Search

Vascularization is undoubtedly one of the greatest challenges in tissue engineering. Its importance is particularly evident when considering the transplantation of (bioengineered) pancreatic islets of Langerhans, which are highly sensitive to the delivery of oxygen and nutrients for their survival and function. Here we studied pseudoislets of Langerhans, which are three-dimensional spheroids composed of β (INS1E), α (alpha TC-1), and endothelial (HUVEC) cells, and were interested in how the location and prevalence of the different cell types affected the presence of endothelial cells in the pseudoislet. We hypothesized that alpha (α) cells play an essential role in islet self-assembly and the incorporation of endothelial cells into the pseudoislet, and are thus important to consider in tissue engineering or regenerative medicine strategies, which typically focuses on the insulin-producing beta (β) cells alone. We first determined the effect of changing the relative ratios of the cells and found the cell distribution converged on a steady state of ∼21% α cells, 74% β cells, and 5% endothelial cells after 10 days of culture regardless of their respective ratios at seeding. We also found that the incorporation of endothelial cells was related to the pseudoislet size, with more endothelial cells found in the core of larger pseudoislets following a concomitant increase of α cells and a decrease in β cells. Finally, we observed that both endothelial and β cells were found adjacent to α cells significantly more frequently than to each other. In conclusion, this study demonstrates that the self-assembly of a pseudoislet is an intrinsically cell-regulated process. The endothelial cells had preferential proximity to the α cells, and this persisted even when challenged with changing the cell ratios and numbers. This study gives insight into the rules governing the self-organization of pseudoislets and suggests an important role for α cells to promote the incorporation of endothelial cells.

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.

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.

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.

Partial replacement of chondrocytes by stem cells has been proposed to improve the performance of autologous chondrocyte implantation (ACI). Our previous studies showed that the increased cartilage production in pellet cocultures of chondrocytes and mesenchymal stem cells (MSCs) is due to a trophic role of the MSCs by stimulating chondrocyte proliferation and matrix production rather than MSCs actively undergoing chondrogenic differentiation. The aim of this study is to compare the trophic effects of stromal vascular fraction cells (SVF) and in vitro expanded adipose stem cells (ASC). SVF and culture-expanded ASC (n = 9) were cocultured with primary human chondrocytes in pellets. By glycosaminoglycan (GAG) and DNA assays, we showed that coculture pellets of SVF and chondrocytes have more GAG deposition than that of ASC and chondrocytes. Results of the short tandem repeats analysis indicated that the increase in the chondrocyte proportion in the coculture pellets is more pronounced in the SVF coculture group than in the ASC coculture group. Using flow cytometry and microarray, we demonstrated that SVF and ASC have different characteristics in cell surface markers and gene expression profiles. SVF is more heterogeneous than ASC, whereas ASC is more enriched in cells from the mesenchymal lineage than SVF. By subcutaneous implantation into nude mice, we showed that constructs of SVF and chondrocytes are better in depositing cartilage matrix than the mixture of ASC and chondrocytes. Taken together, SVF is better than ASC in terms of forming cartilage matrix in pellet coculture and in coimplantation models omitting the need for prior cell expansion. Our study suggests that the SVF in combination with primary human chondrocytes may be a good cell combination for one-stage cartilage repair.

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.

Previously, we showed that mesenchymal stromal cells (MSCs) in co-culture with primary chondrocytes secrete soluble factors that increase chondrocyte proliferation. The objective of this study is to identify these factors. Human primary chondrocytes (hPCs) isolated from late-stage osteoarthritis patients were co-cultured with human bone marrow-derived MSCs (hMSCs) in pellets. Genome-wide mRNA expression analysis and quantitative polymerase chain reactions (qPCR) were used to identify soluble factors that were specifically induced in co-cultures. Immunofluorescent staining combined with cell tracking and enzyme-linked immunosorbent assay (ELISA) were performed to validate up-regulation at the protein level and to identify the cellular origin of the increased proteins. Chemical blockers and neutralizing antibodies were used to elucidate the role of the identified candidate genes in co-cultures. A number of candidate factors were differentially regulated in co-cultures at the mRNA level. Of these, fibroblast growth factor-1 (FGF-1) mRNA and protein expression were markedly increased in co-cultures predominantly due to up-regulated expression in MSCs. Blocking of FGF signaling in co-culture pellets by specific FGF receptor inhibitors or FGF-1 neutralizing antibodies completely blocked hPCs proliferation. We demonstrate that MSCs increase FGF-1 secretion on co-culture with hPCs, which, in turn, is responsible for increased hPCs proliferation in pellet co-cultures.

Bone marrow-derived multipotent mesenchymal stromal cells (MSCs) are the most frequently investigated cell type for potential regenerative strategies because they are relatively easy to isolate and are able to differentiate into several mesenchymal lineages. Unfortunately, during ex vivo culture, MSCs present gradual loss of differentiation potential and reduced clinical efficacy. Reactive oxygen species (ROS) are associated with oxidative damage and accumulate during MSC expansion. Because ROS are believed to be involved in the loss of multipotency, we hypothesized that compounds with antioxidant activity have the capacity to scavenge ROS, prevent cellular damage, and rescue culture-induced loss of multipotency. In this manuscript, we show that antioxidant supplementation can partially rescue the loss of alkaline phosphatase expression induced by oxidizing agents and increases the yield of hMSCs, when supplemented to a fresh bone marrow aspirate. Concomitantly, oxidative DNA damage and ROS levels in hMSCs were reduced by antioxidants. We conclude that antioxidant supplementation during MSC expansion reduces the DNA damage load and increases the MSC yield.

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.

Earlier, we have shown that the increased cartilage production in pellet co-cultures of chondrocytes and bone marrow-derived mesenchymal stem cells (BM-MSCs) is due to a trophic role of the MSC in stimulating chondrocyte proliferation and matrix production rather than MSCs actively undergoing chondrogenic differentiation. These studies were performed in a culture medium that was not compatible with the chondrogenic differentiation of MSCs. In this study, we tested whether the trophic role of the MSCs is dependent on culturing co-culture pellets in a medium that is compatible with the chondrogenic differentiation of MSCs. In addition, we investigated whether the trophic role of the MSCs is dependent on their origins or is a more general characteristic of MSCs. Human BM-MSCs and bovine primary chondrocytes were co-cultured in a medium that was compatible with the chondrogenic differentiation of MSCs. Enhanced matrix production was confirmed by glycosaminoglycans (GAG) quantification. A species-specific quantitative polymerase chain reaction demonstrated that the cartilage matrix was mainly of bovine origin, indicative of a lack of the chondrogenic differentiation of MSCs. In addition, pellet co-cultures were overgrown by bovine cells over time. To test the influence of origin on MSCs' trophic effects, the MSCs isolated from adipose tissue and the synovial membrane were co-cultured with human primary chondrocytes, and their activity was compared with BM-MSCs, which served as control. GAG quantification again confirmed increased cartilage matrix production, irrespective of the source of the MSCs. EdU staining combined with cell tracking revealed an increased proliferation of chondrocytes in each condition. Irrespective of the MSC source, a short tandem repeat analysis of genomic DNA showed a decrease in MSCs in the co-culture over time. Our results clearly demonstrate that in co-culture pellets, the MSCs stimulate cartilage formation due to a trophic effect on the chondrocytes rather than differentiating into chondrocytes, irrespective of culture condition or origin. This implies that the trophic effect of MSCs in co-cultures is a general phenomenon with potential implications for use in cartilage repair strategies.

Osteogenic differentiation of human mesenchymal stromal cells (hMSCs) may potentially be used in cell-based bone tissue-engineering applications to enhance the bone-forming potential of these cells. Osteogenic differentiation and adipogenic differentiation are thought to be mutually exclusive, and although several signaling pathways and cues that induce osteogenic or adipogenic differentiation, respectively, have been identified, there is no general consensus on how to optimally differentiate hMSCs into the osteogenic lineage. Some pathways have also been reported to be involved in both adipogenic and osteogenic differentiation, as for example, the protein kinase A (PKA) pathway, and the aim of this study was to investigate the role of cAMP/PKA signaling in differentiation of hMSCs in more detail. We show that activation of this pathway with dibutyryl-cAMP results in enhanced alkaline phosphatase expression, whereas another cAMP analog induces adipogenesis in long-term mineralization cultures. Adipogenic differentiation, induced by 8-bromo-cAMP, was accompanied by stronger PKA activity and higher expression of cAMP-responsive genes, suggesting that stronger activation correlates with adipogenic differentiation. In addition, a whole-genome expression analysis showed an increase in expression of adipogenic genes in 8-br-cAMP-treated cells. Furthermore, by means of quantitative polymerase chain reaction, we show differences in peroxisome proliferator-activated receptor-γ activation, either alone or in combination with dexamethasone, thus demonstrating differential effects of the PKA pathway, most likely depending on its mode of activation.

Activation of the protein kinase A (PKA) pathway with dibutyryl cyclic adenosine monophosphate (db-cAMP) was recently shown to enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs) in vitro and bone formation in vivo. The major drawback of this compound is its inhibitory effect on proliferation of hMSCs. Therefore, we investigated whether fine-tuning of the dose and timing of PKA activation could enhance bone formation even further, with minimum effects on proliferation. To test this, we selected two different PKA activators (8-bromo-cAMP (8-br-cAMP) and forskolin) and compared their effects on proliferation and osteogenic differentiation with those of db-cAMP. We found that all three compounds induced alkaline phosphatase levels, bone-specific target genes, and secretion of insulin-like growth factor-1, although 8-br-cAMP induced adipogenic differentiation in long-term cultures and was thus considered unsuitable for further in vivo testing. All three compounds inhibited proliferation of hMSCs in a dose-dependent manner, with forskolin inhibiting proliferation most. The effect of forskolin on in vivo bone formation was tested by pretreating hMSCs before implantation, and we observed greater amounts of bone using forskolin than db-cAMP. Our data show forskolin to be a novel agent that can be used to increase bone formation and also suggests a role for PKA in the delicate balance between adipogenic and osteogenic differentiation.

The nutritional requirements of stem cells have not been determined; in particular, the amino acid metabolism of stem cells is largely unknown. In this study, we investigated the amino acid metabolism of human mesenchymal stem cells (hMSCs), with focus on two questions: Which amino acids are consumed and/or secreted by hMSCs and at what rates? To answer these questions, hMSCs were cultured on tissue culture plastic and in a bioreactor, and their amino acid profile was analyzed. The results showed that the kinetics of hMSCs growth and amino acid metabolism were significantly higher for hMSCs in tissue culture plastic than in the bioreactor. Despite differences in culture conditions, 8 essential and 6 nonessential amino acids were consumed by hMSCs in both tissue culture plastic and bioreactor cultures. Glutamine was the most consumed amino acid with significantly higher rates than for any other amino acid. The metabolism of nonessential amino acids by hMSCs deviated significantly from that of other cell lines. The secretion of alanine, glycine, glutamate, and ornithine by hMSCs showed that there is a strong overflow metabolism that can be due to the high concentrations of amino acids provided in the medium. In addition, the data showed that there is a metabolic pattern for proliferating hMSCs, which can contribute to the design of medium without animal serum for stem cells. Further, this study shows how to implement amino acid rates and metabolic principles in three-dimensional stem cell biology.

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.

Previous studies showed that coculture of primary chondrocytes (PCs) with various sources of multipotent cells results in a higher relative amount of cartilage matrix formation than cultures containing only chondrocytes. The aim of this study was to investigate the mechanism underlying this observation. We used coculture pellet models of human mesenchymal stem cells (hMSCs) and human PCs or bovine PCs (bPCs) and studied the fate and the contribution to cartilage formation of the individual cell populations during coculture. Enhanced cartilage matrix deposition was confirmed by histology and quantification of total glycosaminoglycan deposition. Species-specific quantitative polymerase chain reaction demonstrated that cartilage matrix gene expression was mainly from bovine origin when bPCs were used. Short tandem repeat analysis and species-specific quantitative polymerase chain reaction analysis of genomic DNA demonstrated the near-complete loss of MSCs in coculture pellets after 4 weeks of culture. In coculture pellets of immortalized MSCs and bPCs, chondrocyte proliferation was increased, which was partly mimicked using conditioned medium, and simultaneously preferential apoptosis of immortalized MSCs was induced. Taken together, our data clearly demonstrate that in pellet cocultures of MSCs and PCs, the former cells disappear over time. Increased cartilage formation in these cocultures is mainly due to a trophic role of the MSCs in stimulating chondrocyte proliferation and matrix deposition by chondrocytes rather than MSCs actively undergoing chondrogenic differentiation.

Luciferase transgenic mice are a very promising tool for noninvasive, quantitative, and longitudinal evaluation of gene expression. The aim of this study was to validate the Col(I)-Luc transgenic mouse model in which the luciferase gene is driven by bone-specific regulatory elements from the mouse collagen α1(I) gene for bioluminescent imaging of bone development and remodeling. We observed strong luciferase activity in skeletal tissues of Col(I)-Luc mice, and observed that the light intensity declined with postnatal bone development. Luciferase activity was enhanced in a tail bone repair model and we were able to monitor the process of ectopic bone formation induced by recombinant human bone morphogenetic protein 2 using bioluminescent imaging. We conclude that Col(I)-Luc transgenic mice can be applied in the field of bone tissue engineering for monitoring bone repair processes and for investigating osteoinductive molecules or scaffolds.

Enzymatic crosslinking of dextran-tyramine (Dex-TA) conjugates in the presence of horseradish peroxidase and hydrogen peroxide was successively applied in the preparation of hydrogels. Depending on the molecular weight of the dextran (M(n,) (GPC) of 14000 or 31000 g/mol) and the degree of substitution (of 5, 10, or 15) with TA groups, the gelation times ranged from 20 s to 1 min. Hydrogels prepared from Dex31k-TA with a degree of substitution of 10 had storage moduli up to 60 kPa. Similar values were found when chondrocytes were incorporated into the hydrogels. Chondrocyte-seeded Dex-TA hydrogels were prepared at a molar ratio of hydrogen peroxide/TA of 0.2 and cultured in a chondrocyte medium. A live-dead assay and a methylthiazol tetrazolium assay revealed that almost all chondrocytes retained their viability after 2 weeks. Scanning electron microscopy analysis showed that the encapsulated chondrocytes were capable of maintaining their round shape. Histology and immunofluorescent staining demonstrated the production of glycosaminoglycans (GAGs) and collagen type II after culturing for 14 and 21 days. Biochemical analysis showed that GAG accumulation increased with time inside Dex-TA hydrogels. Besides, GAG/DNA for Dex-TA hydrogels was higher than that for agarose at day 28. These results indicate that Dex-TA hydrogels are promising 3D scaffolds for cartilage tissue engineering applications.

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 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.

Bone marrow stromal cells (BMSCs) have been shown to proliferate and produce matrix when seeded onto braided poly(L-lactide/glycolide) acid (PLGA) scaffolds. Mechanical stimulation may be applied to stimulate tissue formation during ligament tissue engineering. This study describes for the first time the effect of constant load on BMSCs seeded onto a braided PLGA scaffold. The seeded scaffolds were subjected to four different loading regimes: Scaffolds were unloaded, loaded during seeding, immediately after seeding, or 2 days after seeding. During the first 5 days, changing the mechanical environment seemed to inhibit proliferation, because cells on scaffolds loaded immediately after seeding or after a 2-day delay, contained fewer cells than on unloaded scaffolds or scaffolds loaded during seeding (p<0.01 for scaffolds loaded after 2 days). During this period, differentiation increased with the period of load applied. After day 5, differences in cell content and collagen production leveled off. After day 11, cell number decreased, whereas collagen production continued to increase. Cell number and differentiation at day 23 were independent of the timing of the mechanical stimulation applied. In conclusion, static load applied to BMSCs cultured on PLGA scaffolds allows for proliferation and differentiation, with loading during seeding yielding the most rapid response. Future research should be aimed at elucidating the biomechanical and biochemical characteristics of tissue formed by BMSCs on PLGA under mechanical stimulation.

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.

The advantage of using anatomically shaped scaffolds as compared to modeled designs was investigated and assessed in terms of cartilage formation in an artificial tracheal construct. Scaffolds were rapid prototyped with a technique named three-dimensional fiber deposition (3DF). Anatomical scaffolds were fabricated from a patient-derived computerized tomography dataset, and compared to cylindrical and toroidal tubular scaffolds. Lewis rat tracheal chondrocytes were seeded on 3DF scaffolds and cultured for 21 days. The 3-(4,5-dimethylthiazol-2yl)-2,5-dyphenyltetrazolium bromide (MTT) and sulfated glycosaminoglycan (GAG) assays were performed to measure the relative number of cells and the extracellular matrix (ECM) formed. After 3 weeks of culture, the anatomical scaffolds revealed a significant increase in ECM synthesis and a higher degree of differentiation as shown by the GAG/MTT ratio and by scanning electron microscopy analysis. Interestingly, a lower scaffold's pore volume and porosity resulted in more tissue formation and a better cell differentiation, as evidenced by GAG and GAG/MTT values. Scaffolds were compliant and did not show any signs of luminal obstruction in vitro. These results may promote anatomical scaffolds as functional matrices for tissue regeneration not only to help regain the original shape, but also for their improved capacity to support larger tissue formation.

During the past decade, there has been much interest in the use of human mesenchymal stem cells (hMSCs) in bone tissue engineering. HMSCs can be obtained relatively easily and expanded rapidly in culture, but for clinical purposes large numbers are often needed and the cost should be kept to a minimum. A rapid and efficient culturing protocol would therefore be beneficial. In this study, we examined the effect of different medium compositions on the expansion and osteogenic differentiation of bone marrow-derived hMSCs from 19 donors. We also investigated the effect of low seeding density and dexamethasone on both hMSCs expansion and their in vitro and in vivo osteogenic differentiation capacity. HMSCs seeded at a density of 100 cells/cm2 had a significantly higher growth rate than at 5000 cell/cm2, which was further improved by the addition of dexamethasone. Expanded hMSCs were characterized in vitro on the basis of positive staining for CD29, CD44, CD105, and CD166. The in vitro osteogenic potential of expanded hMSCs was assessed by flow cytometric staining for alkaline phosphatase. In vivo bone-forming potential of the hMSCs was assessed by seeding the cells in ceramic scaffolds, followed by subcutaneous implantation in nude mice and histopathologic assessment of de novo bone formation after 6-week implantation. Expanded hMSCs from all donors displayed similar osteogenic potential independent of the culture conditions. On the basis of these results we have developed an efficient method to culture hMSCs by seeding the cells at 100 cells/cm2 in an alpha-minimal essential medium-based medium containing dexamethasone.

To engineer tissues with clinically relevant dimensions, one must overcome the challenge of rapidly creating functional blood vessels to supply cells with oxygen and nutrients and to remove waste products. We tested the hypothesis that endothelial cells, cocultured with osteoprogenitor cells, can organize into a prevascular network in vitro. When cultured in a spheroid coculture model with human mesenchymal stem cells, human umbilical vein endothelial cells (HUVECs) form a 3-dimensional prevascular network within 10 days of in vitro culture. The formation of the prevascular network was promoted by seeding 2% or fewer HUVECs. Moreover, the addition of endothelial cells resulted in a 4-fold upregulation of the osteogenic marker alkaline phosphatase. The addition of mouse embryonic fibroblasts did not result in stabilization of the prevascular network. Upon implantation, the prevascular network developed further and structures including lumen could be seen regularly. However, anastomosis with the host vasculature was limited. We conclude that endothelial cells are able to form a 3-dimensional (3D) prevascular network in vitro in a bone tissue engineering setting. This finding is a strong indication that in vitro prevascularization is a promising strategy to improve implant vascularization in bone tissue engineering.

Material-induced bone formation reported in canine, bovid, suid, and primate species does not often occur in lagomorph or rodent models. In this study, we test biphasic calcium phosphate and hydroxyapatite- induced bone formation in subcutaneous pockets of mice and intramuscular pockets in rats, rabbits, and dogs. All scaffolds are of similar size, and all animals were sacrificed at 90 days post-implantation. In dogs (N = 8), all implants showed bone formation with significantly more bone formed in biphasic calcium phosphates (30 +/- 6%, N = 8) as compared to hydroxyapatite (14 +/- 5%, N = 8) (p = 0.003). Hydroxyapatite implants did not induce bone formation in mice, rats, or rabbits. Biphasic calcium phosphate induced bone in 6 of 8 scaffolds implanted in 4 rabbits and 3 of 16 scaffolds implanted in 16 mice, whereas it did not induce bone formation in any of the 8 rats. The results presented herein suggest that the incidence of material-induced bone formation varies with animal species and is related to the implant material used.

Animal studies in cartilage tissue engineering usually include the transfer of cultured cells into chondral or osteochondral defects. Immediately at implantation, the cells are exposed to a dramatically changed environment. The aim of this study was to determine the viability of two cell types currently considered for cellular therapies of cartilage defects-chondrocytes and progenitor cells-shortly after exposure to an osteochondral defect in rabbit knees. To that end, autogenic chondrocytes and periosteal cells were labeled with CM-DiI fluorochrome, seeded or cultured in PEGT/PBT scaffolds for periods up to 2 weeks, transferred into osteochondral defects, harvested 5 days postimplantation, and analyzed for cell viability. In order to further elucidate factors effecting cell viability within our model system, we investigated the effect of serum, 2) extracellular matrix surrounding implanted cells, 3) scaffold interconnectivity, and 4) hyaluronan, as a known cell protectant. Controls included scaffolds with devitalized cells and scaffolds analyzed at implantation. We found that the viability of periosteum cells (14%), but not of chondrocytes (65-95%), was significantly decreased after implantation. The addition of hyaluronan increased periostium cell viability to 44% (p < 0.05). Surprisingly, cell viability in less interconnected compression-molded scaffolds was higher compared to that of fully interconnected scaffolds produced by rapid prototyping. All other factors tested did not affect viability significantly. Our data suggest chondrocytes as a suitable cell source for cartilage repair in line with clinical data on several chondrocyte-based therapies. Although we did not test progenitor cells other the periosteum cells, tissue-engineering approaches using such cell types should take cell viability aspects into consideration.

The potential of porous poly(ether ester) scaffolds made from poly(ethylene glycol) terephthalate: poly(butylene terephthalate) (PEGT:PBT) block copolymers produced by various methods to enable cartilaginous tissue formation in vitro was studied. Scaffolds were fabricated by two different processes: paraffin templating (PT) and compression molding (CM). To determine whether PEGT:PBT scaffolds are able to support chondrogenesis, primary bovine chondrocytes were seeded within cylindrical scaffolds under dynamic seeding conditions. On day 3, constructs were transferred to six-well plates and evaluated for glycosaminoglycan (GAG) distribution (3, 10, and 24 days), type II collagen distribution, cellularity, and total collagen and GAG content (10 and 24 days). It was observed that better cell distribution during infiltration within PT scaffolds allowed greater chondrogenesis, and at later time points, than in CM scaffolds. The amount of GAG remained constant for all groups from 10 to 24 days, whereas collagen content increased significantly. These data suggest that PEGT:PBT scaffolds are suitable for cartilage tissue engineering, with the PT process enabling greater chondrogenesis than CM.

In this study in vitro and in vivo functions were compared between cultured dermal equivalents produced with human fibroblasts isolated either from papillary dermis or adipose tissue of the same donors. Papillary dermal fibroblasts had a normal spindle cell shape; in contrast, adipose tissue fibroblasts had a stellate cell shape, actin stress fibers containing alpha-smooth muscle actin, multiple narrow extensions at their edges, and longer focal adhesion plaques. After dynamic culture for 14 days in PEGT/PBT carrier scaffolds, cell numbers between the two cell sources were comparable, but tissue morphology was different between the cultured groups. In addition, papillary fibroblasts had deposited significantly more glycosaminoglycans (214 +/- 15 versus 159 +/- 21 microg, p < 0.001) and a lower amount of collagen (49 +/- 14 versus 111 +/- 25 microg of hydroxyproline, p < 0.001) than had adipose fibroblasts. Moreover, the latter constructs were significantly more contracted than the papillary fibroblast-cultured constructs (78 +/- 6 versus 96 +/- 3%, p < 0.001). In comparison with the influence of cultured dermal equivalents on wound healing, the transplantation of five groups (control acellular carrier, papillary fibroblast-seeded construct, adipose fibroblast-seeded construct, papillary fibroblast-cultured construct, and adipose fibroblast-cultured construct) to full-thickness wounds on the backs of athymic mice showed clear differences in angiogenesis and tissue ingrowth after 10 days, and in reepithelialization after 21 days. After 10 days, the level of vascular ingrowth in the carrier (von Willebrand staining) for the five groups was as follows: adipose fibroblast-cultured > papillary fibroblast-cultured = adipose fibroblast-seeded > papillary fibroblast-seeded > acellular carrier. After 21 days, only the acellular carriers were not vascularized and the papillary fibroblast-seeded constructs were not completely vascularized. Complete wound reepithelialization (92 +/- 12%) was observed only in the group treated with adipose cultured constructs. Wound contraction was not observed. Staining for HLA-ABC and alpha-smooth muscle actin showed that human fibroblasts had survived and that adipose fibroblasts continued to express the actin isoform. These results showed not only stimulation of skin repair when fibroblasts were present in the carrier, but also significant positive effects of the deposited extracellular matrix (ECM) in the carrier. In addition, the adipose fibroblast-seeded construct, and especially the adipose fibroblast-cultured construct, significantly stimulated angiogenesis and reepithelialization when compared with their corresponding papillary fibroblast constructs. Apparently, tissue source or fibroblast phenotype and the presence of ECM play a crucial role in the stimulation of (impaired) healing and engineering of dermal equivalents.

The engineering of dermal skin substitutes, using autologous fibroblasts, requires high seeding efficiencies, a homogeneous cell distribution in the scaffolds, and optimal culture conditions. Dynamic seeding in spinner flasks was used to seed and subsequently culture fibroblasts in three-dimensional scaffolds. Several seeding and culture variables were investigated. Simulation of medium movement with microspheres showed that three different regions existed in medium (outer, middle, and inner), where overall particle movement was different. In the middle region the flow was turbulent and scaffolds were best placed in this region. After fibroblast seeding, methylene blue staining and scanning electron microscopy analysis of the scaffolds showed that at a low stirring speed (20 rpm) fibroblasts attached mainly onto the upper part of the scaffold, and at 40 and 60 rpm fibroblasts attached and spread throughout the scaffolds. Measurements of total DNA content per scaffold showed that lower stirring speeds (20 and 40 rpm) resulted in significantly higher cell-seeding efficiencies (20 rpm, 99.8 +/- 11.3%; 40 rpm, 93.8 +/- 10.5%) compared with 60 rpm (85.9 +/- 5.3%). Seeding kinetics were comparable for all three speeds investigated. In subsequent studies, 40 rpm was chosen for seeding. Using initial cell numbers ranging from 0.3 x 10(6) to 1.5 x 10(6) fibroblasts per scaffold, seeding efficiencies higher than 85% were consistently found (n = 4). The culture of fibroblast-seeded scaffolds at different stirring speeds (10-80 rpm) showed that stirring speeds higher than 10 rpm significantly stimulated fibroblast proliferation and glycosaminoglycan and collagen deposition as compared with 10 rpm. After 21 days, scaffolds cultured at 80 rpm showed significantly more collagen deposition as compared with those maintained at lower speeds. In conclusion, to achieve high seeding efficiencies, uniform fibroblast distribution and tissue formation in a three-dimensional scaffold, fibroblasts can be dynamically seeded at 40 rpm and subsequently cultured at a stirring speed of 60-80 rpm in spinner flasks. This flexible system shows that it is feasible to tissue engineer autologous dermal substitutes in a clinically acceptable time frame.