Challenges in engineering large customized bone constructs.

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.

High-throughput bone and cartilage micropellet manufacture, followed by assembly of micropellets into biphasic osteochondral tissue.

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.

Human proximal tubule epithelial cells modulate autologous B-cell function.

BACKGROUND: Descriptions of inflammatory cells infiltrating the human kidney rarely mention B cells, other than in the specific scenario of transplantation. In these reports, B cells are localized almost exclusively within the kidney tubulointerstitium where they are ideally placed to interact with proximal tubule epithelial cells (PTEC). We have previously shown that activated PTEC down-modulate autologous T lymphocyte and dendritic cell function. In this report, we extend these prior studies to describe PTEC-B cell interactions. METHODS: Stimulated B cells were cultured in the absence or presence of activated autologous human PTEC and monitored for proliferation, surface antigen expression, cytokine secretion and antibody (Ab) production. RESULTS: PTEC decreased B cell proliferative responses, whilst B cells cultured in the presence of PTEC displayed decreased levels of CD27, a marker of plasma B cells and memory cells. Interestingly, autologous PTEC also significantly decreased the number of B cells secreting both IgG and IgM and overall levels of Ab production. Transwell studies demonstrated that this modulation was primarily contact-dependent, and blocking studies with anti-PD-L1 led to partial restoration in Ab production. Further blocking studies targeting soluble HLA-G (sHLA-G) and IDO, two other immunoinhibitory molecules also up-regulated in our activated PTEC, demonstrated minor restoration of Ab responses. DISCUSSION: We report, for the first time, that PTEC are also able to modulate autologous B-cell phenotype and function via complex contact-dependent (PD-L1), soluble (sHLA-G) and intracellular (IDO) factors. We hypothesize that such mechanisms may have evolved to maintain peripheral immune-homeostasis, especially within the inflammatory milieu that exists within many kidney diseases.

Effects of scaffold architecture on mechanical characteristics and osteoblast response to static and perfusion bioreactor cultures.

Tissue engineering focuses on the repair and regeneration of tissues through the use of biodegradable scaffold systems that structurally support regions of injury while recruiting and/or stimulating cell populations to rebuild the target tissue. Within bone tissue engineering, the effects of scaffold architecture on cellular response have not been conclusively characterized in a controlled-density environment. We present a theoretical and practical assessment of the effects of polycaprolactone (PCL) scaffold architectural modifications on mechanical and flow characteristics as well as MC3T3-E1 preosteoblast cellular response in an in vitro static plate and custom-designed perfusion bioreactor model. Four scaffold architectures were contrasted, which varied in inter-layer lay-down angle and offset between layers, while maintaining a structural porosity of 60 ± 5%. We established that as layer angle was decreased (90° vs. 60°) and offset was introduced (0 vs. 0.5 between layers), structural stiffness, yield stress, strength, pore size, and permeability decreased, while computational fluid dynamics-modeled wall shear stress was increased. Most significant effects were noted with layer offset. Seeding efficiencies in static culture were also dramatically increased due to offset (∼ 45% to ∼ 86%), with static culture exhibiting a much higher seeding efficiency than perfusion culture. Scaffold architecture had minimal effect on cell response in static culture. However, architecture influenced osteogenic differentiation in perfusion culture, likely by modifying the microfluidic environment.

Effect of preculture and loading on expression of matrix molecules, matrix metalloproteinases, and cytokines by expanded osteoarthritic chondrocytes.

OBJECTIVE: One of the pathologic changes that occurs during osteoarthritis (OA) is the degeneration of the pericellular matrix (PCM). Since the PCM is likely to be involved in mechanotransduction, this study was undertaken to investigate the effects of PCM-like matrix accumulation in zonal OA chondrocytes and their influence on chondrocyte response to compression. METHODS: Superficial and middle/deep zone chondrocytes from macroscopically normal cartilage of OA knees were expanded and encapsulated in alginate gels. The effects of compression (short-term or long-term) and preculture on chondrocyte expression of various matrix molecules, cytokines, and matrix metalloproteinases (MMPs) were assessed. Additionally, nonexpanded chondrocytes were encapsulated in alginate and cultured in the presence or absence of transforming growth factor ß1 (TGFß1) and dexamethasone and analyzed following short-term compression experiments. RESULTS: Expanded OA chondrocytes (superficial and middle/deep zone) that were precultured for 2 weeks under free-swelling conditions prior to dynamic compression responded more sensitively to loading and had increased matrix accumulation, increased interleukin-1ß (IL-1ß) and IL-4 levels, and decreased levels of MMP-2 (in the middle/deep zone) compared to the nonloaded controls. Compression also decreased MMP-3 and MMP-13 levels even without preculture. Nonexpanded chondrocytes did not respond to compression, but differences in gene expression were found depending on the zone of harvest, time in culture, and medium composition. CONCLUSION: Our findings demonstrate that with predeposited PCM-like matrix, compressive stimulation can enhance matrix protein accumulation in expanded OA chondrocytes. Investigations into how PCM or other matrix components differentially affect this balance under mechanical loading may provide invaluable insight into OA pathogenesis and the use of expanded cells in tissue engineering and regenerative medicine-based applications.

Addition of Laponite to gelatin methacryloyl bioinks improves the rheological properties and printability to create mechanically tailorable cell culture matrices.

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.

Multifunctional nacre-like materials.

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.

Highly compliant biomimetic scaffolds for small diameter tissue-engineered vascular grafts (TEVGs) produced via melt electrowriting (MEW).

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.

Spray nebulization enables polycaprolactone nanofiber production in a manner suitable for generation of scaffolds or direct deposition of nanofibers onto cells.

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-1polymer 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.

Osteoimmunomodulatory Nanoparticles for Bone Regeneration.

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.

Photo-Cross-Linkable, Injectable, and Highly Adhesive GelMA-Glycol Chitosan Hydrogels for Cartilage Repair.

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.

GelMA-glycol chitosan hydrogels for cartilage regeneration: The role of uniaxial mechanical stimulation in enhancing mechanical, adhesive, and biochemical properties.

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.

Method for manufacture and cryopreservation of cartilage microtissues.

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% O2 atmosphere than a 20% O2 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.

A tumour-spheroid manufacturing and cryopreservation process that yields a highly reproducible product ready for direct use in drug screening assays.

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.

Effects of oxygen and culture system on in vitro propagation and redifferentiation of osteoarthritic human articular chondrocytes.

Regenerative medicine-based approaches for the repair of damaged cartilage rely on the ability to propagate cells while promoting their chondrogenic potential. Thus, conditions for cell expansion should be optimized through careful environmental control. Appropriate oxygen tension and cell expansion substrates and controllable bioreactor systems are probably critical for expansion and subsequent tissue formation during chondrogenic differentiation. We therefore evaluated the effects of oxygen and microcarrier culture on the expansion and subsequent differentiation of human osteoarthritic chondrocytes. Freshly isolated chondrocytes were expanded on tissue culture plastic or CultiSpher-G microcarriers under hypoxic or normoxic conditions (5% or 20% oxygen partial pressure, respectively) followed by cell phenotype analysis with flow cytometry. Cells were redifferentiated in micromass pellet cultures over 4 weeks, under either hypoxia or normoxia. Chondrocytes cultured on tissue culture plastic proliferated faster, expressed higher levels of cell surface markers CD44 and CD105 and demonstrated stronger staining for proteoglycans and collagen type II in pellet cultures compared with microcarrier-cultivated cells. Pellet wet weight, glycosaminoglycan content and expression of chondrogenic genes were significantly increased in cells differentiated under hypoxia. Hypoxia-inducible factor-3α mRNA was up-regulated in these cultures in response to low oxygen tension. These data confirm the beneficial influence of reduced oxygen on ex vivo chondrogenesis. However, hypoxia during cell expansion and microcarrier bioreactor culture does not enhance intrinsic chondrogenic potential. Further improvements in cell culture conditions are therefore required before chondrocytes from osteoarthritic and aged patients can become a useful cell source for cartilage regeneration.

Biofabrication of small diameter tissue-engineered vascular grafts.

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.

Personalized Volumetric Tissue Generation by Enhancing Multiscale Mass Transport through 3D Printed Scaffolds in Perfused Bioreactors.

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.

Collagenase treatment appears to improve cartilage tissue integration but damage to collagen networks is likely permanent.

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.

Hydrogel: A Potential Material for Bone Tissue Engineering Repairing the Segmental Mandibular Defect.

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.

Tissue Engineering Cartilage with Deep Zone Cytoarchitecture by High-Resolution Acoustic Cell Patterning.

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.