Review: bioreactor design towards generation of relevant engineered tissues: focus on clinical translation

In tissue engineering and regenerative medicine, studies that utilize 3D scaffolds for generating voluminous tissues are mostly confined in the realm of in vitro research and preclinical animal model testing. Bioreactors offer an excellent platform to grow and develop 3D tissues by providing conditions that mimic their native microenvironment. Aligning the bioreactor development process with a focus on patient care will aid in the faster translation of the bioreactor technology to clinics. In this review, we discuss the various factors involved in the design of clinically relevant bioreactors in relation to their respective applications. We explore the functional relevance of tissue grafts generated by bioreactors that have been designed to provide physiologically relevant mechanical cues on the growing tissue. The review discusses the recent trends in non‐invasive sensing of the bioreactor culture conditions. It provides an insight to the current technological advancements that enable in situ, non‐invasive, qualitative and quantitative evaluation of the tissue grafts grown in a bioreactor system. We summarize the emerging trends in commercial bioreactor design followed by a short discussion on the aspects that hamper the ‘push’ of bioreactor systems into the commercial market as well as ‘pull’ factors for stakeholders to embrace and adopt widespread utility of bioreactors in the clinical setting. Copyright © 2017 John Wiley & Sons, Ltd.     

The effect of ex vivo dynamic loading on the osteogenic differentiation of genetically engineered mesenchymal stem cell model

Mechanical loading has been described as a highly important stimulus for improvements in the quality and strength of bone. It has also been shown that mechanical stimuli can induce the differentiation of mesenchymal stem cells (MSCs) along the osteogenic lineage. We have previously demonstrated the potent osteogenic effect of MSCs engineered to overexpress the BMP2 gene. In this study we investigated the effect of mechanical loading on BMP2‐expressing MSC‐like cells, using a special bioreactor designed to apply dynamic forces on cell‐seeded hydrogels. Cell viability, alkaline phosphatase (ALP) activity, BMP2 secretion and mineralized substance formation in the hydrogels were quantified. We found that cell metabolism increased as high as 6.8‐fold, ALP activity by 12.5‐fold, BMP2 secretion by 182‐fold and mineralized tissue formation by 1.72‐fold in hydrogels containing MSC‐like cells expressing BMP2, which were cultured in the presence of mechanical loading. We have shown that ex vivo mechanical loading had an additive effect on BMP2‐induced osteogenesis in genetically engineered MSC‐like cells. These data could be valuable for bone tissue‐engineering strategies of the future. Copyright © 2010 John Wiley & Sons, Ltd.     

Load‐induced osteogenic differentiation of mesenchymal stromal cells is caused by mechano‐regulated autocrine signaling

Mechanical boundary conditions critically influence the bone healing process. In this context, previous in vitro studies have demonstrated that cyclic mechanical compression alters migration and triggers osteogenesis of mesenchymal stromal cells (MSC), both processes being relevant to healing. However, it remains unclear whether this mechanosensitivity is a direct consequence of cyclic compression, an indirect effect of altered supply or a specific modulation of autocrine bone morphogenetic protein (BMP) signaling. Here, we investigate the influence of cyclic mechanical compression (ε = 5% and 10%, f = 1 Hz) on human bone marrow MSC (hBMSC) migration and osteogenic differentiation in a 3D biomaterial scaffold, an in vitro system mimicking the mechanical environment of the early bone healing phase. The open‐porous architecture of the scaffold ensured sufficient supply even without cyclic compression, minimizing load‐associated supply alterations. Furthermore, a large culture medium volume in relation to the cell number diminished autocrine signaling. Migration of hBMSCs was significantly downregulated under cyclic compression. Surprisingly, a decrease in migration was not associated with increased osteogenic differentiation of hBMSCs, as the expression of RUNX2 and osteocalcin decreased. In contrast, BMP2 expression was significantly upregulated. Enabling autocrine stimulation by increasing the cell‐to‐medium ratio in the bioreactor finally resulted in a significant upregulation of RUNX2 in response to cyclic compression, which could be reversed by rhNoggin treatment. The results indicate that osteogenesis is promoted by cyclic compression when cells condition their environment with BMP. Our findings highlight the importance of mutual interactions between mechanical forces and BMP signaling in controlling osteogenic differentiation.     

On‐line monitoring of oxygen as a non‐destructive method to quantify cells in engineered 3D tissue constructs

Regulatory guidelines have established the importance of introducing quantitative quality controls during the production and/or at the time of release of cellular grafts for clinical applications. In this study we aimed to determine whether on‐line measurements of oxygen can be used as a non‐destructive method to estimate the number of chondrocytes within an engineered cartilage graft. Human chondrocytes were seeded and cultured in a perfusion bioreactor, and oxygen levels in the culture medium were continuously monitored at the inlet and outlet of the bioreactor chamber throughout the culture period. We found that the drop in oxygen across the perfused construct was linearly correlated with the number of cells per construct (R² = 0.82, p < 0.0001). The method was valid for a wide range of cell numbers, including cell densities currently used in the manufacture of cartilage grafts for clinical applications. Given that few or no non‐destructive assays that quantitatively characterize an engineered construct currently exist, this non‐invasive method could represent a relevant instrument in regulatory compliant manufacturing of engineered grafts meeting specific quality criteria. Copyright © 2012 John Wiley & Sons, Ltd.     

A double chamber rotating bioreactor for enhanced tubular tissue generation from human mesenchymal stem cells: a promising tool for vascular tissue regeneration

Cardiovascular diseases represent a major global health burden, with high rates of mortality and morbidity. Autologous grafts are commonly used to replace damaged or failing blood vessels; however, such approaches are hampered by the scarcity of suitable graft tissue, donor site morbidity and poor long‐term stability. Tissue engineering has been investigated as a means by which exogenous vessel grafts can be produced, with varying levels of success to date, a result of mismatched mechanical properties of these vessel substitutes and inadequate ex vivo vessel tissue genesis. In this work, we describe the development of a novel multifunctional dual‐phase (air/aqueous) bioreactor, designed to both rotate and perfuse small‐diameter tubular scaffolds and encourage enhanced tissue genesis throughout such scaffolds. Within this novel dynamic culture system, an elastomeric nanofibrous, microporous composite tubular scaffold, composed of poly(caprolactone) and acrylated poly(lactide‐co‐trimethylene‐carbonate) and with mechanical properties approaching those of native vessels, was seeded with human mesenchymal stem cells (hMSCs) and cultured for up to 14 days in inductive (smooth muscle) media. This scaffold/bioreactor combination provided a dynamic culture environment that enhanced (compared with static controls) scaffold colonization, cell growth, extracellular matrix deposition and in situ differentiation of the hMSCs into mature smooth muscle cells, representing a concrete step towards our goal of creating a mature ex vivo vascular tissue for implantation. Copyright © 2016 John Wiley & Sons, Ltd.     

Interface integration of layered collagen scaffolds with defined matrix stiffness: implications for sheet‐based tissue engineering

Successful application of sheet‐based engineering for complex tissue reconstruction requires optimal integration of construct components. An important regulator of cellular responses (such as migration and collagen deposition) mediating interface integration is matrix stiffness. In this study we developed a sheet‐based 3D model of interface integration that allows control of interface matrix stiffness. Fluid was removed from acellular or fibroblast‐seeded bilayer collagen hydrogel constructs, using plastic compression to increase collagen density and matrix stiffness. Cell‐seeded constructs were either compressed at day 0 and cultured for 7 days (compressed culture, high stiffness) or left uncompressed during culture and compressed on day 7 (compliant‐compressed culture, low stiffness). Constructs were fitted onto a mechanical testing system to measure interface adhesive strength. Analysis of stresses by finite element modelling predicted a sharp rise of stress and rapid failure at the interface. While cell‐seeded constructs showed a six‐fold increase in interface adhesive strength compared to acellular control constructs (p < 0.05), there was no significant difference between low‐ and high‐stiffness cultures after 1 week. Cell migration across the interface was greater in low‐ compared to high‐stiffness constructs at 24 h (p < 0.05); however, no significant difference was observed after 1 week. Visualization of interfaces showed fusion of the two layers in low‐ but not in high‐stiffness constructs after 1 week of culture. The ability to regulate cellular behaviour at an interface by controlling matrix stiffness could provide an important tool for modelling the integration of sheet‐based bioengineered tissues in bioreactor culture or post‐implantation. Copyright © 2009 John Wiley & Sons, Ltd.     

Pooled thrombin‐activated platelet‐rich plasma: a substitute for fetal bovine serum in the engineering of osteogenic/vasculogenic grafts

The use of fetal bovine serum (FBS) as a culture medium supplement in cell therapy and clinical tissue engineering is challenged by immunological concerns and the risk of disease transmission. Here we tested whether human, thrombin‐activated, pooled, platelet‐rich plasma (tPRP) can be substituted for FBS in the engineering of osteogenic and vasculogenic grafts, using cells from the stromal vascular fraction (SVF) of human adipose tissue. SVF cells were cultured under perfusion flow into porous hydroxyapatite scaffolds for 5 days, with the medium supplemented with either 10% tPRP or 10% FBS and implanted in an ectopic mouse model. Following in vitro culture, as compared to FBS, the use of tPRP did not modify the fraction of clonogenic cells or the different cell phenotypes, but increased by 1.9‐fold the total number of cells. After 8 weeks in vivo, bone tissue was formed more reproducibly and in higher amounts (3.7‐fold increase) in constructs cultured with tPRP. Staining for human‐specific ALU sequences and for the human isoforms of CD31/CD34 revealed the human origin of the bone, the formation of blood vessels by human vascular progenitors and a higher density of human cells in implants cultured with tPRP. In summary, tPRP supports higher efficiency of bone formation by SVF cells than FBS, likely by enhancing cell expansion in vitro while maintaining vasculogenic properties. The use of tPRP may facilitate the clinical translation of osteogenic grafts with intrinsic capacity for vascularization, based on the use of adipose‐derived cells. Copyright © 2015 John Wiley & Sons, Ltd.     

The evolution of simulation techniques for dynamic bone tissue engineering in bioreactors

Bone tissue engineering aims to overcome the drawbacks of current bone regeneration techniques in orthopaedics. Bioreactors are widely used in the field of bone tissue engineering, as they help support efficient nutrition of cultured cells with the possible combination of applying mechanical stimuli. Beneficial influencing parameters of in vitro cultures are difficult to find and are mostly determined by trial and error, which is associated with significant time and money spent. Mathematical simulations can support the finding of optimal parameters. Simulations have evolved over the last 20 years from simple analytical models to complex and detailed computational models. They allow researchers to simulate the mechanical as well as the biological environment experienced by cells seeded on scaffolds in a bioreactor. Based on the simulation results, it is possible to give recommendations about specific parameters for bone bioreactor cultures, such as scaffold geometries, scaffold mechanical properties, the level of applied mechanical loading or nutrient concentrations. This article reviews the evolution in simulating various aspects of dynamic bone culture in bioreactors and reveals future research directions. Copyright © 2013 John Wiley & Sons, Ltd.     

Cartilage graft engineering by co‐culturing primary human articular chondrocytes with human bone marrow stromal cells

Co‐culture of mesenchymal stromal cells (MSCs) with articular chondrocytes (ACs) has been reported to improve the efficiency of utilization of a small number of ACs for the engineering of implantable cartilaginous tissues. However, the use of cells of animal origin and the generation of small‐scale micromass tissues limit the clinical relevance of previous studies. Here we investigated the in vitro and in vivo chondrogenic capacities of scaffold‐based constructs generated by combining primary human ACs with human bone marrow MSCs (BM‐MSCs). The two cell types were cultured in collagen sponges (2 × 6 mm disks) at the BM‐MSCs:ACs ratios: 100:0, 95:5, 75:25 and 0:100 for 3 weeks. Scaffolds freshly seeded or further precultured in vitro for 2 weeks were also implanted subcutaneously in nude mice and harvested after 8 or 6 weeks, respectively. Static co‐culture of ACs (25%) with BM‐MSCs (75%) in scaffolds resulted in up to 1.4‐fold higher glycosaminoglycan (GAG) content than what would be expected based on the relative percentages of the different cell types. In vivo GAG induction was drastically enhanced by the in vitro preculture and maximal at the ratio 95:5 (3.8‐fold higher). Immunostaining analyses revealed enhanced accumulation of type II collagen and reduced accumulation of type X collagen with increasing ACs percentage. Constructs generated in the perfusion bioreactor system were homogeneously cellularized. In summary, human cartilage grafts were successfully generated, culturing BM‐MSCs with a relatively low fraction of non‐expanded ACs in porous scaffolds. The proposed co‐culture strategy is directly relevant towards a single‐stage surgical procedure for cartilage repair. Copyright © 2012 John Wiley & Sons, Ltd.     

Differential effects of cyclic and static pressure on biochemical and morphological properties of chondrocytes from articular cartilage

BACKGROUND: Mechanical stresses are known to play important role on articular cartilage functions in vivo and also on cartilage explants and chondrocytes monolayer culture. This study examined the differential effect of cyclic and static pressures on chondrocytes cultured in alginate matrix, which is physiologically closer to the in vivo environment of cells in cartilage. METHODS: Goat knee joint articular cartilage chondrocytes cultured in alginate beads were exposed to 1.2 and 2.4 MPa cyclic and static loadings via a custom-made cam/follower based machine. Biochemical contents (glycosaminoglycan, collagen, DNA) and protease activity of cells were analyzed separately in cellular matrix, further removed matrix and in culture medium. Morphology of chondrocytes was studied under transmission electron microscopy. FINDINGS: Compared with controls (unloaded cells), cyclic loading increased the glycosaminoglycan content of cells at 1.2 and 2.4 MPa in cellular matrix and further removed matrix (P<0.001) whereas it decreased at similar static loads (P<0.001). In alginate matrix, chondrocytes released a metalloprotease, which required Mn(2+) for activity. Both cyclic load levels inhibited its specific activity in cellular matrix but increased it at static loading (P<0.001). The protease specific activity in further removed matrix increased at both cyclic and static loadings (P<0.001). Transmission electron microscopy data showed improved cells ultrastructure and cell-matrix interactions under cyclic load whereas these deteriorated under static loadings. INTERPRETATION: The study suggests that cyclic load has a positive effect on chondrocytes metabolism and morphology whereas static load has a degenerative effect.     

Investigation into the effects of varying frequency of mechanical stimulation in a cycle‐by‐cycle manner on engineered cardiac construct function

Mechanical stimulation has been used extensively to improve the function of cardiac engineered tissue, as it mimics the physical environment in which the tissue is situated during normal development. However, previous mechanical stimulation has been carried out under a constant frequency that more closely resembles a diseased heart. The goal of this study was to create a bioreactor system that would allow us to control the mechanical stimulation of engineered cardiac tissue on a cycle‐by‐cycle basis. This unique system allows us to determine the effects on cardiac construct function of introducing variability to the mechanical stretch. To test our bioreactor system, constructs created from neonatal rat cardiomyocytes entrapped in fibrin hydrogels were stimulated under various regimes for 2 weeks and then assessed for functional outcomes. No differences were observed in the final cell number in each condition, indicating that variability in frequency did not have a negative effect on viability. The forces were higher for all mechanical stimulation groups compared to static controls, although no differences were observed between the mechanically stimulated conditions, indicating that variable frequency on a cycle‐by‐cycle basis has limited effects on the resulting force. Although differences in the observed twitch force were not observed, differences in the protein expression indicate that variable‐frequency mechanical stimulation had an effect on cell–cell coupling and growth pathway activation in the constructs. Thus, this bioreactor system provides a valuable tool for further development and optimization of engineered myocardial tissue as a repair or replacement strategy for patients undergoing heart failure. Copyright © 2014 John Wiley & Sons, Ltd.     

Industrial approach in developing an advanced therapy product for bone repair

Mesenchymal stem cells (MSCs) are multipotent cells with therapeutic applications. The aim of our work was to develop an advanced therapy product for bone repair, associating autologous human adipose‐derived MSCs (ASCs) with human bone allograft (TBF; Phoenix^®). We drew up specifications that studied: (a) the influence of tissue collection procedures (elective liposuction or non‐invasive resection) and patient age on cell number and function; (b) monolayer cell culture conditions and osteodifferentiation and particularly the possibility of reducing stages of culture; and (c) the bone construct preparation and especially the comparison between two types of cells seeded on bone allograft (number of cultured processed lipoaspirate (PLA) cells and monolayer‐expanded ASCs) and cultured for 1, 2 and 3 weeks. The results showed that tissue harvesting techniques and patient age did not affect PLA cell number and ASC cloning efficiency. PLA cells can be directly osteodifferentiated (instead of culturing them in expansion medium first and then differentiating them) and these cells were able to mineralize when they were cultured in an osteogenic medium containing calcium chloride. PLA cells directly seeded on bone allograft for a minimum of 3 weeks of culture in this osteogenic medium expressed osteocalcin and colonized the matrix better than monolayer‐expanded ASCs. This work detailed the specifications of a pharmaceutical laboratory to develop an advanced therapy product and this current approach is promising for bone repair. Copyright © 2009 John Wiley & Sons, Ltd.     

Chondrogenic differentiation of human chondrocytes cultured in the absence of ascorbic acid

Bioreactor systems will likely play a key role in establishing regulatory compliant and cost‐effective production systems for manufacturing engineered tissue grafts for clinical applications. However, the automation of bioreactor systems could become considerably more complex and costly due to the requirements for additional storage and liquid handling technologies if unstable supplements are added to the culture medium. Ascorbic acid (AA) is a bioactive supplement that is commonly presumed to be essential for the generation of engineered cartilage tissues. However, AA can be rapidly oxidized and degraded. In this work, we addressed whether human nasal chondrocytes can redifferentiate, undergo chondrogenesis, and generate a cartilaginous extracellular matrix when cultured in the absence of AA. We found that when chondrocytes were cultured in 3D micromass pellets either with or without AA, there were no significant differences in their chondrogenic capacity in terms of gene expression or the amount of glycosaminoglycans. Moreover, 3D pellets cultured without AA contained abundant collagen Types II and I extracellular matrix. Although the amounts of Collagens II and I were significantly lower (34% and 50% lower) than in pellets cultured with AA, collagen fibers had similar thicknesses and distributions for both groups, as shown by scanning electron microscopy imaging. Despite the reduced amounts of collagen, if engineered cartilage grafts can be generated with sufficient properties that meet defined quality criteria without the use of unstable supplements such as AA, bioreactor automation requirements can be greatly simplified, thereby facilitating the development of more compact, user‐friendly, and cost‐effective bioreactor‐based manufacturing systems.     

Human tissue‐engineered bone produced in clinically relevant amounts using a semi‐automated perfusion bioreactor system: a preliminary study

The aim of this study was to evaluate a semi‐automated perfusion bioreactor system for the production of clinically relevant amounts of human tissue‐engineered bone. Human bone marrow stromal cells (hBMSCs) of eight donors were dynamically seeded and proliferated in a perfusion bioreactor system in clinically relevant volumes (10 cm³) of macroporous biphasic calcium phosphate scaffolds (BCP particles, 2–6 mm). Cell load and distribution were shown using methylene blue staining. MTT staining was used to demonstrate viability of the present cells. After 20 days of cultivation, the particles were covered with a homogeneous layer of viable cells. Online oxygen measurements confirmed the proliferation of hBMSCs in the bioreactor. After 20 days of cultivation, the hybrid constructs became interconnected and a dense layer of extracellular matrix was present, as visualized by scanning electron microscopy (SEM). Furthermore, the hBMSCs showed differentiation towards the osteogenic lineage as was indicated by collagen type I production and alkaline phosphatase (ALP) expression. We observed no significant differences in osteogenic gene expression profiles between static and dynamic conditions like ALP, BMP2, Id1, Id2, Smad6, collagen type I, osteocalcin, osteonectin and S100A4. For the donors that showed bone formation, dynamically cultured hybrid constructs showed the same amount of bone as the statically cultured hybrid constructs. Based on these results, we conclude that a semi‐automated perfusion bioreactor system is capable of producing clinically relevant and viable amounts of human tissue‐engineered bone that exhibit bone‐forming potential after implantation in nude mice. Copyright © 2009 John Wiley & Sons, Ltd.     

RGD‐functionalized polyurethane scaffolds promote umbilical cord blood mesenchymal stem cell expansion and osteogenic differentiation

Biomimetic materials are essential for the production of clinically relevant bone grafts for bone tissue engineering applications. Their ability to modulate stem cell proliferation and differentiation can be used to harness the regenerative potential of those cells and optimize the efficiency of engineered bone grafts. The arginyl‐glycyl‐aspartic acid (RGD) peptide has been recognized as the adhesion motif of various extracellular matrix proteins and can affect stem cell behaviour in biomaterials. Attempts to functionalize biomaterials with RGD have been limited to a maximum of 1‐ to 3‐mm thickness scaffolds, overlooking the issue of core infiltration that represents a major hurdle in developing real thickness scaffolds. Herein, we present the cross‐linking of RGD on the surface of “real thickness” (5 × 5 × 5 mm) porous polyurethane scaffolds (PU‐RGD), to be used for the expansion and osteogenic differentiation of umbilical cord blood mesenchymal stem cells (UCB MSCs). RGD‐functionalized scaffolds increased initial cell adhesion (1.5‐fold to twofold) and achieved a 3.4‐fold increase in cell numbers at the end of culture compared with a 1.5‐fold increase in non‐functionalized controls. Homogenous cell infiltration to the scaffold core was observed in the PU‐RGD scaffolds. Importantly, PU‐RGD scaffolds were able to enhance the osteogenic differentiation of UCB MSCs. Osteogenic gene and protein expression increased in scaffolds functionalized with 100 μg/ml RGD. Higher RGD concentrations (200 μg/ml) were less efficient in stimulating osteogenic differentiation. We conclude that robust RGD tethering to 3D PU “real thickness” scaffolds is possible and that it promotes core infiltration, expansion, and osteogenic differentiation of UCB MSCs for the purposes of bone regeneration.     

Mechanical compression of articular cartilage induces chondrocyte proliferation and inhibits proteoglycan synthesis by activation of the ERK pathway: implications for tissue engineering and regenerative medicine

Articular cartilage is recalcitrant to endogenous repair and regeneration and is thus a focus of tissue engineering and regenerative medicine strategies. A prerequisite for articular cartilage tissue engineering is an understanding of the signal transduction pathways involved in mechanical compression during trauma or disease. We sought to explore the role of the extracellular signal‐regulated kinase 1/2 (ERK 1/2) pathway in chondrocyte proliferation and proteoglycan synthesis following acute mechanical compression. Bovine articular cartilage explants were cultured with and without the ERK 1/2 pathway inhibitor PD98059. Cartilage explants were statically loaded to 40% strain at a strain rate of 1/s for 5 s. Control explants were cultured under similar conditions but were not loaded. There were four experimental groups: (a) no load, without inhibitor; (b) no load, with the inhibitor PD98059; (c) loaded, without the inhibitor; and (d) loaded, with the inhibitor PD98059. The explants were cultured for varying durations from 5 min to 5 days and were then analysed by biochemical and immunohistochemical methods. Mechanical compression induced phosphorylation of ERK 1/2, and this was attenuated with the ERK 1/2 pathway inhibitor PD98059 in a dose‐dependent manner. Chondrocyte proliferation was increased by mechanical compression. This effect was blocked by the inhibitor of the ERK 1/2 pathway. Mechanical compression also led to a decrease in proteoglycan synthesis that was reversed with inhibitor PD98059. In conclusion, the ERK 1/2 pathway is involved in the proliferative and biosynthetic response of chondrocytes following acute static mechanical compression. Copyright © 2009 John Wiley & Sons, Ltd.     

Examination of the mechanical properties of impacted morsellised bone grafts

Background. In order to successfully apply the technique of impacting morsellised bone grafts in order to fill out bone defects, it is essential to determine the mechanical properties of the grafts. The aim of our study was to determine the type of forces enabling optimal graft impacting (static or dynamic forces), the force values needed for bone graft impacting, the number of impacting cycles needed to obtain a homogenous medium of a definite biomechanical strength, and the properties of the osseous bed after impacting, when exposed to forces similar to those to which the joints are subjected after surgery. Material and methods. The tests were carried out on a strength-testing stand based on the INSTRON 8501 Plus universal strength-testing machine. The tests were performed with both static and dynamic cyclic loads applied to grafts 2 cm thick. Results. In tests of static loads, the greatest displacement of the rammer was observed during the first attempt to knead the grafts with a force of 2 kN. The dynamics of the graft impacting process decreased rapidly in successive cycles and stabilized after ten cycles. A slight increase of rammer displacement was observed. Conclusions. The results from the static and dynamic tests demonstrate that both the value and the character of the force applied influence the mechanical properties of the bed made of frozen bone grafts. Significantly better quality of impacting was obtained with dynamic compacting of the grafts than with application of the same force in a static character. With cyclic impacting of the bed, only the first dozen or so cycles were effective, and further action was of an elastic strain character and failed to improve the quality of impacting.     

A miniaturized bioreactor system for the evaluation of cell interaction with designed substrates in perfusion culture

In tissue engineering, chemical and topographical cues are normally developed using static cell cultures but then applied directly to tissue cultures in three dimensions (3D) and under perfusion. As human cells are very sensitive to changes in the culture environment, it is essential to evaluate the performance of any such cues in a perfused environment before they are applied to tissue engineering. Thus, the aim of this research was to bridge the gap between static and perfusion cultures by addressing the effect of perfusion on cell cultures within 3D scaffolds. For this we developed a scaled‐down bioreactor system, which allows evaluation of the effectiveness of various chemical and topographical cues incorporated into our previously developed tubular ε‐polycaprolactone scaffold under perfused conditions. Investigation of two exemplary cell types (fibroblasts and cortical astrocytes) using the miniaturized bioreactor indicated that: (a) quick and firm cell adhesion in the 3D scaffold was critical for cell survival in perfusion culture compared with static culture; thus, cell‐seeding procedures for static cultures might not be applicable, therefore it was necessary to re‐evaluate cell attachment on different surfaces under perfused conditions before a 3D scaffold was applied for tissue cultures; (b) continuous medium perfusion adversely influenced cell spread and survival, which could be balanced by intermittent perfusion; (c) micro‐grooves still maintained their influences on cell alignment under perfused conditions, while medium perfusion demonstrated additional influence on fibroblast alignment but not on astrocyte alignment on grooved substrates. This research demonstrated that the mini‐bioreactor system is crucial for the development of functional scaffolds with suitable chemical and topographical cues by bridging the gap between static culture and perfusion culture. Copyright © 2011 John Wiley & Sons, Ltd.     

Cyclical strain improves artificial equine tendon constructs in vitro

Tendon injuries are a common cause of morbidity in humans. They also occur frequently in horses, and the horse provides a relevant, large animal model in which to test novel therapies. To develop novel cell therapies that can aid tendon regeneration and reduce subsequent reinjury rates, the mechanisms that control tendon tissue regeneration and matrix remodelling need to be better understood. Although a range of chemical cues have been explored (growth factors, media etc.), the influence of the mechanical environment on tendon cell culture has yet to be fully elucidated. To mimic the in vivo environment, in this study, we have utilised a novel and affordable, custom‐made bioreactor to apply a cyclical strain to tendon‐like constructs generated in three‐dimensional (3D) culture by equine tenocytes. Dynamic shear analysis (DSA), dynamic scanning calorimetry (DSC) and Fourier‐transform infrared (FTIR) spectroscopy were used to determine the mechanical and chemical properties of the resulting tendon‐like constructs. Our results demonstrate that equine tenocytes exposed to a 10% cyclical strain have an increased amount of collagen gel contraction after 7 and 8 days of culture compared with cells cultured in 3D in the absence of external strain. While all the tendon‐like constructs have a very similar chemical composition to native tendon, the application of strain improves their mechanical properties. We envisage that these results will contribute towards the development of improved biomimetic artificial tendon models for the development of novel strategies for equine regenerative therapies.     

Integrated culture platform based on a human platelet lysate supplement for the isolation and scalable manufacturing of umbilical cord matrix‐derived mesenchymal stem/stromal cells

Umbilical cord matrix (UCM)‐derived mesenchymal stem/stromal cells (MSCs) are promising therapeutic candidates for regenerative medicine settings. UCM MSCs have advantages over adult cells as these can be obtained through a non‐invasive harvesting procedure and display a higher proliferative capacity. However, the high cell doses required in the clinical setting make large‐scale manufacturing of UCM MSCs mandatory. A commercially available human platelet lysate‐based culture supplement (UltraGRO^TM, AventaCell BioMedical) (5%(v/v)) was tested to effectively isolate UCM MSCs and to expand these cells under (1) static conditions, using planar culture systems and (2) stirred culture using plastic microcarriers in a spinner flask. The MSC‐like cells were isolated from UCM explant cultures after 11 ± 2 days. After five passages in static culture, UCM MSCs retained their immunophenotype and multilineage differentiation potential. The UCM MSCs cultured under static conditions using UltraGRO^TM‐supplemented medium expanded more rapidly compared with UCM MSCs expanded using a previously established protocol. Importantly, UCM MSCs were successfully expanded under dynamic conditions on plastic microcarriers using UltraGRO^TM‐supplemented medium in spinner flasks. Upon an initial 54% cell adhesion to the beads, UCM MSCs expanded by >13‐fold after 5–6 days, maintaining their immunophenotype and multilineage differentiation ability. The present paper reports the establishment of an easily scalable integrated culture platform based on a human platelet lysate supplement for the effective isolation and expansion of UCM MSCs in a xenogeneic‐free microcarrier‐based system. This platform represents an important advance in obtaining safer and clinically meaningful MSC numbers for clinical translation. Copyright © 2016 John Wiley & Sons, Ltd.