-like tissues, they are an ideal cell population for use in the manufacture of osteochondral tissues. Effective differentiation of MSC to bone-like and cartilage-like tissues requires two unique medium formulations and this presents a challenge both in achieving initial MSC differentiation and in maintaining tissue stability when the unified osteochondral tissue is subsequently cultured in a single medium formulation. In this proof-of-principle study, we used an in-house fabricated microwell platform to manufacture thousands of micropellets formed from 166 MSC each. We then characterized the development of bone-like and cartilage-like tissue formation in the micropellets maintained for 8-14 days in sequential combinations of osteogenic or chondrogenic induction medium. When bone-like or cartilage-like micropellets were induced for only 8 days, they displayed significant phenotypic changes when the osteogenic or chondrogenic induction medium, respectively, was swapped. Based on these data, we developed an extended 14-day protocol for the pre-culture of bone-like and cartilage-like micropellets in their respective induction medium. Unified osteochondral tissues were formed by layering 12,000 osteogenic micropellets and 12,000 chondrogenic micropellets into a biphasic structure and then further culture in chondrogenic induction medium. The assembled tissue was cultured for a further 8 days and characterized via histology. The micropellets had amalgamated into a continuous structure with distinctive bone-like and cartilage-like regions. This proof-of-concept study demonstrates the feasibility of micropellet assembly for the formation of osteochondral-like tissues for possible use in osteochondral defect repair.

Appropriate selection of scaffold architecture is a key challenge in cartilage tissue engineering. Gap junction-mediated intercellular contacts play important roles in precartilage condensation of mesenchymal cells. However, scaffold architecture could potentially restrict cell-cell communication and differentiation. This is particularly important when choosing the appropriate culture platform as well as scaffold-based strategy for clinical translation, that is, hydrogel or microtissues, for investigating differentiation of chondroprogenitor cells in cartilage tissue engineering. We, therefore, studied the influence of gap junction-mediated cell-cell communication on chondrogenesis of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and articular chondrocytes. Expanded human chondrocytes and BM-MSCs were either (re-) differentiated in micromass cell pellets or encapsulated as isolated cells in alginate hydrogels. Samples were treated with and without the gap junction inhibitor 18-α glycyrrhetinic acid (18αGCA). DNA and glycosaminoglycan (GAG) content and gene expression levels (collagen I/II/X, aggrecan, and connexin 43) were quantified at various time points. Protein localization was determined using immunofluorescence, and adenosine-5'-triphosphate (ATP) was measured in conditioned media. While GAG/DNA was higher in alginate compared with pellets for chondrocytes, there were no differences in chondrogenic gene expression between culture models. Gap junction blocking reduced collagen II and extracellular ATP in all chondrocyte cultures and in BM-MSC hydrogels. However, differentiation capacity was not abolished completely by 18αGCA. Connexin 43 levels were high throughout chondrocyte cultures and peaked only later during BM-MSC differentiation, consistent with the delayed response of BM-MSCs to 18αGCA. Alginate hydrogels and microtissues are equally suited culture platforms for the chondrogenic (re-)differentiation of expanded human articular chondrocytes and BM-MSCs. Therefore, reducing direct cell-cell contacts does not affect in vitro chondrogenesis. However, blocking gap junctions compromises cell differentiation, pointing to a prominent role for hemichannel function in this process. Therefore, scaffold design strategies that promote an increasing distance between single chondroprogenitor cells do not restrict their differentiation potential in tissue-engineered constructs.

Cartilage defects heal imperfectly and osteoarthritic changes develop frequently as a result. Although the existence of specific behaviours of chondrocytes derived from various depth-related zones in vitro has been known for over 20 years, only a relatively small body of in vitro studies has been performed with zonal chondrocytes and current clinical treatment strategies do not reflect these native depth-dependent (zonal) differences. This is surprising since mimicking the zonal organization of articular cartilage in neo-tissue by the use of zonal chondrocyte subpopulations could enhance the functionality of the graft. Although some research groups including our own have made considerable progress in tailoring culture conditions using specific growth factors and biomechanical loading protocols, we conclude that an optimal regime has not yet been determined. Other unmet challenges include the lack of specific zonal cell sorting protocols and limited amounts of cells harvested per zone. As a result, the engineering of functional tissue has not yet been realized and no long-term in vivo studies using zonal chondrocytes have been described. This paper critically reviews the research performed to date and outlines our view of the potential future significance of zonal chondrocyte populations in regenerative approaches for the treatment of cartilage defects. Secondly, we briefly discuss the capabilities of additive manufacturing technologies that can not only create patient-specific grafts directly from medical imaging data sets but could also more accurately reproduce the complex 3D zonal extracellular matrix architecture using techniques such as hydrogel-based cell printing., (Copyright © 2012 John Wiley & Sons, Ltd.)

We and others have published on the rapid manufacture of micropellet tissues, typically formed from 100-500 cells each. The micropellet geometry enhances cellular biological properties, and in many cases the micropellets can subsequently be utilized as building blocks to assemble complex macrotissues. Generally, micropellets are formed from cells alone, however when replicating matrix-rich tissues such as cartilage it would be ideal if matrix or biomaterials supplements could be incorporated directly into the micropellet during the manufacturing process. Herein we describe a method to efficiently incorporate donor cartilage matrix into tissue engineered cartilage micropellets. We lyophilized bovine cartilage matrix, and then shattered it into microscopic pieces having average dimensions < 10 μm diameter; we termed this microscopic donor matrix "cartilage dust (CD)". Using a microwell platform, we show that ~0.83 μg CD can be rapidly and efficiently incorporated into single multicellular aggregates formed from 180 bone marrow mesenchymal stem/stromal cells (MSC) each. The microwell platform enabled the rapid manufacture of thousands of replica composite micropellets, with each micropellet having a material/CD core and a cellular surface. This micropellet organization enabled the rapid bulking up of the micropellet core matrix content, and left an adhesive cellular outer surface. This morphological organization enabled the ready assembly of the composite micropellets into macroscopic tissues. Generically, this is a versatile method that enables the rapid and uniform integration of biomaterials into multicellular micropellets that can then be used as tissue building blocks. In this study, the addition of CD resulted in an approximate 8-fold volume increase in the micropellets, with the donor matrix functioning to contribute to an increase in total cartilage matrix content. Composite micropellets were readily assembled into macroscopic cartilage tissues; the incorporation of CD enhanced tissue size and matrix content, but did not enhance chondrogenic gene expression.