Hydrodynamic control of titania nanotube formation on Ti-6Al-4V alloys enhances osteogenic differentiation of human mesenchymal stromal cells.

In order to obtain bioactive bone-implant interfaces with enhanced osteogenic capacity, various approaches have been developed to modify surface physicochemical properties of bio-inert titanium and titanium alloys. One promising strategy involves fabricating highly ordered nanotubes (NT) on implant surfaces via electrochemical anodization. However, few studies have applied this technique to Ti-6Al-4V alloys most commonly adopted for the fabrication of osteo-integrated surfaces on orthopedic implants. In this study, we investigated the influence of electrolyte hydrodynamics to NT fabrication on Ti-6Al-4V in ethylene glycol based electrolyte and evaluated the osteogenic differentiation capacity of human mesenchymal stromal cells (hMSCs) on different diameter NT surfaces. Computational Fluid Dynamics (CFD) analysis was used to simulate electrolyte flow profiles under various stirring conditions (e.g. stirrer bar location and flow direction) and their correlation to NT formation. Polished Ti-6Al-4V disks (240 grit) were anodized at 20 and 40 V under optimal electrolyte flow conditions for comparison of NT diameter-controlled osteogenic differentiation and mineralization potential of hMSCs over 21 days culture in osteogenic media. Ti-6Al-4V surfaces anodized with 20 and 40 V resulted with NTs diameter approx. 39 and 83 nm, respectively. Electrolyte hydrodynamics (flow profile) significantly influenced the uniformity of NT formation. Here, a uniform velocity and shear stress profile at the surface promoted homogeneous NT growth, whereas large variation in either flow velocity or shear stress to the surface impaired mature NT formation. After 21 days of culture, fluorescence staining demonstrated significantly greater osteocalcin and osteopontin expression, and increased mineralized deposits (xylenol orange staining) on fluctuating NT surfaces anodized under 20 V (Ø 39 nm) relative to flat NT layer anodized with 40 V (Ø 83 nm) and polished controls. This study provides a systematic investigation of NT formation with respect to the electrolyte hydrodynamic effects to NT growth on Ti-6Al-4V alloys, demonstrating the feasibility of a one-step anodization process for generating uniform NT under optimal hydrodynamics. Optimized wavy micro-/nano-topography with Ø 39 nm NT stimulated osteogenic differentiation capacity of hMSCs on Ti-6Al-4V alloys and confirmed the potential application of anodization to improve osteo-integrative surfaces in orthopedic implants.

Intact vitreous humor as a potential extracellular matrix hydrogel for cartilage tissue engineering applications.

Decellularisation of tissues, utilising their biochemical cues, poses exciting tissue engineering (TE) opportunities. However, removing DNA from cartilage (dCart) requires harsh treatments due to its dense structure, causing loss of bioactivity and limiting its application as a cartilaginous extra cellular matrix (ECM). In this study, we demonstrate for the first time the successful application of vitreous humor (VH), a highly hydrated tissue closely resembling the glycosaminoglycan (GAG) and collagen composition of cartilage, as an ECM hydrogel to support chondrogenic differentiation. Equine VH was extracted followed by biochemical quantifications, histological examinations, cytotoxicity (human mesenchymal stromal cells, hMSCs and human articular chondrocytes, hACs) and U937 cell proliferation studies. VH was further seeded with hACs or hMSCs and cultured for 3-weeks to study chondrogenesis compared to scaffold-free micro-tissue pellet cultures and collagen-I hydrogels. Viability, metabolic activity, GAG and DNA content, chondrogenic gene expression (aggrecan, collagen I/II mRNA) and mechanical properties were quantified and matrix deposition was visualised using immunohistochemistry (Safranin-O, collagen I/II). VH was successfully extracted, exhibiting negligible amounts of DNA (0.4 ±â€¯0.4 µg/mg dry-weight) and notable preservation of ECM components. VH displayed neither cytotoxic responses nor proliferation of macrophage-like U937 cells, instead enhancing both hMSC and hAC proliferation. Interestingly, encapsulated cells self-assembled the VH-hydrogel into spheroids, resulting in uniform distribution of both GAGs and collagen type II with increased compressive mechanical properties, rendering VH a permissive native ECM source to fabricate cartilaginous hydrogels for potential TE applications. STATEMENT OF SIGNIFICANCE: Fabricating bioactive and cell-instructive cartilage extracellular matrix (ECM) derived biomaterials and hydrogels has over recent years proven to be a challenging task, often limited by poor retention of inherent environmental cues post decellularisation due to the dense and avascular nature of native cartilage. In this study, we present an alternative route to fabricate highly permissive and bioactive ECM hydrogels from vitreous humor (VH) tissue. This paper specifically reports the discovery of optimal VH extraction protocols and cell seeding strategy enabling fabrication of cartilaginous matrix components into a hydrogel support material for promoting chondrogenic differentiation. The work showcases a naturally intact and unmodified hydrogel design that improves cellular responses and may help guide the development of cell instructive and stimuli responsive hybrid biomaterials in a number of TERM applications.

A definition of bioinks and their distinction from biomaterial inks.

Biofabrication aims to fabricate biologically functional products through bioprinting or bioassembly (Groll et al 2016 Biofabrication 8 013001). In biofabrication processes, cells are positioned at defined coordinates in three-dimensional space using automated and computer controlled techniques (Moroni et al 2018 Trends Biotechnol. 36 384-402), usually with the aid of biomaterials that are either (i) directly processed with the cells as suspensions/dispersions, (ii) deposited simultaneously in a separate printing process, or (iii) used as a transient support material. Materials that are suited for biofabrication are often referred to as bioinks and have become an important area of research within the field. In view of this special issue on bioinks, we aim herein to briefly summarize the historic evolution of this term within the field of biofabrication. Furthermore, we propose a simple but general definition of bioinks, and clarify its distinction from biomaterial inks.

A 96-well microplate bioreactor platform supporting individual dual perfusion and high-throughput assessment of simple or biofabricated 3D tissue models.

Traditional 2D monolayer cell cultures and submillimeter 3D tissue construct cultures used widely in tissue engineering are limited in their ability to extrapolate experimental data to predict in vivo responses due to their simplistic organization and lack of stimuli. The rise of biofabrication and bioreactor technologies has sought to address this through the development of techniques to spatially organize components of a tissue construct, and devices to supply these tissue constructs with an increasingly in vivo-like environment. Current bioreactors supporting both parenchymal and barrier tissue constructs in interconnected systems for body-on-a-chip platforms have chosen to emphasize study throughput or system/tissue complexity. Here, we report a platform to address this disparity in throughput and both system complexity (by supporting multiple in situ assessment methods) and tissue complexity (by adopting a construct-agnostic format). We introduce an ANSI/SLAS-compliant microplate and docking station fabricated via stereolithography (SLA), or precision machining, to provide up to 96 samples (Ø6 × 10 mm) with two individually-addressable fluid circuits (192 total), loading access, and inspection window for imaging during perfusion. Biofabricated ovarian cancer models were developed to demonstrate the in situ assessment capabilities via microscopy and a perfused resazurin-based metabolic activity assay. In situ microscopy highlighted flexibility of the sample housing to accommodate a range of sample geometries. Utility for drug screening was demonstrated by exposing the ovarian cancer models to an anticancer drug (doxorubicin) and generating the dose-response curve in situ, while achieving an assay quality similar to static wellplate culture. The potential for quantitative analysis of temporal tissue development and screening studies was confirmed by imaging soft- (gelatin) and hard-tissue (calcium chloride) analogs inside the bioreactor via spectral computed tomography (CT) scanning. As a proof-of-concept for particle tracing studies, flowing microparticles were visualized to inform the design of hydrogel constructs. Finally, the ability for mechanistic yet high-throughput screening was demonstrated in a vascular coculture model adopting endothelial and mesenchymal stem cells (HUVEC-MSC), encapsulated in gelatin-norbornene (gel-NOR) hydrogel cast into SLA-printed well inserts. This study illustrates the potential of a scalable dual perfusion bioreactor platform for parenchymal and barrier tissue constructs to support a broad range of multi-organ-on-a-chip applications.

Engineering of a complex bone tissue model with endothelialised channels and capillary-like networks.

In engineering of tissue analogues, upscaling to clinically-relevant sized constructs remains a significant challenge. The successful integration of a vascular network throughout the engineered tissue is anticipated to overcome the lack of nutrient and oxygen supply to residing cells. This work aimed at developing a multiscale bone-tissue-specific vascularisation strategy. Engineering pre-vascularised bone leads to biological and fabrication dilemmas. To fabricate channels endowed with an endothelium and suitable for osteogenesis, rather stiff materials are preferable, while capillarisation requires soft matrices. To overcome this challenge, gelatine-methacryloyl hydrogels were tailored by changing the degree of functionalisation to allow for cell spreading within the hydrogel, while still enabling endothelialisation on the hydrogel surface. An additional challenge was the combination of the multiple required cell-types within one biomaterial, sharing the same culture medium. Consequently, a new medium composition was investigated that simultaneously allowed for endothelialisation, capillarisation and osteogenesis. Integrated multipotent mesenchymal stromal cells, which give rise to pericyte-like and osteogenic cells, and endothelial-colony-forming cells (ECFCs) which form capillaries and endothelium, were used. Based on the aforementioned optimisation, a construct of 8 × 8 × 3 mm, with a central channel of 600 µm in diameter, was engineered. In this construct, ECFCs covered the channel with endothelium and osteogenic cells resided in the hydrogel, adjacent to self-assembled capillary-like networks. This study showed the promise of engineering complex tissue constructs by means of human primary cells, paving the way for scaling-up and finally overcoming the challenge of engineering vascularised tissues.

Is tranexamic acid toxic to articular cartilage when administered topically? What is the safe dose?

Aims: The intra-articular administration of tranexamic acid (TXA) has been shown to be effective in reducing blood loss in unicompartmental knee arthroplasty and anterior cruciate reconstruction. The effects on human articular cartilage, however, remains unknown. Our aim, in this study, was to investigate any detrimental effect of TXA on chondrocytes, and to establish if there was a safe dose for its use in clinical practice. The hypothesis was that TXA would cause a dose-dependent damage to human articular cartilage. Materials and Methods: The cellular morphology, adhesion, metabolic activity, and viability of human chondrocytes when increasing the concentration (0 mg/ml to 40 mg/ml) and length of exposure to TXA (0 to 12 hours) were analyzed in a 2D model. This was then repeated, excluding cellular adhesion, in a 3D model and confirmed in viable samples of articular cartilage. Results: Increasing concentrations above 20 mg/ml resulted in atypical morphology, reduced cellular adhesion and metabolic activity associated with increased chondrocyte death. However, the cell matrix was not affected by the concentration of TXA or the length of exposure, and offered cellular protection for concentrations below 20 mg/ml. Conclusion: These results show that when in vitro chondrocytes are exposed to higher concentrations of TXA, such as that expected following recommended intra-articular administration, cytotoxicity is observed. This effect is dose-dependent, such that a tissue concentration of 10 mg/ml to 20 mg/ml could be expected to be safe. Cite this article: Bone Joint J 2018;100-B:404-12.

Automated 3D bioassembly of micro-tissues for biofabrication of hybrid tissue engineered constructs.

Bottom-up biofabrication approaches combining micro-tissue fabrication techniques with extrusion-based 3D printing of thermoplastic polymer scaffolds are emerging strategies in tissue engineering. These biofabrication strategies support native self-assembly mechanisms observed in developmental stages of tissue or organoid growth as well as promoting cell-cell interactions and cell differentiation capacity. Few technologies have been developed to automate the precise assembly of micro-tissues or tissue modules into structural scaffolds. We describe an automated 3D bioassembly platform capable of fabricating simple hybrid constructs via a two-step bottom-up bioassembly strategy, as well as complex hybrid hierarchical constructs via a multistep bottom-up bioassembly strategy. The bioassembly system consisted of a fluidic-based singularisation and injection module incorporated into a commercial 3D bioprinter. The singularisation module delivers individual micro-tissues to an injection module, for insertion into precise locations within a 3D plotted scaffold. To demonstrate applicability for cartilage tissue engineering, human chondrocytes were isolated and micro-tissues of 1 mm diameter were generated utilising a high throughput 96-well plate format. Micro-tissues were singularised with an efficiency of 96.0 ± 5.1%. There was no significant difference in size, shape or viability of micro-tissues before and after automated singularisation and injection. A layer-by-layer approach or aforementioned bottom-up bioassembly strategy was employed to fabricate a bilayered construct by alternatively 3D plotting a thermoplastic (PEGT/PBT) polymer scaffold and inserting pre-differentiated chondrogenic micro-tissues or cell-laden gelatin-based (GelMA) hydrogel micro-spheres, both formed via high-throughput fabrication techniques. No significant difference in viability between the construct assembled utilising the automated bioassembly system and manually assembled construct was observed. Bioassembly of pre-differentiated micro-tissues as well as chondrocyte-laden hydrogel micro-spheres demonstrated the flexibility of the platform while supporting tissue fusion, long-term cell viability, and deposition of cartilage-specific extracellular matrix proteins. This technology provides an automated and scalable pathway for bioassembly of both simple and complex 3D tissue constructs of clinically relevant shape and size, with demonstrated capability to facilitate direct spatial organisation and hierarchical 3D assembly of micro-tissue modules, ranging from biomaterial free cell pellets to cell-laden hydrogel formulations.

Modular Tissue Assembly Strategies for Biofabrication of Engineered Cartilage.

This review describes the prospects of applying modular assembly techniques and strategies for fabrication of advanced tissue engineered cartilage constructs. Articular cartilage is a tissue that has important functions in preserving and enabling locomotion. However, its limited intrinsic repair capacity and lack of current long-term clinical solutions makes it a candidate for repair or regeneration via tissue engineering strategies. Key advances in biofabrication and 3D bioprinting techniques allowing the specific placement of cells and tissues enable novel strategies to be adopted with increased chances of success. In particular, modular assembly, where separate biological components such as microtissue units, cellular building blocks or spheroids are combined with structural scaffold components to create a functional whole, offers potential as a new strategy for engineering of articular cartilage. Various modular assembly or bottom-up fabrication strategies have been investigated or applied for engineering of a range of tissues and cell types, however, modular approaches to cartilage engineering have been limited thus far. The integrative nature of many current approaches to engineering of articular cartilage means optimization of separate components (such as the scaffold and cells) is challenging, resulting in strategies which are less amenable to clinical scale-up or modification. In addition, current tissue engineering strategies may not replicate the function and complex structure of native tissue. This review outlines recent developments in fabrication of cellular or tissue modules as well as scaffold design where it impacts modular biofabrication, and discusses existing modular approaches applicable to articular cartilage regeneration and repair. Modular tissue assembly approaches allow complex hybrid constructs to be fabricated with direct control over both structural and cellular organization of pre-formed tissue units. The combination of modular assembly with automated biofabrication technologies may offer solutions to the development of optimal tissue-engineered cartilage constructs.

Three-dimensional assembly of tissue-engineered cartilage constructs results in cartilaginous tissue formation without retainment of zonal characteristics.

Articular cartilage has limited regenerative capabilities. Chondrocytes from different layers of cartilage have specific properties, and regenerative approaches using zonal chondrocytes may yield better replication of the architecture of native cartilage than when using a single cell population. To obtain high seeding efficiency while still mimicking zonal architecture, cell pellets of expanded deep zone and superficial zone equine chondrocytes were seeded and cultured in two layers on poly(ethylene glycol)-terephthalate-poly(butylene terephthalate) (PEGT-PBT) scaffolds. Scaffolds seeded with cell pellets consisting of a 1:1 mixture of both cell sources served as controls. Parallel to this, pellets of superficial or deep zone chondrocytes, and combinations of the two cell populations, were cultured without the scaffold. Pellet cultures of zonal chondrocytes in scaffolds resulted in a high seeding efficiency and abundant cartilaginous tissue formation, containing collagen type II and glycosaminoglycans (GAGs) in all groups, irrespective of the donor (n = 3), zonal population or stratified scaffold-seeding approach used. However, whereas total GAG production was similar, the constructs retained significantly more GAG compared to pellet cultures, in which a high percentage of the produced GAGs were secreted into the culture medium. Immunohistochemistry for zonal markers did not show any differences between the conditions. We conclude that spatially defined pellet culture in 3D scaffolds is associated with high seeding efficiency and supports cartilaginous tissue formation, but did not result in the maintenance or restoration of the original zonal phenotype. The use of pellet-assembled constructs leads to a better retainment of newly produced GAGs than the use of pellet cultures alone.

The rates of wear of X3 highly cross-linked polyethylene at five years when coupled with a 36 mm diameter ceramic femoral head in young patients.

Polyethylene wear debris can cause osteolysis and the failure of total hip arthroplasty. We present the five-year wear rates of a highly cross-linked polyethylene (X3) bearing surface when used in conjunction with a 36 mm ceramic femoral head. This was a prospective study of a cohort of 100 THAs in 93 patients. Pain and activity scores were measured pre- and post-operatively. Femoral head penetration was measured at two months, one year, two years and at five years using validated edge-detecting software (PolyWare Auto). At a mean of 5.08 years (3.93 to 6.01), 85 hips in 78 patients were available for study. The mean age of these patients was 59.08 years (42 to 73, the mean age of males (n = 34) was 59.15 years, and females (n = 44) was 59.02 years). All patients had significant improvement in their functional scores (p < 0.001). The steady state two-dimensional linear wear rate was 0.109 mm/year. The steady state volumetric wear rate was 29.61 mm(3)/year. No significant correlation was found between rate of wear and age (p = 0.34), acetabular component size (p = 0.12) or clinical score (p = 0.74). Our study shows low steady state wear rates at five years in X3 highly cross-linked polyethylene in conjunction with a 36 mm ceramic femoral head. The linear wear rate was almost identical to the osteolysis threshold of 0.1 mm/year recommended in the literature.

Positive and negative bioimprinted polymeric substrates: new platforms for cell culture.

Bioimprinting, which involves capturing cell morphological details into a polymer matrix, provides a new class of patterned surfaces which opens an opportunity to investigate how cells respond to their own signatures and may introduce possibilities for regulating their behaviour. In this study, phenotypic details of human nasal chondrocytes (HNCs) were replicated in soft polydimethylsiloxane (PDMS) mould resulting in inverse replicas of cells, which have been termed here as 'negative bioimprint'. For the first time, the information from this negative bioimprint was then transferred into another PDMS layer resulting in surfaces which resemble cell morphology and were called 'positive bioimprints'. Soft lithography was used to transfer these details from PDMS into different polymers like polystyrene, tissue culture polystyrene and clinically used block co-polymer poly (ethylene glycol) terephthalate-poly (butylene terephthalate) (PEGT-PBT). Results obtained from surface characterization confirmed that fine details of cells were successfully replicated from cells to different polymer matrices without any significant loss of information during the different steps of pattern transfer. HNCs seeded on different polymer surfaces with positive and negative bioimprints exhibited distinct behaviour. Cells cultured on positive bioimprints were more spread out and displayed high levels of proliferation compared to those on negative bioimprints, where cells were more compact with lower proliferation.

The in vitro and in vivo evaluation of the biocompatibility of Mg alloys.

Magnesium (Mg) and its alloys are being widely investigated for their potential use as resorbable biomaterials for orthopaedic applications. However, the natural corrosion of the metals results in potentially harmful perturbations to the physiological environment, which requires a comprehensive understanding of their biocompatibility. Currently, most investigations proceed directly from in vitro biocompatibility studies to intraosseous implantation. However, this can result in the unnecessary elimination of appropriate materials due to over sensitive in vitro methods or the implantation of potentially harmful materials. This study involved the development of a relevant in vitro cell culture method, and an in vivo soft tissue implantation technique to provide an intermediate step between basic cell culture methods and large animal intraosseous investigations. A Live/Dead fluorescent assay was used to investigate the viability of both L929 and SaOS-2 cells exposed to Mg alloys, with the results compared to those seen with the intramuscular implantation of the same materials in Lewis rats. These methods were able to successfully provide data on the corrosion of Mg alloys, allowing the identification of slowly and safely corroding materials that may be used in future intraosseous investigations.

Tissue-engineered constructs: the effect of scaffold architecture in osteochondral repair.

Cartilage has a poor regenerative capacity. Tissue-engineering approaches using porous scaffolds seeded with chondrocytes may improve cartilage repair. The aim of this study was to examine the effect of pore size and pore interconnectivity on cartilage repair in osteochondral defects treated with different scaffolds seeded with allogenic chondrocytes. Scaffolds consisting of 55 wt% poly(ethylene oxide terephthalate) and 45 wt% poly(butylene terephthalate) (PEOT/PBT) with different pore sizes and interconnectivities were made, using a compression moulding (CM) and a three-dimensional fibre (3DF) deposition technique. In these scaffolds, allogenic chondrocytes were seeded, cultured for 3 weeks and implanted in osteochondral defects of skeletally mature rabbits. At 3 weeks no difference in cartilage repair between an empty osteochondral defect, CM or 3DF scaffolds was found. Three months post-implantation, cartilage repair was significantly improved after implantation of a 3DF scaffold compared to a CM scaffold. Although not significant, Mankin scores for osteoarthritis (OA) indicated less OA in the 3DF scaffold group compared to empty defects and CM-treated defects. It is concluded that scaffold pore size and pore interconnectivity influences osteochondral repair and a decreased pore interconnectivity seems to impair osteochondral repair.

On the role of surface roughness in the corrosion of pure magnesium in vitro.

The relationship between surface roughness and degradation behavior in magnesium (Mg) biomaterials is still a controversial issue. This study aims to clarify the relationship between surface roughness and corrosion rate of pure Mg. Pure Mg samples with surface roughness values (Ra) of 0.59, 2.68, and 9.12 µm were cast using an indirect solid-free form fabrication method. The in vitro corrosion behavior was evaluated using hydrogen evolution, mass loss, potentiodynamic polarization, and electrochemical impedance spectroscopy. It was confirmed that surface roughness had a significant influence on the corrosion rate of pure Mg, with increasing roughness resulting in an accelerated corrosion rate. However, pitting corrosion was not observed, suggesting that surface roughness does not affect the pitting potential of Mg.

The early radiological results of the uncemented Oxford medial compartment knee replacement.

We carried out a prospective investigation into the radiological outcomes of uncemented Oxford medial compartment unicondylar replacement in 220 consecutive patients (231 knees) performed in a single centre with a minimum two-year follow-up. The functional outcomes using the mean Oxford knee score and the mean high-activity arthroplasty score were significantly improved over the pre-operative scores (p < 0.001). There were 196 patients with a two-year radiological examination performed under fluoroscopic guidance, aiming to provide images acceptable for analysis of the bone-implant interface. Of the six tibial zones examined on each knee on the anteroposterior radiograph, only three had a partial radiolucent line. All were in the medial aspect of the tibial base plate (zone 1) and all measured < 1 mm. All of these patients were asymptomatic. There were no radiolucent lines seen around the femoral component or on the lateral view. There was one revision for loosening at one year due to initial inadequate seating of the tibial component. These results confirm that the early uncemented Oxford medial unicompartmental compartmental knee replacements were reliable and the incidence of radiolucent lines was significantly decreased compared with the reported results of cemented versions of this implant. These independent results confirm those of the designing centre.

Validation of a high-throughput microtissue fabrication process for 3D assembly of tissue engineered cartilage constructs.

Described here is a simple, high-throughput process to fabricate pellets with regular size and shape and the assembly of pre-cultured pellets in a controlled manner into specifically designed 3D plotted porous scaffolds. Culture of cartilage pellets is a well-established process for inducing re-differentiation in expanded chondrocytes. Commonly adopted pellet culture methods using conical tubes are inconvenient, time-consuming and space-intensive. We compared the conventional 15-mL tube pellet culture method with 96-well plate-based methods, examining two different well geometries (round- and v-bottom plates). The high-throughput production method was then used to demonstrate guided placement of pellets within a scaffold of defined pore size and geometry for the 3D assembly of tissue engineered cartilage constructs. While minor differences were observed in tissue quality and size, the chondrogenic re-differentiation capacity of human chondrocytes, as assessed by GAG/DNA, collagen type I and II immunohistochemistry and collagen type I, II and aggrecan mRNA expression, was maintained in the 96-well plate format and pellets of regular size and spheroidal shape were produced. This allowed for simple production of large numbers of reproducible tissue spheroids. Furthermore, the pellet-assembly method successfully allowed fluorescently labelled pellets to be individually visualised in 3D. During subsequent culture of 3D assembled tissue engineered constructs in vitro, pellets fused to form a coherent tissue, promoting chondrogenic differentiation and GAG accumulation.

Rapid prototyping of anatomically shaped, tissue-engineered implants for restoring congruent articulating surfaces in small joints.

BACKGROUND: Preliminary studies investigated advanced scaffold design and tissue engineering approaches towards restoring congruent articulating surfaces in small joints. MATERIALS AND METHODS: Anatomical femoral and tibial cartilage constructs, fabricated by three-dimensional fibre deposition (3DF) or compression moulding/particulate leaching (CM), were evaluated in vitro and in vivo in an autologous rabbit model. Effects of scaffold pore architecture on rabbit chondrocyte differentiation and mechanical properties were evaluated following in vitro culture and subcutaneous implantation in nude mice. After femoral and tibial osteotomy and autologous implantation of tissue-engineered constructs in rabbit knee joints, implant fixation and joint articulation were evaluated. RESULTS: Rapid prototyping of 3DF architectures with 100% interconnecting pores promoted homogeneous distribution of viable cells, glycosaminoglycan (GAG) and collagen type II; significantly greater GAG content and differentiation capacity (GAG/DNA) in vitro compared to CM architectures; and higher mechanical equilibrium modulus and dynamic stiffness (at 0.1 Hz). Six weeks after implantation, femoral and tibial constructs had integrated with rabbit bone and knee flexion/extension and partial load bearing were regained. Histology demonstrated articulating surfaces between femoral and tibial constructs for CM and 3DF architectures; however, repair tissue appeared fibrocartilage-like and did not resemble implanted cartilage. CONCLUSIONS: Anatomically shaped, tissue-engineered constructs with designed mechanical properties and internal pore architectures may offer alternatives for reconstruction or restoration of congruent articulating surfaces in small joints.

Polymer scaffolds fabricated with pore-size gradients as a model for studying the zonal organization within tissue-engineered cartilage constructs.

The zonal organization of cells and extracellular matrix (ECM) constituents within articular cartilage is important for its biomechanical function in diarthroidal joints. Tissue-engineering strategies adopting porous three-dimensional (3D) scaffolds offer significant promise for the repair of articular cartilage defects, yet few approaches have accounted for the zonal structural organization as in native articular cartilage. In this study, the ability of anisotropic pore architectures to influence the zonal organization of chondrocytes and ECM components was investigated. Using a novel 3D fiber deposition (3DF) technique, we designed and produced 100% interconnecting scaffolds containing either homogeneously spaced pores (fiber spacing, 1 mm; pore size, about 680 microm in diameter) or pore-size gradients (fiber spacing, 0.5-2.0 mm; pore size range, about 200-1650 microm in diameter), but with similar overall porosity (about 80%) and volume fraction available for cell attachment and ECM formation. In vitro cell seeding showed that pore-size gradients promoted anisotropic cell distribution like that in the superficial, middle, and lower zones of immature bovine articular cartilage, irrespective of dynamic or static seeding methods. There was a direct correlation between zonal scaffold volume fraction and both DNA and glycosaminoglycan (GAG) content. Prolonged tissue culture in vitro showed similar inhomogeneous distributions of zonal GAG and collagen type II accumulation but not of GAG:DNA content, and levels were an order of magnitude less than in native cartilage. In this model system, we illustrated how scaffold design and novel processing techniques can be used to develop anisotropic pore architectures for instructing zonal cell and tissue distribution in tissue-engineered cartilage constructs.

The effect of PEGT/PBT scaffold architecture on the composition of tissue engineered cartilage.

A highly interconnecting and accessible pore network has been suggested as one of a number of prerequisites in the design of scaffolds for tissue engineering. In the present study, two processing techniques, compression-molding/particulate-leaching (CM), and 3D fiber deposition (3DF), were used to develop porous scaffolds from biodegradable poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) co-polymers with varying pore architectures. Three-dimensional micro-computed tomography (microCT) was used to characterize scaffold architectures and scaffolds were seeded with articular chondrocytes to evaluate tissue formation. Scaffold porosity ranged between 75% and 80%. Average pore size of tortuous CM scaffolds (182 microm) was lower than those of organized 3DF scaffolds (525 microm). The weight ratio of glycosaminoglycans (GAG)/DNA, as a measure of cartilage-like tissue formation, did not change after 14 days of culture whereas, following subcutaneous implantation, GAG/DNA increased significantly and was significantly higher in 3DF constructs than in CM constructs, whilst collagen type II was present within both constructs. In conclusion, 3DF PEGT/PBT scaffolds create an environment in vivo that enhances cartilaginous matrix deposition and hold particular promise for treatment of articular cartilage defects.

The effect of PEGT/PBT scaffold architecture on oxygen gradients in tissue engineered cartilaginous constructs.

Repair of articular cartilage defects using tissue engineered constructs composed of a scaffold and cultured autologous cells holds promise for future treatments. However, nutrient limitation (e.g. oxygen) has been suggested as a cause of the onset of chondrogenesis solely within the peripheral boundaries of larger constructs. In the present study, oxygen gradients were evaluated by microelectrode measurements in two porous polyethylene glycol terephthalate/polybutylene terephthalate (PEGT/PBT) scaffold architectures, a compression-molded and particle-leached sponge (CM) and a 3D-deposited fiber (3DF) scaffold. During the first 14 days in vitro, gradients intensified, after which a gradual decrease of the gradients was observed in vitro. In vivo, however, gradients changed instantly and became less pronounced. Although similar gradients were observed regardless of scaffold type, significantly more cells were present in the center of 3DF constructs after 2 weeks of in vivo culture. Our results stress the importance of a rationally designed scaffold for tissue-engineering applications. Organized structures, such as the 3DF PEGT/PBT polymer scaffolds, offer possibilities for regulation of nutrient supply and, therefore, hold promise for clinical approaches for cartilage repair.