Dr Manus Biggs, lecturer within the Department of Biomedical Engineering and Investigator at CÚRAM, the Science Foundation Ireland Centre for Research in Medical Devices at NUI Galway, has just published two separate research papers on the role of nanomechanical cues in cell biology in top-tier international journals, one in the Nature journal Nature Biomedical Engineering and another in the prestigious materials journal, Advanced Materials.

Both research papers by Dr Biggs describe advances made in elucidating the roles of nanomechanical processes in directing skeletal stem-cell function. Advances in nanofabrication processes, bioreactor design and laser interferometry have created unique opportunities to fabricate tissues in the laboratory from combinations of engineered extracellular matrices (scaffolds), cells and biologically relevant stimulation or cues. Specifically, Dr Biggs’ research has a focus on fabricating biomimetic environments and conditions with nanoscale resolution to drive cellular differentiation in vitro.

Growing 3D living bone grafts in the lab

In the study published in Nature Biomedical Engineering, the researchers from the University of Glasgow, the University of Strathclyde, and NUI Galway describe how they have used nanoscale vibrations or 'nanokicks' to grow 3D living bone grafts in the laboratory. Ordinarily, stem cells reside in a niche environment within the bone marrow and have the potential to differentiate into a range of specialised cell types, responsible for generating bone, cartilage, ligament, tendon and muscle tissues. The team began research into employing high frequency, low-amplitude vibrations to drive osteogenesis back in 2009, observing that skeletal stem cells cultured on vibration plates would differentiate into osteoblast and begin to form bone tissue. Subsequent research identified that the optimal vibrational kicks for the production of bone tissue were about 20 nanometers in amplitude with a frequency of 1kHz. The development of the nanokicking technology employed tools borrowed from sophisticated laser interferometer systems built for gravitational wave detection of astrophysical objects, to grow three-dimensional constructs of mineralised bone in the laboratory for the first time. These 3D living-bone grafts resemble a sort of bone putty and, when implanted into patients in the future, will be able to repair or replace damaged sections of bone with a focus on treating bone loss due to trauma.

Critically, by using a patient's own skeletal cells, surgeons will be able to negate problems associated with rejection and can bridge larger bone defects. “After blood, bone is the most transplanted tissue used in patients in the form of bone graft. Autologous graft [bone grafts taken from the patient’s own body and commonly employed for the treatments of bone cancer, trauma or infection] is in short supply and can be associated with pain and donor site morbidity. Tissue engineered, bone-like graft would help meet this clinical demand as well as provide researchers with a potential tissue model for drug screening,” Dr Biggs explained. This collaborative research has shown, for the first time, that high-frequency vibrations of nanoscale amplitude alone can be used to differentiate patient-derived stem cells, to form mineralised tissue in 3D. To achieve this, a totally new genre of vibrational bioreactor was designed and developed. Using this bioreactor, it was demonstrated that nanoscale mechano-transduction is independent of other environmental factors, such as matrix rigidity. By doing this, the teams from Glasgow and Galway have provided a scalable pathway to control the differentiation of stem cells to bone cells across a broad range of applications.

Nanoscale mechanical phenomena and stem-cell differentiation

In his second study, which has been published in Advanced Materials, Dr Biggs and his team collaborated with Prof Shalom Wind at Columbia University to further probe the roles of nanoscale mechanical phenomena on stem cell differentiation. Speaking about the study, Dr Biggs said: “Recent studies indicate that stem cells are highly responsive to nanoscale external cues and undergo differentiation to a specific tissue lineage when subjected to physicomechanical conditions which mimic those of tissue. A well-described phenomenon is that of rigidity-driven differentiation, whereby a cell with multipotent capability will adopt the phenotype of a specialised cell type, if exposed to the mechanical conditions of that cell type's tissue. "For example, the compressive modulus of bone osteoid is within the kPa range and a skeletal stem-cell grown in the lab within a rigid matrix with a compressive modulus in this range will spontaneously differentiate into a specialised bone cell. But although tissues are associated with generalised bulk rigidity values, at the sub-cellular level, and particularly at the micro- and nanoscale levels, tissues are comprised of multiple elements [such as fibres, cells and crystals] with widely differing rigidity.”

To address this observation, Dr Biggs and his team investigated whether the use of electron-beam patterning could alter the rigidity of a viscoelastic PDMS substrate at discrete regions, thereby enabling the development of a new class of 2D substrates possessing patterned features of controlled rigidity, with dimensions ranging from the micron to the nanoscale. Electron-beam patterning allows for the fabrication of devices with nanoscale resolution and has been used extensively in the microelectronics industry for the production of integrated circuits or microchips. In this work, the team showed for the first time that direct-write electron-beam exposure can significantly alter the rigidity of an elastomeric material, increasing the compressive modulus of the material by up to four orders of magnitude. In order to assess the response of human mesenchymal stem cells to electron-beam patterned heterogeneous rigidity, the team created viscoelastic PDMS substrates patterned with discrete spots with dimensions ranging from 100-2000 nm and with elastic moduli ranging from 35 kPa up to 350 MPa. These studies showed that rigidity patterning could significantly affect how skeletal stem-cell populations adhere to a 2D surface (i.e. a polymeric implant) through the modulation of cellular focal adhesion formation (specialised supramolecular protein structures that intimately couple a cell to a substrate). Furthermore, these heterogeneous rigidity materials significantly influenced osteochondral lineage commitment as a function of both feature diameter and rigidity, driving skeletal stem-cells to the bone and cartilage lineages.

Bioreactor design and biomimetic nanomaterial formulations

In combination, these studies will establish the groundwork for a new generation of bioreactor design and biomimetic nanobiomaterial formulations. Tissue engineering and regenerative medicine is a key area of research within the Biggs lab and at CÚRAM, with a goal of finding solutions to chronic health problems and addressing unmet medical need. Ultimately, the use of these technologies to develop clinically translatable reparative and regenerative approaches to chronic illnesses is a major goal and, in conjunction with musculoskeletal research directions, future studies at the Biggs lab are focusing on elucidating the roles of nanoscale physicomechanical cues on differential processes in neural systems, and specifically exploring how these processes can be employed in driving regenerative processes within the central nervous system.

To read the Advanced Materials paper in full, visit: http://onlinelibrary.wiley.com/doi/10.1002/adma.201702119/full To read the Nature Biomedical Engineering paper in full, visit: http://rdcu.be/vMwt See www.curamdevices.ie for more information or follow CÚRAM on Twitter @curamdevices.