Achieving ceramics that combine stiffness, strength, and toughness in complex geometries remains a major challenge, limiting their use in applications such as bone implants. Bio-inspired architectures – composed of stiff, strong building blocks connected by deformable interfaces – offer a promising solution. However, their implementation is constrained by limited control over the arrangement and composition of blocks, especially in constructs with complex shapes at centimeter- to meter-scale. We present a light-based printing method that deposits and assembles interlocking ceramic bricks onto connecting micro-pins, inspired by nacre’s growth mechanism. A custom alumina resin was developed and printed using a tabletop LCD 3D printer, sintered, and subsequently infiltrated with polycaprolactone, a tough and biocompatible polymer that forms the deformable interfaces. The resulting nacre-like constructs, with 76-78 wt% (50-52 vol%) ceramic, showed flexural stiffness of 2.27±0.44 GPa, strength of 41.4±1.33 MPa, and energy absorption of at least 6.35±0.97 MJ/m 3 . These values match the energy absorption of cortical bone, exceed the rule-of-mixtures value by fivefold, and surpass that of monolithic ceramic by over 4,000 times. Finally, a 5 cm-long nacre-like construct replicating the geometry of the human tibia was fabricated and tested in flexion, demonstrating scalability and potential for biomedical applications.

Hydrogels are frequently used in regenerative medicine due to their hydrated, tissue-compatible nature, and tuneable mechanics. While many strategies enable bulk mechanical modulation, little attention is given to tuning surface tribology, and its impact on cellular behavior under mechanical stimuli. Here, we demonstrate that photocrosslinking hydrogels on hydrophobic substrates leads to significant, long-lasting reductions in surface friction, ideal for cartilage tissue regeneration. Gelatin methacryloyl and hyaluronic acid methacrylate hydrogels photocrosslinked on polytetrafluoroethylene possess more hydrated, lubricious surfaces, with lower friction coefficients and crosslinking densities than those crosslinked on glass. This facilitated self-lubrication via water exudation, limiting shear during biaxial stimulation. When subject to intermittent biaxial loading mimicking joint movement, low-friction chondrocyte-laden neo-tissues formed superior hyaline cartilage, confirming the benefits of reduced friction on tissue development. Finally, in situ photocrosslinking enabled precise hydrogel formation in a full-thickness cartilage defect, highlighting the clinical potential and emphasizing the importance of crosslinking substrate in regenerative medicine.

Biofabrication techniques represent a promising avenue for the production of small diameter vascular grafts. However, while current tissue-engineered vascular grafts (TEVGs) fulfil certain functional requirements of native blood vessels, most exhibit very poor mechanical compliance, directly reducing patency in vivo. Here, highly compliant TEVGs were cultured in a dynamic pulsatile bioreactor which ensured enhanced compliance, using biomimetic melt electrowritten (MEW) tubular scaffolds as substrates for tissue growth. Through 6-week in vitro culture, we investigated differences in extracellular matrix (ECM) production and mechanical performance of TEVGs cultured with placental mesenchymal stem cells (MSCs) and smooth muscle cells (SMCs) in static and dynamic conditions. Pulsatile stimulation successfully maintained the high compliance (12.4 ± 0.8 % per 100 mmHg) of our biomimetic scaffolds, substantially greater than existing small diameter grafts. Dynamic TEVGs demonstrated physiologically relevant burst pressure (1125 ± 212 mmHg) and suture pull-out force (3.0 ± 0.4 N), while also accumulating greater ECM components than static TEVGs. To assess off-the-shelf suitability, grafts were decellularized and lyophilised to produce d-TEVGs, which exhibited negligible loss of mechanics or ECM integrity. Finally, rehydrated d-TEVGs were seeded with endothelial cells in vitro, with an intimal endothelial lining forming after 7 days. These findings demonstrate the production of TEVGs with specifically engineered mechanical compliance which has been maintained by dynamic in vitro culture, supporting continued work toward biofabrication of the next generation of vascular grafts.

Osteoarthritis (OA) is a prevalent degenerative joint disease affecting over 530 million individuals worldwide. Recent studies suggest a potential link between iron overload, a condition characterised by the excessive accumulation of iron in the body, and the onset of OA. Iron is essential for various biological processes, and any disruption in its homeostasis can trigger significant health effects, including OA. This study aimed to elucidate the effects of excess iron on joint tissue and the underlying mechanisms associated with excess iron and OA development. Human articular cartilage (n = 6) and synovium (n = 4) were collected from patients who underwent total knee arthroplasty. Cartilage and synovium explants were incubated with a gradually increasing concentration of ferric ammonium citrate for 3 days respectively. The effects of iron homeostasis in tissue explants were analysed using a Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS). To further study the effects of iron excess on OA initiation and development, male 3-week-old Hfe−/− and 5-week-old Tfr2−/− mice, animal models of hereditary haemochromatosis were established. Littermate wild-type mice were fed a high-iron diet to induce dietary overload. All animals were sacrificed at 8 weeks of age, and knee joints were harvested for histological analysis. The LA-ICP-MS analysis unveiled changes in the elemental composition related to iron metabolism, which included alterations in FTH1, FPN1, and HAMP within iron(III)-treated cartilage explants. While chondrocyte viability remained stable under different iron concentrations, ex vivo treatment with a high concentration of Fe3+ increased the catabolic gene expression of MMP13. Similar alterations were observed in the synovium, with added increases in GAG content and inflammation markers. In vivo studies further supported the role of iron overload in OA development as evidenced by spontaneous OA symptoms, proteoglycan loss, increased Mankin scores, synovial thickening, and enhanced immunohistochemical expression of MMP13, ADAMTS5, and P21 in Hfe−/−, Tfr2−/−, and diet-induced iron overload mouse models. Our findings elucidate the specific pathways through which excess iron accelerates OA progression and highlights potential targets for therapeutic intervention aimed at modulating iron levels to mitigate OA symptoms.

Despite the growing significance of generating highly porous, high-precision 3D printed scaffolds using melt electrowriting (MEW), the absence of easy-to-use and robust design and g-code generation tools hinders its effectiveness and widespread adoption. This article introduces a versatile scaffold design workflow for MEW fabrication, drawing on learnings in scaffold design and fabrication optimization from pioneers in the field and unifies innovative approaches into one user-friendly platform. The application contains a library of validated flat and tubular patterns, as well as incorporating automated solutions to common challenges such as scaffold dimension fitting and toolpath corrections previously reported in the literature and historically challenging to deploy into new g-codes. Available to users via GitHub, this application readily generates a g-code that compensates for MEW fiber lag across diverse geometries on a variety of flat and cylindrical collectors. The application's capabilities are demonstrated through proof-of-concept prints, including layer-shifting strategies, and their effects on scaffold mechanical properties. By integrating design flexibility, layer-shifting strategies, and advanced visualization, this application streamlines the MEW scaffold design process, offering a valuable tool for researchers and promises to both advance the field and enable greater access to tools required to produce high-quality, reproducible scaffolds. This study presents a versatile scaffold design software for melt electrowriting (MEW) fabrication. Consolidating a broad library of scaffold design patterns and fabrication correction tools presented in the MEW literature, this software enables rapid design iterations and automatic g-code generation. Using this software, researchers can incorporate best practice scaffold fabrication features to improve print accuracy, and reproducibility, and accelerate MEW innovation. image

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.

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 250µm pore sizes, demonstrating minimal deviation of less than 4% from the intended architecture, with consistent fibre diameter of 15 ± 2µm 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 250µm pores exhibited physiologically relevant burst pressures of 1078 ± 236 mmHg, compared to 631 ± 105 mmHg for corresponding 500µm controls. Similar trends were observed for strength and failure, indicating enhanced mechanical performance of scaffolds with reduced pore spacing. Preliminaryin vitroculture 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.

Tissue engineering is a promising approach for the production of small-diameter vascular grafts; however, there are limited data directly comparing the suitability of applicable cell types for vessel biofabrication. Here, we investigated the potential of adult smooth muscle cells (SMCs), placental mesenchymal stem cells (MSCs), placental endothelial colony-forming cells (ECFCs), and a combination of MSCs and ECFCs on highly porous biocompatible poly(ɛ-caprolactone) (PCL) scaffolds produced via melt electrowriting (MEW) for the biofabrication of tissue-engineered vascular grafts (TEVGs). Cellular attachment, proliferation, and deposition of essential extracellular matrix (ECM) components were analysed in vitro over four weeks. TEVGs cultured with MSCs accumulated the highest levels of collagenous components within a dense ECM, while SMCs and the coculture were more sparsely populated, ascertained via histological and immunofluorescence imaging, and biochemical assessment. Scanning electron microscopy (SEM) enabled visualisation of morphological differences in cell attachment and growth, with MSCs and SMCs infiltrating and covering scaffolds completely within the 28-day culture period. Coverage and matrix deposition by ECFCs was limited. However, ECFCs lined the ECM formed by MSCs in coculture, visualised via immunostaining. Thus, of cells investigated, placental MSCs were identified as the preferred cell source for the fabrication of tissue-engineered constructs, exhibiting extensive population of porous polymer scaffolds and production of ECM components; with the inclusion of ECFCs for luminal endothelialisation, an encouraging outcome warranting further consideration in future studies. In combination, these findings represent a substantial step toward the development of the next generation of small-diameter vascular grafts in the management of cardiovascular disease.

The field of melt electrowriting (MEW) has seen significant progress, bringing innovative advancements to the fabrication of biomaterial scaffolds, and creating new possibilities for applications in tissue engineering and beyond. Multidisciplinary collaboration across materials science, computational modeling, AI, bioprinting, microfluidics, and dynamic culture systems offers promising new opportunities to gain deeper insights into complex biological systems. As the focus shifts towards personalized medicine and reduced reliance on animal models, the multidisciplinary approach becomes indispensable. This review provides a concise overview of current strategies and innovations in controlling and optimizing cellular responses to MEW scaffolds, highlighting the potential of scaffold material, MEW architecture, and computational modeling tools to accelerate the development of efficient biomimetic systems. Innovations in material science and the incorporation of biologics into MEW scaffolds have shown great potential in adding biomimetic complexity to engineered biological systems. These techniques pave the way for exciting possibilities for tissue modeling and regeneration, personalized drug screening, and cell therapies.

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.

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.

Nacre, the iridescent inner layer of seashells, displays an exceptional combination of strength and toughness due to its 'brick-wall' architecture. Significant research has been devoted to replicating nacre's architecture and its associated deformation and failure mechanisms. Using the resulting materials in applications necessitates adding functionalities such as self-healing, force sensing, bioactivity, heat conductivity and resistance, transparency, and electromagnetic interference shielding. Herein, progress in the fabrication, mechanics, and multi-functionality of nacre-like materials, particularly over the past three years is systematically and critically reviewed. The fabrication techniques reviewed include 3D printing, freeze-casting, mixing/coating-assembling, and laser engraving. The mechanical properties of the resulting materials are discussed in comparison with their constituents and previously developed nacre mimics. Subsequently, the progress in incorporating multifunctionalities and the resulting physical, chemical, and biological properties are evaluated. We finally provide suggestions based on 3D/4D printing, advanced modelling techniques, and machine elements to make reprogrammable nacre-like components with complex shapes and small building blocks, tackling some of the main challenges in the science and translation of these materials.

Treatment of large bone fractures remains a challenge for orthopedists. Bone regeneration is a complex process that includes skeletal cells such as osteoblasts, osteoclasts, and immune cells to regulate bone formation and resorption. Osteoimmunology, studying this complicated process, has recently been used to develop biomaterials for advanced bone regeneration. Ideally, a biomaterial shall enable a timely switch from early stage inflammatory (to recruit osteogenic progenitor cells) to later-stage anti-inflammatory (to promote differentiation and terminal osteogenic mineralization and model the microstructure of bone tissue) in immune cells, especially the M1-to-M2 phenotype switch in macrophage populations, for bone regeneration. Nanoparticle (NP)-based advanced drug delivery systems can enable the controlled release of therapeutic reagents and the delivery of therapeutics into specific cell types, thereby benefiting bone regeneration through osteoimmunomodulation. In this review, we briefly describe the significance of osteoimmunology in bone regeneration, the advancement of NP-based approaches for bone regeneration, and the application of NPs in macrophage-targeting drug delivery for advanced osteoimmunomodulation.

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.

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.

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.

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.

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.

Articular cartilage is comprised of zones that vary in architecture, extracellular matrix composition, and mechanical properties. Here, we designed and engineered a porous zonal microstructured scaffold from a single biocompatible polymer (poly [epsilon-caprolactone]) using multiple fabrication strategies: electrospinning, spherical porogen leaching, directional freezing, and melt electrowriting. With this approach we mimicked the zonal structure of articular cartilage and produced a stiffness gradient through the scaffold which aligns with the mechanics of the native tissue. Chondrocyte-seeded scaffolds accumulated extracellular matrix including glycosaminoglycans and collagen II over four weeks in vitro. This prompted us to further study the repair efficacy in a skeletally mature porcine model. Two osteochondral lesions were produced in the trochlear groove of 12 animals and repaired using four treatment conditions: (1) microstructured scaffold, (2) chondrocyte seeded microstructured scaffold, (3) MaioRegen (TM), and (4) empty defect. After 6 months the defect sites were harvested and analyzed using histology, micro computed tomography, and Raman microspectroscopy mapping. Overall, the scaffolds were retained in the defect space, repair quality was repeatable, and there was clear evidence of osteointegration. The repair quality of the microstructured scaffolds was not superior to the control based on histological scoring; however, the lower score was biased by the lack of histological staining due to the limited degradation of the implant at 6 months. Longer follow up studies (e.g., 1 yr) will be required to fully evaluate the efficacy of the microstructured scaffold. In conclusion, we found consistent scaffold retention, osteointegration, and prolonged degradation of the microstructured scaffold, which we propose may have beneficial effects for the long-term repair of osteochondral defects.

When repairing cartilage defects a major challenge is achieving high-quality integration between the repair tissue and adjacent native cartilage. Matrix-rich cartilage is not easily remodeled, motivating several studies to trial enzyme treatment of the tissue interface to facilitate remodeling and integration. Studying and optimizing such processes is tedious, as well as potentially expensive, and thus simpler models are needed to evaluate the merits of enzyme treatment on cartilage tissue integration. Herein, we used engineered cartilage microtissues formed from bone marrow-derived stromal cells (BMSC) or expanded articular chondrocytes (ACh) to study the impact of enzyme treatment on cartilage tissue integration and matrix remodeling. A 5-min treatment with collagenase appeared to improve cartilage microtissue integration, while up to 48 h treatment with hyaluronidase did not. Alcian blue and anti-collagen II staining suggested that collagenase treatment did facilitate near seamless integration of cartilage microtissues. Microtissue sections were stained with Picrosirius red and characterized using polarized light microscopy, revealing that individual microtissues contained a collagen network organized in concentric shells. While collagenase treatment appeared to improve tissue integration, assessment of the collagen fibers with polarized light indicated that enzymatically damaged networks were not remodeled nor restored during subsequent culture. This model and these data paradoxically suggest that collagen network disruption is required to improve cartilage tissue integration, but that the disrupted collagen networks are unlikely to subsequently be restored. Future studies should attempt to limit collagen network disruption to the surface of the cartilage, and we recommend using Picrosirius red staining and polarized light to assess the quality of matrix remodeling and integration.