Biomaterials Science Blog

Induced pluripotent stem (iPS) cells continue to hold great promise for cell transplantation therapies for numerous debilitating diseases. For instance, a patient’s skin cells can be “re-programmed” into iPS cells and converted into heart, liver, or bone cells (to name a few). These cells can then be used to regenerate specific tissues, and would theoretically bypass the patient’s immune response since the original cells come directly from the patient.

However, the scalability and growth of iPS cell in vitro continues to be a significant challenge. To address this problem, researchers at the University of Tokyo and École Polytechnique Fédérale de Lausanne have engineered bioactive hydrogel beads to significantly increase the expansion of iPS cells in vitro while maintaining their pluripotency.

Mouse iPS cells were readily encapsulated and expanded in alginate/polyethylene glycol (PEG) gel beads using an effective three-step protocol.  First, iPS cells were added to a precursor solution of functionalized PEG and alginate. The precursor solution was loaded into a linear extrusion vessel and allowed to drip into a calcium chloride bath to crosslink the alginate and form gel beads. Second, the maleimide-functionalized 4-armed PEG in the beads was further crosslinked with di-thiolated linear PEG using a Michael addition reaction. Finally, the fully-crosslinked beads were coated with poly(L-lysine) to prevent volumetric swelling of the beads, since swelling imparts harmful mechanical stress to the encapsulated cells during the culture process.

Interestingly, the encapsulated iPS cells were pluripotent after 8 days in culture (confirmed with Nanog expression and colony morphology). To make the beads “bioactive,” the PEG networks were functionalized with cell adhesion components (RGD peptide or E-cadherin) to enhance iPS cell expansion. Overall, increasing the concentration of RGD binding sites in the bead offers up to an 80% increase in iPS cell proliferation compared to non-bioactive bead controls.

Additional experiments demonstrate that the expression of pluripotency markers and primitive endoderm markers is a function of bead design. Notably, the pluripotency marker Rex1 was significantly more expressed in beads containing RGD or E-Cadherin compared to non-bioactive beads. Furthermore, endoderm markers such as Gata4 and HNF4 were down regulated in beads containing specific concentrations of RGD. These results support the conclusion that the design of the bioactive bead has direct effects on the pluripotency of encapsulated iPS cells.

Ultimately, bioactive hydrogel beads are a promising solution for increasing iPS cell expansion in vitro, and are well suited for use in future regenerative medicine applications.

Development of bioactive hydrogel capsules for the 3D expansion of pluripotent stem cells in bioreactors
Yoji Tabata, Ikki Horiguchi, Matthias P. Lutolf and Yasuyuki Sakai  
Biomater. Sci., 2014, Advance Article DOI: 10.1039/C3BM60183H

Brian Aguado is currently a Ph.D. Candidate and NSF Fellow in the Biomedical Engineering department at Northwestern University. He holds a B.S. degree in Biomechanical Engineering from Stanford University and a M.S. degree in Biomedical Engineering from Northwestern University. When he’s not in the lab, Brian enjoys traveling, cooking, swimming, and spending time with family and friends. Read more about Brian’s research publications here.

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Hickman et al. have developed methods to successfully differentiate human muscle progenitor cells into functional, multinucleated myofibers. 

The first step towards replacing muscle lost due to diseases such as muscular dystrophy is to build functional model systems of healthy muscle tissue.  In order to do this with human cells, human muscle progenitor cells must be removed from the body, cultured in a dish, and treated with appropriate signaling molecules in order to appropriately differentiate into functional muscle fibers, known as myotubes or myofibers.  Hickman et al. have done precisely this – the authors demonstrate that it is possible to differentiate muscle progenitor cells into human myofibers outside of the body.  These myofibers are also shown to contract both in response to external electrical stimulation as well as spontaneously.

The researchers developed a special media cocktail that allowed these myofibers to grow in culture over a period of more than two weeks.  Importantly, the media cocktail did not contain serum, an undefined extract from animal blood that is often used to keep grow cells in culture outside of the body.  This has important implications for eventual clinical translation.  The researchers found that their differentiation medium produced myofibers that contained organized sarcomeres (the internal cell machinery that allows fibers to contract and do work). Muscle contraction generally occurs through the uptake of calcium by the cells from their surroundings.  Calcium uptake requires the presence of two different types of calcium channels, DHPR and RyR.  The researchers generated myofibers that demonstrated the presence of both of these channels.  Finally, these myofibers also contracted on their own, as well as in response to an applied electrical potential. 

This research marks an important step forward in muscle development research, and can be found here.

Spontaneous contraction of human myotubes in culture

In vitro differentiation of functional human skeletal myotubes in a defined system
Xiufang Guo, Keshel Greene, Nesar Akanda, Alec S. T. Smith, Maria Stancescu, Stephen Lambert, Herman Vandenburgh and James J. Hickman  
Biomater. Sci., 2013, Advance Article  DOI: 10.1039/C3BM60166H

Debanti Sengupta recently completed her PhD in Chemistry from Stanford University.  She is currently a Siebel postdoctoral scholar at the University of California, Berkeley.  

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Bioactive ceramics are commonly used for the repair and replacement of damaged bone tissues.  However, there is still a limited understanding of how the inherent material properties of these ceramics can be used to predict their osteoinductive properties.  The development of models which relate these quantities could lead to the more efficient and intelligent design of materials that promote bone formation.

In a study that pushes this goal one step closer to reality, researchers from the University of Wisconsin, Madison, the University of Akron and the University of Michigan investigate the role which crystal structure plays in controlling the attachment and osteogenic differentiation of human mesenchymal stem cells (hMSCs).  The group studied hMSC viability and differentiation on two CaSiO₃ polymorphs: pseudowollastonite (psw) and wollastonite (wol).  As polymorphs, these materials have identical chemical compositions and stoichiometries, and can be fabricated to have nearly equivalent surface roughness.  The only significant difference between these two materials is their crystal structure.  While the wol polymorph has a stable, open silicate chain structure, the psw polymorph has a highly strained silicate ring structure. 

Interestingly, significant differences in hMSC growth and differentiation were observed, with the psw polymorph exhibiting superior osteoinductive properties.  Although the hMSCs cultured on psw initially exhibited poor viability, they were able to recover and, after 20 days, exhibited a statistically higher cell count than the hMSCs cultured on wol.  Fine-grained calcium phosphate precipitate developed on the surfaces of both polymorphs but only the psw polymorph exhibited polycrystalline calcite aggregates as well as nodular amorphous precipitates which were identified as CaP, or bone nodules.

The group attributed these differences in hMSC behavior to the different soluble factor release rates exhibited by the two materials.  Psw has a much higher initial dissolution rate than wol, presumably because its highly strained structure makes it more susceptible to hydrolysis.  This resulted in a large initial release of silicon atoms, which caused the initial low cell viability, and calcium atoms, which caused increased calcite and calcium phosphate formation.

Overall, the study proved that psw is more osteoinductive than wol, thereby showing that crystal structure can play an enormous role in determining the osteoconductive and osteoinductive properties of a bioceramic.  The collected data mark a promising step forward in the creation of a model that can successfully predict osteoinductivity based on a bioceramic’s inherent material properties.

Crystal structures of CaSiO3 polymorphs control growth and osteogenic differentiation of human mesenchymal stem cells on bioceramic surfaces
Nianli Zhang, James A. Molenda, Steven Mankoci, Xianfeng Zhou, William L. Murphy and Nita Sahai  
Biomater. Sci., 2013, 1, 1101-1110 DOI: 10.1039/C3BM60034C

Ellen Tworkoski is a guest web-writer for Biomaterials Science.  She is currently a second year Ph.D. student in the biomedical engineering department at Northwestern University (Evanston, IL, USA).

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Collagen has been widely used in scaffold fabrication protocols in the tissue-engineering field for decades, since the material is biocompatible and biodegradable. However, the structure of collagen molecules is often disrupted during various processing techniques (e.g. decellularization or electrospinning), which decreases the mechanical stiffness of the material. There is an urgent need to generate mechanically robust materials that integrate with the host tissue for soft tissue engineering applications.

To address this issue, Elliot Chaikof’s group at Harvard Medical School has taken a “bottom-up approach” to fabricate thick collagen mats without damaging the structure of collagen molecules. Collagen gels were cast in a rectangular mold and dried to generate a sheet of collagen approximately 15-40 microns thick. Using a layer-by-layer fabrication strategy, subsequent collagen layers were cast on top of the previous layer and dried to form 120 μm thick sheets. The casting method successfully preserves the collagen molecule microstructure, and allows for tunable mechanical properties over several orders of magnitude as a function of concentration, thickness, and number of layers.

After elegantly characterizing the material properties, the collagen mats were used in an in vivo model of ventral hernia repair. The mats were compared to commercially available Permacol^TM collagen sheets currently used for hernia repair. After surgically forming a hernia in the abdomen of rats, the collagen mats were sutured on the abdominal wall. After 3 months, the collagen mats showed significantly enhanced integration at the implant site over the Permacol^TM controls.

Vivek Kumar, lead author on the study, explains that the collagen mats were 65% thinner than the 1 mm thick Permacol^TM matrices – potentially accounting for the enhanced integration of the mat at the implant site. Even though the collagen mats were thinner than the commercially available collagen sheets, they were able to prevent re-herniation in all the rats tested due to the comparable mechanical properties of the collagen mat to the Permacol^TM matrices.

Additionally, the layer-by-layer stacking method could be used to incorporate specific drugs in between the layers (much like making a sandwich) to reduce the risk of re-herniation and stimulate new tissue formation. Kumar also claims that these collagen mats can be used for a wide array of applications, such as vascular tissue replacements, artificial skin, and dura mater replacements.

 Interestingly, Kumar also suggests the method he developed is scalable. “We have cast much larger gels and much smaller gels successfully,” Kumar says. “The fabrication scheme is amenable to a variety of other geometries such as tubes and other intricate mold designs. The simplicity of our approach suggests good potential for use in industry – where often times a simpler fabrication method is better.”

This innovative research shows a simple method to create mechanically robust collagen mats that integrate exceptionally well with the surrounding tissue, and competes with current commercially available materials.

Collagen-based substrates with tunable strength for soft tissue engineering
Vivek A. Kumar, Jeffrey M. Caves, Carolyn A. Haller, Erbin Dai, Liying Liu, Stephanie Grainger and Elliot L. Chaikof 
Biomater. Sci., 2013, 1, 1193-1202 DOI: 10.1039/C3BM60129C

Brian Aguado is currently a Ph.D. Candidate and NSF Fellow in the Biomedical Engineering department at Northwestern University. He holds a B.S. degree in Biomechanical Engineering from Stanford University and a M.S. degree in Biomedical Engineering from Northwestern University. When he’s not in the lab, Brian enjoys traveling, cooking, swimming, and spending time with family and friends. Read more about Brian’s research publications here.

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