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To exploit the usability of Digital Light Processing (DLP) in regenerative medicine, biodegradable, mechanically customizable and well-defined polyester urethane acrylate resins were synthesized based on poly(caprolactone-co-trimethlenecarbonate). By controlling the monomer ratio, the resultant fabricated constructs showed tunable mechanical properties, degradation and attached hMSC morphologies.

Cells are inherently conferred with the ability to self-organize into the tissues and organs comprising the human body. Self-organization can be recapitulated in vitro and recent advances in the organoid field are just one example of how we can generate small functioning elements of organs. Tissue engineers can benefit from the power of self-organization and should consider how they can harness and enhance the process with their constructs. For example, aggregates of stem cells and tissue-specific cells benefit from the input of carefully selected biomolecules to guide their differentiation toward a mature phenotype. This can be further enhanced by the use of technologies to provide a physiological microenvironment for self-organization, enhance the size of the constructs, and enable the long-term culture of self-organized structures. Of importance, conducting self-organization should be limited to fine-tuning and should avoid over-engineering that could counteract the power of inherent cellular self-organization. Impact Statement Self-organization is a powerful innate feature of cells that can be fine-tuned but not over-engineered to create new tissues and organs.

The field of tissue engineering is making great strides in developing replacement tissue grafts for clinical use, marked by the rapid development of novel biomaterials, their improved integration with cells, better-directed growth and differentiation of cells, and improved three-dimensional tissue mass culturing. One major obstacle that remains, however, is the lack of graft vascularization, which in turn renders many grafts to fail upon clinical application. With that, graft vascularization has turned into one of the holy grails of tissue engineering, and for the majority of tissues, it will be imperative to achieve adequate vascularization if tissue graft implantation is to succeed. Many different approaches have been developed to induce or augment graft vascularization, both in vitro and in vivo. In this review, we highlight the importance of vascularization in tissue engineering and outline various approaches inspired by both biology and engineering to achieve and augment graft vascularization., (© 2019 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.)

One of the biggest challenges in tissue engineering and regenerative medicine is to incorporate a functioning vasculature to overcome the consequences of a lack of oxygen and nutrients in the tissue construct. Otherwise, decreased oxygen tension leads to incomplete metabolism and the formation of the so-called reactive oxygen species (ROS). Cells have many endogenous antioxidant systems to ensure a balance between ROS and antioxidants, but if this balance is disrupted by factors such as high levels of ROS due to long-term hypoxia, there will be tissue damage and dysfunction. Current attempts to solve the oxygen problem in the field rarely take into account the importance of the redox balance and are instead centred on releasing or generating oxygen. The first problem with this approach is that although oxygen is necessary for life, it is paradoxically also a highly toxic molecule. Furthermore, although some oxygen-generating biomaterials produce oxygen, they also generate hydrogen peroxide, a ROS, as an intermediate product. In this review, we discuss why it would be a superior strategy to supplement oxygen delivery with molecules to safeguard the important redox balance. Redox sensor proteins that can stimulate the anaerobic metabolism, angiogenesis, and enhancement of endogenous antioxidant systems are discussed as promising targets. We propose that redox regulating biomaterials have the potential to tackle some of the challenges related to angiogenesis and that the knowledge in this review will help scientists in tissue engineering and regenerative medicine realize this aim., (© 2018 The Authors. Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.)

The fabrication of bioactive scaffolds able to mimic the in vivo cellular microenvironment is a challenge for regenerative medicine. The creation of sites for the selective binding of specific endogenous proteins represents an attractive strategy to fabricate scaffolds able to elicit specific cell response. Here, electrospinning (ESP) and soft-molecular imprinting (soft-MI) techniques were combined to fabricate a soft-molecular imprinted electrospun bioactive scaffold (SMIES) for tissue regeneration. Scaffolds functionalized using different proteins and growth factors (GFs) arranged onto the surface were designed, fabricated and validated with different polyesters, demonstrating the versatility of the developed approach. The scaffolds bound selectively each of the different proteins used, indicating that the soft-MI method allowed fabricating high affinity binding sites on ESP fibers compared to non-imprinted ones. The imprinting of ESP fibers with several GFs resulted in a significant effect on cell behavior. FGF-2 imprinted SMIES promoted cell proliferation and metabolic activity. BMP-2 and TGF-β3 imprinted SMIES promoted cellular differentiation. These scaffolds hold the potential to be used in a cell-free approach to steer endogenous tissue regeneration in several regenerative medicine applications.

When tissue engineering strategies rely on the combination of three-dimensional (3D) polymeric or ceramic scaffolds with cells to culture implantable tissue constructs in vitro, it is desirable to monitor tissue growth and cell fate to be able to more rationally predict the quality and success of the construct upon implantation. Such a 3D construct is often referred to as a 'black-box' since the properties of the scaffolds material limit the applicability of most imaging modalities to assess important construct parameters. These parameters include the number of cells, the amount and type of tissue formed and the distribution of cells and tissue throughout the construct. Immunolabeling enables the spatial and temporal identification of multiple tissue types within one scaffold without the need to sacrifice the construct. In this report, we concisely review the applicability of antibodies (Abs) and their conjugation chemistries in tissue engineered constructs. With some preliminary experiments, we show an efficient conjugation strategy to couple extracellular matrix Abs to fluorophores. The conjugated probes proved to be effective in determining the presence of collagen type I and type II on electrospun and additive manufactured 3D scaffolds seeded with adult human bone marrow derived mesenchymal stromal cells. The conjugation chemistry applied in our proof of concept study is expected to be applicable in the coupling of any other fluorophore or particle to the Abs. This could ultimately lead to a library of probes to permit high-contrast imaging by several imaging modalities.

Engineering the osteochondral tissue presents some challenges mainly relying in its function of transition from the subchondral bone to articular cartilage and the gradual variation in several biological, mechanical, and structural features. A possible solution for osteochondral regeneration might be the design and fabrication of scaffolds presenting a gradient able to mimic this transition. Covalent binding of biological factors proved to enhance cell adhesion and differentiation in two-dimensional culture substrates. Here, we used polymer brushes as selective linkers of bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β3 (TGF-β3) on the surface of 3D scaffolds fabricated via additive manufacturing (AM) and subsequent controlled radical polymerization. These growth factors (GFs) are known to stimulate the differentiation of human mesenchymal stromal cells (hMSCs) toward the osteogenic and chondrogenic lineages, respectively. BMP-2 and TGF-β3 were covalently bound both homogeneously within a poly(ethylene glycol) (PEG)-based brush-functionalized scaffolds, and following a gradient composition by varying their concentration along the axial section of the 3D constructs. Following an approach previously developed by our group and proved to be successful to generate fibronectin gradients, opposite brush-supported gradients of BMP-2 and TGF-β3 were finally generated and subsequently tested to differentiate cells in a gradient fashion. The brush-supported GFs significantly influenced hMSCs osteochondral differentiation when the scaffolds were homogenously modified, yet no effect was observed in the gradient scaffolds. Therefore, this technique seems promising to maintain the biological activity of growth factors covalently linked to 3D scaffolds, but needs to be further optimized in case biological gradients are desired., (© 2017 The Authors. Biotechnology Journal published by Wiley-VCH GmbH & Co. KGaA, Weinheim.)

Tissue engineering needs innovative solutions to better fit the requirements of a minimally invasive approach, providing at the same time instructive cues to cells. The use of shape memory polyurethane has been investigated by producing 4D scaffolds via additive manufacturing technology. Scaffolds with two different pore network configurations (0/90° and 0/45°) were characterized by dynamic-mechanical analysis. The thermo-mechanical analysis showed a T g at about 32 °C (T g = T trans ), indicating no influence of the fabrication process on the transition temperature. In addition, shape recovery tests showed a good recovery of the permanent shape for both scaffold configurations. When cells were seeded onto the scaffolds in the temporary shape and the permanent shape was recovered, cells were significantly more elongated after shape recovery. Thus, the mechanical stimulus imparted by shape recovery is able to influence the shape of cells and nuclei. The obtained results indicate that a single mechanical stimulus is sufficient to initiate changes in the morphology of adherent cells.

The functionalization of biomaterials substrates used for cell culture is gearing towards an increasing control over cell activity. Although a number of biomaterials have been successfully modified by different strategies to display tailored physical and chemical surface properties, it is still challenging to step from 2D substrates to 3D scaffolds with instructive surface properties for cell culture and tissue regeneration. In this study, additive manufacturing and thermally induced phase separation are combined to create 3D scaffolds with tunable surface morphology from polymer gels. Surface features vary depending on the gel concentration, the exchanging temperature, and the nonsolvent used. When preosteoblasts (MC-3T3 cells) are cultured on these scaffolds, a significant increase in alkaline phosphatase activity is measured for submicron surface topography, suggesting a potential role on early cell differentiation., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chemical and mechanical cues are well-established influencers of in vitro chondrogenic differentiation of ATDC5 cells. Here, we investigate the role of topographical cues in this differentiation process, a study not been explored before. Previously, using a library of surface micro-topographies we found some distinct patterns that induced alkaline phosphatase (ALP) production in human mesenchymal stromal cells. ALP is also a marker for hypertrophy, the end stage of chondrogenic differentiation preceding bone formation. Thus, we hypothesized that these patterns could influence end-stage chondrogenic differentiation of ATDC5 cells. In this study, we randomly selected seven topographies among the ALP influencing hits. Cells grown on these surfaces displayed varying nuclear shape and actin filament structure. When stimulated with insulin-transferrin-selenium (ITS) medium, nodule formation occurred and in some cases showed alignment to the topographical patterns. Gene expression analysis of cells growing on topographical surfaces in the presence of ITS medium revealed a downregulation of early markers and upregulation of late markers of chondrogenic differentiation compared to cells grown on a flat surface. In conclusion, we demonstrated that surface topography in addition to other cues can promote hypertrophic differentiation suitable for bone tissue engineering.

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

In the field of tissue engineering, there is a need for methods that allow assessing the performance of tissue-engineered constructs noninvasively in vitro and in vivo. To date, histological analysis is the golden standard to retrieve information on tissue growth, cellular distribution, and cell fate on tissue-engineered constructs after in vitro cell culture or on explanted specimens after in vivo applications. Yet, many advances have been made to optimize imaging techniques for monitoring tissue-engineered constructs with a sub-mm or μm resolution. Many imaging modalities have first been developed for clinical applications, in which a high penetration depth has been often more important than lateral resolution. In this study, we have reviewed the current state of the art in several imaging approaches that have shown to be promising in monitoring cell fate and tissue growth upon in vitro culture. Depending on the aimed tissue type and scaffold properties, some imaging methods are more applicable than others. Optical methods are mostly suited for transparent materials such as hydrogels, whereas magnetic resonance-based methods are mostly applied to obtain contrast between hard and soft tissues regardless of their transparency. Overall, this review shows that the field of imaging in scaffold-based tissue engineering is developing at a fast pace and has the potential to overcome the limitations of destructive endpoint analysis.

Background: There's a large clinical need for novel vascular grafts. Tissue engineered blood vessels (TEBVs) have great potential to improve the outcome of vascular grafting procedures. Here, we present a novel approach to generate autologous TEBV in vivo. Polymer rods were engineered and implanted, evoking an inflammatory response that culminates in encapsulation by a fibrocellular capsule. We hypothesized that, after extrusion of the rod, the fibrocellular capsule differentiates into an adequate vascular conduit once grafted into the vasculature., Methods and Results: Rods were implanted subcutaneously in pigs. After 4 weeks, rods with tissue capsules grown around it were harvested. Tissue capsules were grafted bilaterally as carotid artery interposition. One and 4-week patency were evaluated by angiography whereupon pigs were sacrificed. Tissue capsules before and after grafting were evaluated on tissue remodeling using immunohistochemistry, RNA profiling and mechanical testing. Rods were encapsulated by thick, well-vascularized tissue capsules, composed of circumferentially aligned fibroblasts, collagen and few leukocytes, with adequate mechanical strength. Patency was 100% after 1 week and 87.5% after 4 weeks. After grafting, tissue capsules remodeled towards a vascular phenotype. Gene profiles of TEBVs gained more similarity with carotid artery. Wall thickness and αSMA-positive area significantly increased. Interestingly, a substantial portion of (myo)fibroblasts present before grafting expressed smooth muscle cell markers. While leukocytes were hardly present anymore, the lumen was largely covered with endothelial cells. Burst pressure remained stable after grafting., Conclusions: Autologous TEBVs were created in vivo with sufficient mechanical strength enabling vascular grafting. Grafts differentiated towards a vascular phenotype upon grafting., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Limited nutrient diffusion in three-dimensional (3D) constructs is a major concern in tissue engineering. Therefore, monitoring nutrient availability and diffusion within a scaffold is an important asset. Since nutrients come in various forms, we have investigated the diffusion of the oxygen, luciferin and dextran molecules within tissue-engineered constructs using optical imaging technologies. First, oxygen availability and diffusion were investigated, using transgenic cell lines in which a hypoxia-responsive element drives expression of the green fluorescent protein gene. Using confocal imaging, we observed oxygen limitation, starting at around 200 µm from the periphery in the context of agarose gel with 1 million CHO cells. Diffusion of luciferin was monitored real-time in agarose gels using a cell line in which the luciferase gene was driven by a constitutively active CMV promoter. Gel concentration affected the diffusion rate of luciferin. Furthermore, we assessed the diffusion rates of fluorescent dextran molecules of different molecular weights in biomaterials by fluorescence recovery after photobleaching (FRAP) and observed that diffusion depended on both molecular size and gel concentration. In conclusion, we have validated a set of efficient tools to investigate molecular diffusion of a range of molecules and to optimize biomaterials design in order to improve nutrient delivery., (Copyright © 2013 John Wiley & Sons, Ltd.)

Coupling of rapid prototyping techniques and surface-confined polymerizations allows the fabrication of 3D multidirectional gradients of biomolecules within microporous scaffolds. The compositional gradients can be tailored by polymer-brush-assisted diffusion of protein solutions. This technique allows spatial control over stem cells manipulation within 3D environments., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This work aims to design a synthetic construct that mimics the natural bone extracellular matrix through innovative approaches based on simultaneous type I collagen electrospinning and nanophased hydroxyapatite (nanoHA) electrospraying using non-denaturating conditions and non-toxic reagents. The morphological results, assessed using scanning electron microscopy and atomic force microscopy (AFM), showed a mesh of collagen nanofibers embedded with crystals of HA with fiber diameters within the nanometer range (30 nm), thus significantly lower than those reported in the literature, over 200 nm. The mechanical properties, assessed by nanoindentation using AFM, exhibited elastic moduli between 0.3 and 2 GPa. Fourier transformed infrared spectrometry confirmed the collagenous integrity as well as the presence of nanoHA in the composite. The network architecture allows cell access to both collagen nanofibers and HA crystals as in the natural bone environment. The inclusion of nanoHA agglomerates by electrospraying in type I collagen nanofibers improved the adhesion and metabolic activity of MC3T3-E1 osteoblasts. This new nanostructured collagen-nanoHA composite holds great potential for healing bone defects or as a functional membrane for guided bone tissue regeneration and in treating bone diseases.

In cartilage, tissue engineering bioreactors can create a controlled environment to study chondrocyte behavior under mechanical stimulation or produce chondrogenic grafts of clinically relevant size. Here we present a novel bioreactor that combines mechanical stimulation with a two compartment system through which nutrients can be supplied solely by diffusion from opposite sides of a tissue-engineered construct. This design is based on the hypothesis that creating gradients of nutrients, growth factors, and growth factor antagonists can aid in the generation of zonal tissue-engineered cartilage. Computational modeling predicted that the design facilitates the creation of a biologically relevant glucose gradient. This was confirmed by quantitative glucose measurements in cartilage explants. In this system, it is not only possible to create gradients of nutrients, but also of anabolic or catabolic factors. Therefore, the bioreactor design allows control over nutrient supply and mechanical stimulation useful for in vitro generation of cartilage constructs that can be used for the resurfacing of articulated joints or as a model for studying osteoarthritis disease progression.

Animal experiments help to progress and ensure safety of an increasing number of novel therapies, drug development and chemicals. Unfortunately, these also lead to major ethical concerns, costs and limited experimental capacity. We foresee a coercion of all these issues by implantation of well systems directly into vertebrate animals. Here, we used rapid prototyping to create wells with biomaterials to create a three-dimensional (3D) well-system that can be used in vitro and in vivo. First, the well sizes and numbers were adjusted for 3D cell culture and in vitro screening of molecules. Then, the functionality of the wells was evaluated in vivo under 36 conditions for tissue regeneration involving human mesenchymal stem cells (hMSCs) and bovine primary chondrocytes (bPCs) screened in one animal. Each biocompatible well was controlled to contain μl-size volumes of tissue, which led to tissue penetration from the host and tissue formation under implanted conditions. We quantified both physically and biologically the amounts of extracellular matrix (ECM) components found in each well. Using this new concept the co-culture of hMSCs and bPCs was identified as a positive hit for cartilage tissue repair, which was a comparable result using conventional methods. The in vivo screening of candidate conditions opens an entirely new range of experimental possibilities, which significantly abates experimental animal use and increases the pace of discovery of medical treatments.

Mesenchymal stromal cells (hMSCs) are advancing into the clinic but the therapeutic efficacy of hMSCs faces the problem of donor variability. In bone tissue engineering, no reliable markers have been identified which are able to predict the bone-forming capacity of hMSCs prior to implantation. To this end, we isolated hMSCs from 62 donors and characterized systematically their in vitro lineage differentiation capacity, gene expression signature and in vivo capacity for ectopic bone formation. Our data confirms the large variability of in vitro differentiation capacity which did not correlate with in vivo ectopic bone formation. Using DNA microarray analysis of early passage hMSCs we identified a diagnostic bone-forming classifier. In fact, a single gene, CADM1, strongly correlated with the bone-forming capacity of hMSCs and could be used as a reliable in vitro diagnostic marker. Furthermore, data mining of genes expressed correlating with in vivo bone formation represented involvement in neurogenic processes and Wnt signaling. We will apply our data set to predict therapeutic efficacy of hMSCs and to gain novel insight in the process of bone regeneration. Our bio-informatics driven approach may be used in other fields of cell therapy to establish diagnostic markers for clinical efficacy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The repertoire of growth factors determines the biological engagement of human mesenchymal stromal cells (hMSCs) in processes such as immunomodulation and tissue repair. Hypoxia is a strong modulator of the secretome and well known stimuli to increase the secretion of pro-angiogenic molecules. In this manuscript, we employed a high throughput screening assay on an hMSCs cell line in order to identify small molecules that mimic hypoxia. Importantly, we show that the effect of these small molecules was cell type/species dependent, but we identified phenanthroline as a robust hit in several cell types. We show that phenanthroline induces high expression of hypoxia-target genes in hMSCs when compared with desferoxamine (DFO) (a known hypoxia mimic) and hypoxia incubator (2% O(2)). Interestingly, our microarray and proteomics analysis show that only phenanthroline induced high expression and secretion of another angiogenic cytokine, interleukin-8, suggesting that the mechanism of phenanthroline-induced hypoxia is distinct from DFO and hypoxia and involves the activation of other signaling pathways. We showed that phenanthroline alone was sufficient to induce blood vessel formation in a Matrigel plug assay in vivo paving the way to its application in ischeamic-related diseases., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The repair of articular cartilage is challenging due to the sparse native cell population combined with the avascular and aneural nature of the tissue. In recent years, cartilage tissue engineering has shown great promise. As with all tissue engineering strategies, the possible therapeutic outcome is intimately linked with the used combination of cells, growth factors, and biomaterials. However, the optimal combination has remained a controversial topic and no consensus has been reached. In consequence, much effort has been dedicated, to further design, investigate, and optimize cartilage repair strategies. Specifically, various research groups have performed intensive investigations attempting to identify the single most optimal cell source for articular cartilage repair strategies. However, recent findings indicate that not the heavily investigated monocell source, but the less studied combinations of cell sources in coculture might be more attractive for cartilage repair strategies. This review will give a comprehensive overview on the cell sources that have been investigated for articular cartilage repair strategies. In particular, the advantages and disadvantages of investigated cell sources are comprehensively discussed with emphasis on the potential of cocultures in which benefits are combined, while the disadvantages of single-cell sources for cartilage repair are mitigated.

There is a rise in the popularity of arthroscopic procedures in orthopedics. However, the majority of cell-based bone tissue-engineered constructs (TECs) rely on solid preformed scaffolding materials, which require large incisions and extensive dissections for placement at the defect site. Thus, they are not suitable for minimally invasive techniques. The aim of this study was to develop a clinically relevant, easily moldable, bone TEC, amenable to minimally invasive techniques, using human mesenchymal stromal cells (hMSCs) and calcium phosphate microparticles in combination with an in situ forming platelet-rich plasma gel obtained from human platelets. Most conventional TECs rely on seeding and culturing single-cell suspensions of hMSCs on scaffolds. However, for generating TECs amenable to the minimally invasive approach, it was essential to aggregate the hMSCs in vitro before seeding them on the scaffolds as unaggregated MSCs did not generate any bone. Twenty four hours of in vitro aggregation was determined to be optimal for maintaining cell viability in vitro and bone formation in vivo. Moreover, no statistically significant difference was observed in the amount of bone formed when the TECs were implanted via an open approach or a minimally invasive route. TECs generated using MSCs from three different human donors generated new bone through the minimally invasive route in a reproducible manner, suggesting that these TECs could be a viable alternative to preformed scaffolds employed through an open surgery for treating bone defects.

Nanostructured three-dimensional constructs combining layer-by-layer technology (LbL) and template leaching were processed and evaluated as possible support structures for cartilage tissue engineering. Multilayered constructs were formed by depositing the polyelectrolytes chitosan (CHT) and chondroitin sulphate (CS) on either bidimensional glass surfaces or 3D packet of paraffin spheres. 2D CHT/CS multi-layered constructs proved to support the attachment and proliferation of bovine chondrocytes (BCH). The technology was transposed to 3D level and CHT/CS multi-layered hierarchical scaffolds were retrieved after paraffin leaching. The obtained nanostructured 3D constructs had a high porosity and water uptake capacity of about 300%. Dynamical mechanical analysis (DMA) showed the viscoelastic nature of the scaffolds. Cellular tests were performed with the culture of BCH and multipotent bone marrow derived stromal cells (hMSCs) up to 21 days in chondrogenic differentiation media. Together with scanning electronic microscopy analysis, viability tests and DNA quantification, our results clearly showed that cells attached, proliferated and were metabolically active over the entire scaffold. Cartilaginous extracellular matrix (ECM) formation was further assessed and results showed that GAG secretion occurred indicating the maintenance of the chondrogenic phenotype and the chondrogenic differentiation of hMSCs.

Tissue engineering techniques using a combination of polymeric scaffolds and cells represent a promising approach for nerve regeneration. We fabricated electrospun scaffolds by blending of Poly (3-hydroxybutyrate) (PHB) and Poly (3-hydroxy butyrate-co-3- hydroxyvalerate) (PHBV) in different compositions in order to investigate their potential for the regeneration of the myelinic membrane. The thermal properties of the nanofibrous blends was analyzed by differential scanning calorimetry (DSC), which indicated that the melting and glass temperatures, and crystallization degree of the blends decreased as the PHBV weight ratio increased. Raman spectroscopy also revealed that the full width at half height of the band centered at 1725 cm(-1) can be used to estimate the crystalline degree of the electrospun meshes. Random and aligned nanofibrous scaffolds were also fabricated by electrospinning of PHB and PHBV with or without type I collagen. The influence of blend composition, fiber alignment and collagen incorporation on Schwann cell (SCs) organization and function was investigated. SCs attached and proliferated over all scaffolds formulations up to 14 days. SCs grown on aligned PHB/PHBV/collagen fibers exhibited a bipolar morphology that oriented along the fiber direction, while SCs grown on the randomly oriented fibers had a multipolar morphology. Incorporation of collagen within nanofibers increased SCs proliferation on day 14, GDNF gene expression on day 7 and NGF secretion on day 6. The results of this study demonstrate that aligned PHB/PHBV electrospun nanofibers could find potential use as scaffolds for nerve tissue engineering applications and that the presence of type I collagen in the nanofibers improves cell differentiation.

Large bone defects naturally regenerate via a highly vascularized tissue which progressively remodels into cartilage and bone. Current approaches in bone tissue engineering are restricted by delayed vascularization and fail to recapitulate this stepwise differentiation toward bone tissue. Here, we use the morphogen Sonic Hedgehog (Shh) to induce the in vitro organization of an endothelial capillary network in an artificial tissue. We show that endogenous Hedgehog activity regulates angiogenic genes and the formation of vascular lumens. Exogenous Shh further induces the in vitro development of the vasculature (vascular lumen formation, size, distribution). Upon implantation, the in vitro development of the vasculature improves the in vivo perfusion of the artificial tissue and is necessary to contribute to, and enhance, the formation of de novo mature bone tissue. Similar to the regenerating callus, the artificial tissue undergoes intramembranous and endochondral ossification and forms a trabecular-like bone organ including bone-marrow-like cavities. These findings open the door for new strategies to treat large bone defects by closely mimicking natural endochondral bone repair.

State-of-the-art bioactive hydrogels can easily and efficiently be formed by enzyme-catalyzed mild-crosslinking reactions in situ. Yet this cell-friendly and substrate-specific method remains under explored. Hydrogels prepared by using enzyme systems like tyrosinases, transferases and lysyl oxidases show interesting characteristics as dynamic scaffolds and as systems for controlled release. Increased attention is currently paid to hydrogels obtained via crosslinking of precursors by transferases or peroxidases as catalysts. Enzyme-mediated crosslinking has proven its efficiency and attention has now shifted to the development of enzymatically crosslinked hydrogels with higher degrees of complexity, mimicking extracellular matrices. Moreover, bottom-up approaches combining biocatalysts and self-assembly are being explored for the development of complex nano-scale architectures. In this review, the use of enzymatic crosslinking for the preparation of hydrogels as an innovative alternative to other crosslinking methods, such as the commonly used UV-mediated photo-crosslinking or physical crosslinking, will be discussed. Photo-initiator-based crosslinking may induce cytotoxicity in the formed gels, whereas physical crosslinking may lead to gels which do not have sufficient mechanical strength and stability. These limitations can be overcome using enzymes to form covalently crosslinked hydrogels. Herewith, we report the mechanisms involved and current applications, focusing on emerging strategies for tissue engineering and regenerative medicine., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Mesenchymal stromal cells are present in very low numbers in the bone marrow, necessitating their selective expansion on tissue culture plastic prior to their use in tissue-engineering applications. MSC expansion is laborious, time consuming, unphysiological and not economical, thus calling for automated bioreactor-based strategies. We and others have shown that osteogenic grafts can be cultured in bioreactors by seeding either 2D-expanded cells or by direct seeding of the mononuclear fraction of bone marrow. To further streamline this protocol, we assessed in this study the possibility of seeding the cells onto porous calcium phosphate ceramics directly from unprocessed bone marrow. Using predetermined volumes of bone marrow from multiple human donors with different nucleated cell counts, we were able to grow a confluent cell sheath on the scaffold surface in 3 weeks. Cells of stromal, endothelial and haematopoietic origin were detected, in contrast to grafts grown from 2D expanded cells, where only stromal cells could be seen. Upon implantation in nude mice, similar quantities of bone tissue were generated as compared to that obtained by using the conventional number of culture expanded cells from the same donor. We conclude that human osteogenic grafts can be efficiently prepared by direct seeding of cells from unprocessed bone marrow., (Copyright © 2011 John Wiley & Sons, Ltd.)

Scaffolds derived from natural polysaccharides are very promising in tissue engineering applications and regenerative medicine, as they resemble glycosaminoglycans in the extracellular matrix (ECM). In this study, we have prepared freeze-dried composite scaffolds of chitosan (CHT) and hyaluronic acid (HA) in different weight ratios containing either no HA (control) or 1%, 5%, or 10% of HA. We hypothesized that HA could enhance structural and biological properties of CHT scaffolds. To test this hypothesis, physicochemical and biological properties of CHT/HA scaffolds were evaluated. Scanning electron microscopy micrographs, mechanical properties, swelling tests, enzymatic degradation, and Fourier transform infrared (FTIR) chemical maps were performed. To test the ability of the CHT/HA scaffolds to support chondrocyte adhesion and proliferation, live-dead and MTT assays were performed. Results showed that CHT/HA composite scaffolds are noncytotoxic and promote cell adhesion. ECM formation was further evaluated with safranin-O and alcian blue staining methods, and glycosaminoglycan and DNA quantifications were performed. The incorporation of HA enhanced cartilage ECM production. CHT/5HA had a better pore network configuration and exhibited enhanced ECM cartilage formation. On the basis of our results, we believe that CHT/HA composite matrixes have potential use in cartilage repair.

In this study, injectable hydrogels were prepared by horseradish peroxidase-mediated co-crosslinking of dextran-tyramine (Dex-TA) and heparin-tyramine (Hep-TA) conjugates and used as scaffolds for cartilage tissue engineering. The swelling and mechanical properties of these hydrogels can be easily controlled by the Dex-TA/Hep-TA weight ratio. When chondrocytes were incorporated in these gels, cell viability and proliferation were highest for gels with a 50/50 weight ratio of Dex-TA/Hep-TA. Moreover, these hydrogels induced an enhanced production of chondroitin sulfate and a more abundant presence of collagen as compared to Dex-TA hydrogels. The results indicate that injectable Dex-TA/Hep-TA hydrogels are promising scaffolds for cartilage regeneration., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Electrospun scaffolds are widely used for various biomedical applications. In this study, we prepared electrospun bioactive composite scaffolds combining hydroxyapatite, collagen (Col) and a synthetic polymer-PolyActive™-to mimic naturally occurring extracellular matrix for in situ bone regeneration. Human mesenchymal stem cells (hMSCs) adhered and proliferated on these scaffolds. Cells on all scaffold types showed an increased metabolic activity with time. On day 4, the metabolic activity of cells cultured on PolyActive™ (PA)-hydroxyapatite (HA)-Col in 1,1,1,3,3,3-hexafluoro-2-propanolhexafluoro-2-propanol (HFIP) was significantly higher than that of cells grown on PA-Col samples. Furthermore, on day 6, cells on PA-HA-Col in HFIP showed significantly higher metabolic activity than those on PA and PA-Col scaffolds. Quantitative PCR analysis for a panel of osteogenic genes showed statistically significant differences between scaffolds. Cells cultured on PA-HA scaffolds had a significantly higher osteonectin and RunX2 expression compared to those on PA-HA-Col scaffolds. Cells on PA-HA-Col in HFIP scaffolds had significantly higher expression of alkaline phosphatase (ALP) and Col 1 compared to PA and PA-Col scaffolds respectively. The bone morphogenetic protein-2 and S100A4 expression of PA-Col and PA-HA-Col constructs was significantly lower than the basal level expression of cells on PA scaffolds. Although not statistically significant in all cases, cells cultured on PA-HA-Col in HFIP and PA-HA scaffolds had the highest expression for most of the genes analysed. The results of the study demonstrate that bioactive composite scaffolds prepared by electrospinning could find potential use in bone regeneration applications.

The advance of rapid prototyping techniques has significantly improved control over the pore network architecture of tissue engineering scaffolds. In this work, we have assessed the influence of scaffold pore architecture on cell seeding and static culturing, by comparing a computer designed gyroid architecture fabricated by stereolithography with a random pore architecture resulting from salt leaching. The scaffold types showed comparable porosity and pore size values, but the gyroid type showed a more than 10-fold higher permeability due to the absence of size-limiting pore interconnections. The higher permeability significantly improved the wetting properties of the hydrophobic scaffolds and increased the settling speed of cells upon static seeding of immortalised mesenchymal stem cells. After dynamic seeding followed by 5 days of static culture gyroid scaffolds showed large cell populations in the centre of the scaffold, while salt-leached scaffolds were covered with a cell sheet on the outside and no cells were found in the scaffold centre. It was shown that interconnectivity of the pores and permeability of the scaffold prolonged the time of static culture before overgrowth of cells at the scaffold periphery occurred. Furthermore, novel scaffold designs are proposed to further improve the transport of oxygen and nutrients throughout the scaffolds and to create tissue engineering grafts with a designed, pre-fabricated vasculature., (Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

A practical method of photocrosslinking high molecular weight poly(trimethylene carbonate)(PTMC) is presented. Flexible, elastomeric and biodegradable networks could be readily prepared by UV irradiating PTMC films containing pentaerythritol triacrylate (PETA) and a photoinitiator. The network characteristics, mechanical properties, wettability, and in vitro enzymatic erosion of the photocrosslinked PTMC films were investigated. Densely crosslinked networks with gel contents up to 98% could be obtained in this manner. Upon photocrosslinking, flexible and tough networks with excellent elastomeric properties were obtained. To illustrate the ease with which the properties of the networks can be tailored, blends of PTMC with mPEG-PTMC or with PTMC-PCL-PTMC were also photocrosslinked. The wettability and the enzymatic erosion rate of the networks could be tuned by blending with block copolymers. Tissue engineering scaffolds were also fabricated using these flexible photocrosslinkable materials. After crosslinking, the fabricated PTMC-based scaffolds showed inter-connected pores and extensive microporosity. Human mesenchymal stem cell (hMSC) culturing studies showed that the photocrosslinked scaffolds prepared from PTMC and PTMC/PTMC-PCL-PTMC blends are well-suited for tissue engineering applications., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

To form tissues with uniform cell distribution and extracellular matrix arrangement is of great relevance to obtain the desirable function and maintain structural integrity. Scaffold configuration is believed to play a critical role in regulating cell spatial distribution and consequently tissue formation. In this study, three types of poly(ethyleneglycol-terephthalate)-poly (butylenes terephthalate) (PEGT/PBT) scaffolds [compression molded scaffold (CM), compression molded scaffold after chloroform/isopropanol reticulation (CMR), 3D rapid prototyped fibrous scaffold (RP)] with various configurations were used to support the tissue formation of adipose stromal cells for up to 21 days. Characterization of the scaffolds with muCT revealed that RP scaffolds were composed of repeating structural units with well controlled interconnected pores, in contrast to the irregular pore morphology in CM or CMR. Cell seeding efficacy onto various scaffolds was comparable (from 67 +/- 4% to 82 +/- 3%), while only RP scaffold led to even cell attachment onto the inner fibers of the scaffolds. Continuous cell proliferation and deposition of new collagen and glycosaminoglycans (GAG) were measured for all three scaffolds, while with a significant amount measured in RP at 21 days. By 21 days, complete uniform tissue formation was only achieved in RP scaffolds under a dynamic cell culture in spinner flasks. The present study successfully demonstrates the feasibility of controlling uniform tissue formation at a microscale by manipulating the structural configuration of the scaffold., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Various cell types have been investigated as candidate cell sources for cartilage and bone tissue engineering. In this review, we focused on chondrogenic and osteogenic differentiation of mouse and human embryonic stem cells (ESCs) and their potential in cartilage and bone tissue engineering. A decade ago, mouse ESCs were first used as a model to study cartilage and bone development and essential genes, factors and conditions for chondrogenesis and osteogenesis were unravelled. This knowledge, combined with data from the differentiation of adult stem cells, led to successful chondrogenic and osteogenic differentiation of mouse ESCs and later also human ESCs. Next, researchers focused on the use of ESCs for skeletal tissue engineering. Cartilage and bone tissue was formed in vivo using ESCs. However, the amount, homogeneity and stability of the cartilage and bone formed were still insufficient for clinical application. The current protocols require improvement not only in differentiation efficiency but also in ESC-specific hurdles, such as tumourigenicity and immunorejection. In addition, some of the general tissue engineering challenges, such as cell seeding and nutrient limitation in larger constructs, will also apply for ESCs. In conclusion, there are still many challenges, but there is potential for ESCs in skeletal tissue engineering., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

Cartilage tissue engineering, in which chondrogenic cells are combined with a scaffold, is a cell-based approach to regenerate damaged cartilage. Various scaffold materials have been investigated, among which are hydrogels. Previously, we have developed dextran-based hydrogels that form under physiological conditions via a Michael-type addition reaction. Hydrogels can be formed in situ by mixing a thiol-functionalized dextran with a tetra-acrylated star poly(ethylene glycol) solution. In this article we describe how the degradation time of dextran-poly(ethylene glycol) hydrogels can be varied from 3 to 7 weeks by changing the degree of substitution of thiol groups on dextran. The degradation times increased slightly after encapsulation of chondrocytes in the gels. The effect of the gelation reaction on cell viability and cartilage formation in the hydrogels was investigated. Chondrocytes or embryonic stem cells were mixed in the aqueous dextran solution, and we confirmed that the cells survived gelation. After a 3-week culturing period, chondrocytes and embryonic stem cell-derived embryoid bodies were still viable and both cell types produced cartilaginous tissue. Our data demonstrate the potential of dextran hydrogels for cartilage tissue engineering strategies.

The aim of this study was to investigate the effect of implant location on bone formation in goats using autologous bone marrow-derived stromal cells in porous calcium phosphate scaffolds. Intramuscular locations were compared to posterolateral spine fusion locations in eight goats. As scaffolds, we used biphasic calcium phosphate porous blocks of 5 x 5 x 5 mm. Cell-seeded implants were compared to empty controls. Bone marrow-derived stromal cells were seeded at 8 million cells per cm(3) scaffold and cultured for 1 week. The follow-up time was 12 weeks. Fluorochromes were administered intravenously at 4, 6, and 8 weeks. Ectopic implants showed 21 +/- 3.6% bone formation for the cell seeded and 2.0 +/- 3.0% for the controls (p < 0.001). Paraspinal implants, however, showed 0.10 +/- 0.13% in the cell seeded compared to 0.023 +/- 0.027% in the control group (p = 0.09). A benefit of the cells was only found in the area closest to the paraspinal muscles (p < 0.01). Bone formation in the control samples was of later onset compared to the cell-seeded implants. In conclusion, cell-based bone tissue engineering in an ectopic environment was clearly effective. Similar constructs implanted in a posterolateral spine fusion location hardly showed any effect.

The development of 3D scaffolds consisting of stacked multi-layered porous sheets featuring microchannels is proposed and investigated in this work. In this concept, the inner-porosity of the sheets allows diffusion of nutrients and signalling products between the layers whereas the microchannels facilitate nutrient supply on all layers as they provide space for the culture medium to be perfused throughout the scaffold. Besides the above, these scaffolds have excellent distribution of the cells as seeding and attaching of the cells occurs on individual layers that are subsequently stacked. In addition, these scaffolds enable gaining local data from within the scaffolds as unstacking of the stacked layers allows for determination of various parameters per layer. Here, we show the proof of this concept by culturing C2C12 pre-myoblasts and A4-4 cells on stacked Poly(l-lactic acid) (PLLA) sheets featuring microchannels. The results obtained for culturing under static conditions clearly indicate that despite inhibited cell proliferation due to nutrient limitations, diffusion between the layers takes place and cells on various layers stay viable and also affect each other. Under dynamic conditions, medium flow through the channels improves nutrient availability to the cells on the various layers, drastically increasing cell proliferation on all layers.

In vitro-generated tissues hold significant promise in modern biology since they can potentially mimic physiological and pathological tissues. However, these are currently structurally and functionally of limited complexity and necessitate self-organization and recapitulation of tissue development mechanisms in vitro. Tools derived from nano- and microfabrications along with bottom-up strategies are emerging to allow the fabrication of primitive tissues structures that can remodel overtime. Subsequently, clues are accumulating to show that, beyond genetic material, both intrinsic tissue architectures and microenvironmental cues can lead to morphogenesis related mechanisms in vitro. The question arises, however, as how we may design and assemble structures prone to adequate tissue remodeling, predict and manipulate those developmental mechanisms in vitro? Systems integrating architectural, physical and molecular cues will allow more systematic investigation of basic principles of tissue morphogenesis, differentiation or maintenance and will feedback to reproduce the dynamic of tissue development in vitro and form more complex tissues.

The biological effect of the perfluorocarbon-based artificial oxygen carrier (Oxygent) was investigated in tissue-engineered trachea (TET) construction. Media supplemented with and without 10% Oxygent were compared in all assessments. Partial tissue oxygen tension (PtO(2)) was measured with polarographic microprobes; epithelial metabolism was monitored by microdialysis inside the TET epithelium perfused with the medium underneath. Chondrocyte-DegraPol constructs were cultured for 1 month with the medium before glycosaminoglycan assessment and histology. Tissue reaction of TET epithelial scaffolds immersed with the medium was evaluated on the chick embryo chorioallantoic membrane. Oxygent perfusion medium increased the TET epithelial PtO(2) (51.2 +/- 0.3 mm Hg vs. 33.4 +/- 0.3 mm Hg at 200 microm thickness; 12.5 +/- 0.1 mm Hg vs. 3.1 +/- 0.1 mm Hg at 400 microm thickness, p < 0.01) and decreased the lactate concentration (0.63 +/- 0.08 vs. 0.80 +/- 0.06 mmol/L, p < 0.05), lactate/pyruvate (1.87 +/- 0.26 vs. 3.36 +/- 10.13, p < 0.05), and lactate/glucose ratios (0.10 +/- 0.00 vs. 0.29 +/- 0.14, p < 0.05). Chondrocyte-DegraPol in Oxygent group presented lower glycosaminoglycan value (0.03 +/- 0.00 vs. 0.13 +/- 0.00, p < 0.05); histology slides showed poor acid mucopolysaccharides formation. Orthogonal polarization spectral imaging showed no difference in functional capillary density between the scaffolds cultured on chorioallantoic membranes. The foreign body reaction was similar in both groups. We conclude that Oxygent increases TET epithelial PtO(2), improves epithelial metabolism, does not impair angiogenesis, and tends to slow cartilage tissue formation.

In vitro vascularization is an upcoming strategy to solve the problem of insufficient blood supply after implantation. Although recent publications show promising results, these studies were generally performed with clinically irrelevant endothelial cell model systems. We tested the use of endothelial progenitor cells (EPC) obtained from umbilical cord blood and human mesenchymal stem cells (hMSC) from the bone marrow for their use in a prevascularized bone tissue engineering setting. MSC were differentiated toward endothelial cells. They formed capillary-like structures containing lumen, stained positive for CD31, attained the ability to take up acetylated low-density lipoproteins, and formed perfused vessels in vivo. However, in a three-dimensional coculture setting with undifferentiated hMSC, the cells stopped expressing CD31 and did not form prevascular structures. EPC from the cord blood were able to form prevascular structures in the same coculture setting, but only when the state of endothelial differentiation was mature. The amount of prevascular structures formed when using EPC was less than when human umbilical vein endothelial cells or human dermal microvascular endothelial cells were used. The degree of organization, however, was higher. We conclude that EPC can be used for complex tissue engineering applications, but the differentiation stage of these cells is important.

Application of hydrogels in tissue engineering and innovative strategies such as organ printing, which is based on layered 3D deposition of cell-laden hydrogels, requires design of novel hydrogel matrices. Hydrogel demands for 3D printing include: 1) preservation of the printed shape after the deposition; 2) maintaining cell viability and cell function and 3) easy handling of the printed construct. In this study we analyze the applicability of a novel, photosensitive hydrogel (Lutrol) for printing of 3D structured bone grafts. We benefit from the fast temperature-responsive gelation ability of thermosensitive Lutrol-F127, ensuring organized 3D extrusion, and the additional stability provided by covalent photocrosslinking allows handling of the printed scaffolds. We studied the cytotoxicity of the hydrogel and osteogenic differentiation of embedded osteogenic progenitor cells. After photopolymerization of the modified Lutrol hydrogel, cells remain viable for up to three weeks and retain the ability to differentiate. Encapsulation of cells does not compromise the mechanical properties of the formed gels and multilayered porous Lutrol structures were successfully printed.

In this study we tested the possibility of seeding chondrocytes into poly (ethylene glycol)-terephthalate-poly (butylene terephthalate) PEOT/PBT scaffold through an intra-scaffold medium flow and the impact of this continuous medium flow on subsequent chondrocyte-scaffold culture. Eight cubic PEOT/PBT co-polymers (1 cm(3)) were assigned into two groups. In the semi-dynamic seeding group a continuous medium flow was created inside the scaffolds by a pump system. Around six million chondrocytes were harvested each day, suspended in 1 ml medium and delivered onto the scaffold through the perfusion for a sequential five days. Traditional chondrocytes directly seeding and static culture method was performed as control. Scanning electron microscopy (SEM) and histology assessments were performed to evaluate the distribution of chondrocytes inside the scaffolds and MTT test was chosen to check cell vitality. SEM pictures and histology slices from the perfusion group showed a better three-dimensional cell growth and extensive cell distribution inside the scaffolds; while in the control group chondrocytes only dispersedly formed a monolayer on the surface of scaffolds. Accordingly, MTT results from the perfusion group were much higher than those from control group (0.123 vs. 0.067, P<0.01). Continuous medium perfusion inside PEOT/PBT scaffold effectively combines chondrocyte seeding and culture systems for the reconstruction of tissue engineered trachea.

Tissue engineering has been an active field of research for several decades now. However, the amount of clinical applications in the field of tissue engineering is still limited. One of the current limitations of tissue engineering is its inability to provide sufficient blood supply in the initial phase after implantation. Insufficient vascularization can lead to improper cell integration or cell death in tissue-engineered constructs. This review will discuss the advantages and limitations of recent strategies aimed at enhancing the vascularization of tissue-engineered constructs. We will illustrate that combining the efforts of different research lines might be necessary to obtain optimal results in the field.

In 6 patients the potency of bone tissue engineering to reconstruct jaw defects was tested. After a bone marrow aspirate was taken, stem cells were cultured, expanded and grown for 7 days on a bone substitute in an osteogenic culture medium to allow formation of a layer of extracellular bone matrix. At the end of the procedure, this viable bone substitute was not only re-implanted in the patient, but also simultaneously subcutaneously implanted in mice to prove its osteogenic potency. In all patients, a viable bone substitute was successfully constructed, which was proven by bone formation after subcutaneous implantation in mice (ectopic bone formation). However, the same construct was reluctant to form bone in patients with intra-oral osseous defects (orthotopic bone formation). Although biopsies, taken 4 months after reconstructing the intra-oral bone defect, showed bone formation in 3 patients, only in 1 patient bone formation was induced by the tissue-engineered construct. Although bone tissue engineering has proven its value in animal studies, extra effort is needed to make it a predictable method for reconstruction jaw defects in humans. To judge its benefit, it is important to differentiate between bone formation induced by cells from the border of the osseous defect (osteoconduction) in relation to bone matrix produced by the implanted cells (osteogenesis).

Embryonic stem cells can provide an unlimited supply of pluripotent cells for tissue engineering applications. Bone tissue engineering by directly differentiating ES cells (ESCs) into osteoblasts has been unsuccessful so far. Therefore, we investigated an alternative approach, based on the process of endochondral ossification. A cartilage matrix was formed in vitro by mouse ESCs seeded on a scaffold. When these cartilage tissue-engineered constructs (CTECs) were implanted s.c., the cartilage matured, became hypertrophic, calcified, and was ultimately replaced by bone tissue in the course of 21 days. Bone aligning hypertrophic cartilage was observed frequently. Using various chondrogenic differentiation periods in vitro, we demonstrated that a cartilage matrix is required for bone formation by ESCs. Chondrogenic differentiation of mesenchymal stem cells and articular chondrocytes showed that a cartilage matrix alone was not sufficient to drive endochondral bone formation. Moreover, when CTECs were implanted orthotopically into critical-size cranial defects in rats, efficient bone formation was observed. We report previously undescribed ESC-based bone tissue engineering under controlled reproducible conditions. Furthermore, our data indicate that ESCs can also be used as a model system to study endochondral bone formation.

Embryonic stem (ES) cells are a potential source for cartilage tissue engineering because they provide an unlimited supply of cells that can be differentiated into chondrocytes. So far, chondrogenic differentiation of both mouse and human ES cells has only been demonstrated in two-dimensional cultures, in pellet cultures, in a hydrogel, or on thin biomaterials. The next challenge will be to form cartilage on a load-bearing, clinically relevant-sized scaffold in vitro and in vivo, to regenerate defects in patients suffering from articular cartilage disorders. For a successful implant, cells have to be seeded efficiently and homogenously throughout the scaffold. Parameters investigated were the scaffold architecture, seeding method, and cellular condition. Seeding in a three-dimensional fiber-deposited (3DF) scaffold was more homogenous than in a compression-molded scaffold. The seeding efficiency on bare scaffolds was compromised by the absence of serum in the chondrogenic medium, but could be improved by combining the cells with a gel and subsequent injection into the 3DF scaffolds. However, the viability of the cells was unsatisfactory in the interior of the graft. Cell aggregates, the so-called embryoid bodies (EBs), were seeded with increased survival rate. Mouse ES cells readily underwent chondrogenic differentiation in vitro in pellets, on bare scaffolds, in Matrigel, and in agarose, both as single cells and in EBs. The differentiation protocol requires further improvement to achieve homogenous differentiation and abolish teratoma formation in vivo. We conclude that ES cells can be used as a cell source for cartilage tissue engineering, pending further optimization of the strategy.

For biotechnological research in vitro in general and tissue engineering specifically, it is essential to mimic the natural conditions of the cellular environment as much as possible. In choosing a model system for in vitro experiments, the investigator always has to balance between being able to observe, measure or manipulate cell behaviour and copying the in situ environment of that cell. Most tissues in the body consist of more than one cell type. The organization of the cells in the tissue is essential for the tissue's normal development, homeostasis and repair reaction. In a co-culture system, two or more cell types brought together in the same culture environment very likely interact and communicate. Co-culture has proved to be a powerful in vitro tool in unravelling the importance of cellular interactions during normal physiology, homeostasis, repair and regeneration. The first co-culture studies focused mainly on the influence of cellular interactions on oocytes maturation to a pre-implantation blastocyst. Therefore, a brief overview of these studies is given here. Later on in the history of co-culture studies, it was applied to study cell-cell communication, after which, almost immediately as the field of tissue engineering was recognized, it was introduced in tissue engineering to study cellular interactions and their influence on tissue formation. This review discusses the introduction and applications of co-culture systems in cell biology research, with the emphasis on tissue engineering and its possible application for studying cartilage regeneration.

Despite decades of extensive research, the application of cell-based bone tissue engineering in clinically relevant models remains challenging. To improve effectiveness, a better understanding of how the technique should work is crucial. In the current study, we investigated the onset time, rate, location and direction of bone formation in ectopically and orthotopically implanted clinically sized tissue-engineered constructs to gain insight the mechanism behind it. Bone marrow stromal cells (BMSCs) were obtained from 10 goats, culture expanded and cryopreserved. Porous biphasic calcium phosphate (BCP) disks of 17mmx6mm were per-operatively seeded with BMSCs or left empty. Both conditions were implanted intramuscularly and in bilateral critical-sized iliac wing defects. Fluorochromes were administered at 3, 5 and 7 weeks and samples were retrieved after 9 weeks. Histology showed abundant and homogeneous bone formation throughout the intramuscular BMSC samples and little bone in the controls. Histomorphometry and measurements of the fluorochrome labels of the ectopical BMSC samples indicated that osteogenesis started at the periphery and subsequent osteoconduction filled the whole scaffold within 7 weeks. In the orthotopically implanted disks, there was good integration with the surrounding bone, but minimal bone in the center of the implants, in both conditions. Bone was only derived from the interface with the surrounding bone, there was no early bone at the surfaces in contact to soft tissue as was seen in the ectopical samples. Apparently cell survival was minimal and insufficient for relevant additional bone formation. However, the speed of integration with surrounding bone and subsequent bone apposition on the BMSC-seeded orthotopic scaffolds were found to be significantly enhanced, which may be relevant especially in challenging environments.