Jan de Boer

An algorithm-based topographical biomaterials library to instruct cell fate

It is increasingly recognized that material surface topography is able to evoke specific cellular responses, endowing materials with instructive properties that were formerly reserved for growth factors. This opens the window to improve upon, in a cost-effective manner, biological performance of any surface used in the human body. Unfortunately, the interplay between surface topographies and cell behavior is complex and still incompletely understood. Rational approaches to search for bioactive surfaces will therefore omit previously unperceived interactions. Hence, in the present study, we use mathematical algorithms to design nonbiased, random surface features and produce chips of poly(lactic acid) with 2,176 different topographies. With human mesenchymal stromal cells (hMSCs) grown on the chips and using high-content imaging, we reveal unique, formerly unknown, surface topographies that are able to induce MSC proliferation or osteogenic differentiation. Moreover, we correlate parameters of the mathematical algorithms to cellular responses, which yield novel design criteria for these particular parameters. In conclusion, we demonstrate that randomized libraries of surface topographies can be broadly applied to unravel the interplay between cells and surface topography and to find improved material surfaces.

Effects of Wnt signaling on proliferation and differentiation of human mesenchymal stem cells

Mesenchymal stem cells are pluripotent cells from bone marrow, which can be differentiated into the osteogenic, chondrogenic, and adipogenic lineages in vitro and are a source of cells in bone and cartilage tissue engineering. An improvement in current tissue-engineering protocols requires more detailed insight into the molecular cues that regulate the distinct steps of osteochondral differentiation. Because Wnt signaling has been widely implicated in mesenchymal differentiation, we analyzed the role of Wnt signaling in human mesenchymal stem cell (hMSC) biology by stimulation of the pathway with lithium chloride and Wnt3A-conditioned medium. We demonstrate a role for low levels of Wnt signaling in proliferation of uncommitted hMSCs and confirm that Wnt signaling controls osteoprogenitor proliferation. On the other hand, at high Wnt levels we observed a block in adipogenic differentiation and an increase in the expression of alkaline phosphatase, suggesting a role in the initiation of osteogenesis. The results of this study suggest that bone tissue engineering could benefit from the activation of critical levels of Wnt signaling at defined stages of differentiation. Moreover, our data suggest that hMSCs provide a valid in vitro model to study the role of Wnt signaling in mesenchymal biology.

The use of endothelial progenitor cells for prevascularized bone tissue engineering.

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.

Critical Steps toward a tissue-engineered cartilage implant using embryonic stem cells.

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.

Analysis of high-throughput screening reveals the effect of surface topographies on cellular morphology

Surface topographies of materials considerably impact cellular behavior as they have been shown to affect cell growth, provide cell guidance, and even induce cell differentiation. Consequently, for successful application in tissue engineering, the contact interface of biomaterials needs to be optimized to induce the required cell behavior. However, a rational design of biomaterial surfaces is severely hampered because knowledge is lacking on the underlying biological mechanisms. Therefore, we previously developed a high-throughput screening device (TopoChip) that measures cell responses to large libraries of parameterized topographical material surfaces. Here, we introduce a computational analysis of high-throughput materiome data to capture the relationship between the surface topographies of materials and cellular morphology. We apply robust statistical techniques to find surface topographies that best promote a certain specified cellular response. By augmenting surface screening with data-driven modeling, we determine which properties of the surface topographies influence the morphological properties of the cells. With this information, we build models that predict the cellular response to surface topographies that have not yet been measured. We analyze cellular morphology on 2176 surfaces, and find that the surface topography significantly affects various cellular properties, including the roundness and size of the nucleus, as well as the perimeter and orientation of the cells. Our learned models capture and accurately predict these relationships and reveal a spectrum of topographies that induce various levels of cellular morphologies. Taken together, this novel approach of high-throughput screening of materials and subsequent analysis opens up possibilities for a rational design of biomaterial surfaces.

A relativity concept in mesenchymal stromal cell manufacturing.

Mesenchymal stromal cells (MSCs) are being experimentally tested in several biological systems and clinical settings with the aim of verifying possible therapeutic effects for a variety of indications. MSCs are also known to be heterogeneous populations, with phenotypic and functional features that depend heavily on the individual donor, the harvest site, and the culture conditions. In the context of this multidimensional complexity, a recurrent question is whether it is feasible to produce MSC batches as standard therapeutics, possibly within scalable manufacturing systems. Here, we provide a short overview of the literature on different culture methods for MSCs, including those employing innovative technologies, and of some typically assessed functional features (e.g., growth, senescence, genomic stability, clonogenicity, etc.). We then offer our perspective of a roadmap on how to identify and refine manufacturing systems for MSCs intended for specific clinical indications. We submit that the vision of producing MSCs according to a unique standard, although commercially attractive, cannot yet be scientifically substantiated. Instead, efforts should be concentrated on standardizing methods for characterization of MSCs generated by different groups, possibly covering a vast gamut of functionalities. Such assessments, combined with hypotheses on the therapeutic mode of action and associated clinical data, should ultimately allow definition of in-process controls and measurable release criteria for MSC manufacturing. These will have to be validated as predictive of potency in suitable pre-clinical models and of therapeutic efficacy in patients.

Differential bone-forming capacity of osteogenic cells from either embryonic stem cells or bone marrow-derived mesenchymal stem cells

For more than a decade, human mesenchymal stem cells (hMSCs) have been used in bone tissue‐engineering research. More recently some of the focus in this field has shifted towards the use of embryonic stem cells. While it is well known that hMSCs are able to form bone when implanted subcutaneously in immune‐deficient mice, the osteogenic potential of embryonic stem cells has been mainly assessed in vitro. Therefore, we performed a series of studies to compare the in vitro and in vivo osteogenic capacities of human and mouse embryonic stem cells to those of hMSCs. Embryonic and mesenchymal stem cells showed all characteristic signs of osteogenic differentiation in vitro when cultured in osteogenic medium, including the deposition of a mineralized matrix and expression of genes involved in osteogenic differentiation. As such, based on the in vitro results, osteogenic ES cells could not be discriminated from osteogenic hMSCs. Nevertheless, although osteogenic hMSCs formed bone upon implantation, osteogenic cells derived from both human and mouse embryonic stem cells did not form functional bone, indicated by absence of osteocytes, bone marrow and lamellar bone. Although embryonic stem cells show all signs of osteogenic differentiation in vitro, it appears that, in contrast to mesenchymal stem cells, they do not possess the ability to form bone in vivo when a similar culture method and osteogenic differentiation protocol was applied. Copyright

In-depth clinico-pathological examination of RNA foci in a large cohort of C9ORF72 expansion carriers

A growing body of evidence suggests that a loss of chromosome 9 open reading frame 72 (C9ORF72) expression, formation of dipeptide-repeat proteins, and generation of RNA foci contribute to disease pathogenesis in amyotrophic lateral sclerosis and frontotemporal dementia. Although the levels of C9ORF72 transcripts and dipeptide-repeat proteins have already been examined thoroughly, much remains unknown about the role of RNA foci in C9ORF72-linked diseases. As such, we performed a comprehensive RNA foci study in an extensive pathological cohort of C9ORF72 expansion carriers (nxa0=xa063). We evaluated two brain regions using a newly developed computer-automated pipeline allowing recognition of cell nuclei and RNA foci (sense and antisense) supplemented by manual counting. In the frontal cortex, the percentage of cells with sense or antisense RNA foci was 26 or 12%, respectively. In the cerebellum, 23% of granule cells contained sense RNA foci and 1% antisense RNA foci. Interestingly, the highest percentage of cells with RNA foci was observed in cerebellar Purkinje cells (~70%). In general, more cells contained sense RNA foci than antisense RNA foci; however, when antisense RNA foci were present, they were usually more abundant. We also observed that an increase in the percentage of cells with antisense RNA foci was associated with a delayed age at onset in the frontal cortex (rxa0=xa00.43, pxa0=xa00.003), whereas no other associations with clinico-pathological features were seen. Importantly, our large-scale study is the first to provide conclusive evidence that RNA foci are not the determining factor of the clinico-pathological variability observed in C9ORF72 expansion carriers and it emphasizes that the distribution of RNA foci does not follow the pattern of neurodegeneration, stressing the complex interplay between different aspects of C9ORF72-related diseases.

Stepping into the omics era: Opportunities and challenges for biomaterials science and engineering.

UNLABELLEDnThe research paradigm in biomaterials science and engineering is evolving from using low-throughput and iterative experimental designs towards high-throughput experimental designs for materials optimization and the evaluation of materials properties. Computational science plays an important role in this transition. With the emergence of the omics approach in the biomaterials field, referred to as materiomics, high-throughput approaches hold the promise of tackling the complexity of materials and understanding correlations between material properties and their effects on complex biological systems. The intrinsic complexity of biological systems is an important factor that is often oversimplified when characterizing biological responses to materials and establishing property-activity relationships. Indeed, in vitro tests designed to predict in vivo performance of a given biomaterial are largely lacking as we are not able to capture the biological complexity of whole tissues in an in vitro model. In this opinion paper, we explain how we reached our opinion that converging genomics and materiomics into a new field would enable a significant acceleration of the development of new and improved medical devices. The use of computational modeling to correlate high-throughput gene expression profiling with high throughput combinatorial material design strategies would add power to the analysis of biological effects induced by material properties. We believe that this extra layer of complexity on top of high-throughput material experimentation is necessary to tackle the biological complexity and further advance the biomaterials field.nnnSTATEMENT OF SIGNIFICANCEnIn this opinion paper, we postulate that converging genomics and materiomics into a new field would enable a significant acceleration of the development of new and improved medical devices. The use of computational modeling to correlate high-throughput gene expression profiling with high throughput combinatorial material design strategies would add power to the analysis of biological effects induced by material properties. We believe that this extra layer of complexity on top of high-throughput material experimentation is necessary to tackle the biological complexity and further advance the biomaterials field.

Stimulatory effect of cobalt ions incorporated into calcium phosphate coatings on neovascularization in an in vivo intramuscular model in goats

UNLABELLEDnRapid vascularization of bone graft substitutes upon implantation is one of the most important challenges to overcome in order to achieve successful regeneration of large, critical-size bone defects. One strategy for stimulating vascularization during the regeneration process is to create a hypoxic microenvironment by either directly lowering the local oxygen tension, or by applying hypoxia-mimicking factors. Cells compensate for the hypoxic condition by releasing angiogenic factors leading to new blood vessel formation. In the present study, we explored the potential of cobalt ions (Co(2+)), known chemical mimickers of hypoxia, to stimulate vascularization within a bone graft substitute in vivo. To this end, Co(2+) ions were incorporated into calcium phosphate (CaPs) coatings deposited on poly(lactic acid) (PLA) particles with their effect on the formation of new blood vessels studied upon intramuscular implantation in goats. PLA particles and CaP-coated particles without Co(2+) ions served as controls. Pathological scoring of the inflammatory response following a 12-week implantation period showed no significant differences between the four types of materials. Based on histological and immunohistochemical analyses, both blood vessel area and number of blood vessels in CaP-coated PLA particles containing Co(2+) were higher than in the uncoated PLA particles and CaP-coated PLA particles without Co(2+). Analysis of blood vessel size distribution indicated abundant formation of small blood vessels in all the samples, while large blood vessels were predominantly found in PLA particles coated with CaP containing Co(2+) ions. The results of this study support the use of CaPs containing Co(2+) ions to enhance vascularization in vivo.nnnSTATEMENT OF SIGNIFICANCEnIn this work, we have investigated the potential of cobalt ions, incorporated into thin calcium phosphate (CaP) coatings that were deposited on particles of poly(lactic acid) (PLA), to induce neovascularization in vivo. Qualitative and quantitative histological and immunohistochemical analyses showed that both the number of blood vessels and the total blood vessel area were higher in CaP-coated PLA particles containing cobalt ions as compared to the uncoated PLA particles and CaP-coated PLA particles without the metallic additive. Furthermore, a wider distribution of blood vessel sizes, varying from very small to large vessels was specifically observed in samples containing cobalt ions. This in vivo study will significantly contribute to the existing knowledge on the use of bioinorganics, which are simple and inexpensive inorganic factors that can be used to control relevant biological process during tissue regeneration, such as vascularization. As such, we are convinced that this manuscript will be of interest to the readers of Acta Biomaterialia.