Leaps and bones

Evidence of bone grafts date back 2,000 years to a single skull that was discovered in Peru. There is ongoing debate about its authenticity, but archaeologists agree the specimen shows evidence of an attempt to insert a gold plate. The first recorded medical use of a graft dates, however, from 1668. Job van Meekeren, a Dutch surgeon, is credited with using animal bone to repair the skull of an injured soldier. Fast forward 400 years, and bone grafting technology is ubiquitous in reconstructive orthopaedic surgery. Allografts (donated bone tissue) and autografts (samples of a patient’s own healthy bone tissue) are an everyday occurrence in hospitals. But they do have their disadvantages, not least the risk of infection, blood loss and nerve damage.

The “holy grail” in the field of artificial bone material is a composite that demonstrates an internal architecture capable of fully integrating with natural bone, while also stimulating the healing process as efficiently as an autologous graft. After decades of innovation in the fields of biochemistry and bioengineering, along with the assistance of 3D printing and computer aided design technology, that holy grail may soon be within reach. In order to reach this outcome and produce the formation of new bone – a process known as osteogenesis – a material must overcome two obstacles. It needs to be able to stimulate the body to attract, proliferate and differentiate mesenchymal stem cells or immature bone cells into osteoblasts to form healthy bone tissue (osteoinduction); and it needs a suitable scaffold for new bone to regrow (osteoconduction).

Materials used in bone replacement fall into two categories: inorganic ceramics and organic synthesised polymers – both of which have different roles. Biologically inert ceramics have star quality in helping build scaffolding for new bone formation. On the other hand, synthetic polymers like poly methyl acrylate, polyethyl acrylate (PEA) and polycaprolactone – a tough biodegradable, semicrystalline and hydrophobic polyester with biocompatibility – stimulate new bone structure using stem cells and growth factors cultivated three dimensionally.

The University of Glasgow has a long tradition in bone research and Manuel Salmeron-Sanchez, the chair of its Centre for the Cellular Microenvironment, says it’s a challenge designing a material that is both osteinductive and osteoconductive. “Ceramics are fine for osteoconducting bone, but they lack the bioactivity to generate bone formation,” he says. “They cannot properly differentiate stem cells into osteoblasts.” At the Centre, Salmeron-Sanchez has been developing a material that is both osteoinductive and osteoconductive. “In our lab, we have been able to combine synthetic material with the activity provided by growth factor by using a combination of PEA, fibronectin (FN) and bone morphogenetic protein 2 (BMP-2),” he explains. “Fibronectin is an adhesive glycoprotein that is crucial for tissue repair and for regulating cell motility and also in embryogenesis. BMP-2 is associated with the genetic coding mechanism and cell differentiation for bone and cartilage.”

Good vibrations

Perhaps the most fascinating part of the process is the use of nanovibrational stimulation technology (NST) on the three-dimensional culture of PEA FN and BMP- 2. Centred on the apparent ability of cells to react in their microenvironment through mechanotransduction systems, studies have shown that the growth and differentiation of stem cells can be controlled by NST – even in the absence of biochemical stimuli such as growth factors. “We find that low levels of BMP-2 bound to PEA+FN to allow local presentation of BMP-2 in a way that is highly bioactive yet does not compromise safety,” explains Salmeron-Sanchez. “Local BMP-2 triggers stem cells in the bone marrow to differentiate into osteoblasts, it is the trigger of the process of bone formation, and we’ve shown that it leads to bone regeneration. The late Adam Curtis, emeritus professor of cell biology at the University of Glasgow, first suggested the idea that nanovibration might kick-start activity at the cellular level, and it has been shown to be true in vitro.”

Original attempts were on 2D-layered culture plates and focused on vibrating single petri dishes with cells growing in monolayers. Rapid advances have given access to 3D skeletal tissue engineering that are bumped by the so-called “nanokick”. However, the vibration of culture plates and 3D hydrogels may in theory seem a simple task, but achieving consistent vibration transmission is problematic because creating the devices for inducing nanovibrations is a huge technical challenge.

A whole sector of engineering design has evolved to deliver “good vibrations”. Experiments under vibrational conditions of an amplitude of 25μm and frequencies of 20–60Hz have been tried in pursuit of the ideal frequency. In 2011, when these collaborations on nanovibration in biological systems began, the first detection of gravitational waves was still in the future. Since their discovery, these links between gravitational-wave research and biology have given huge impetus to the work being carried out in Glasgow, and has enabled Salmeron-Sanchez and his research team to reach the stage they’re at now. “We are currently working with a company based in the Basque region of Spain to manufacture PEA in a way that is compatible with medical devices and so that we can go to the regulators and apply for clinical trials,” he says, adding that he is hopeful for the start of trials in the next three years if the project can secure funding from the EU amid the post-Brexit politics.

Bone on demand

In Australia, research addressing the challenge of bone replacement is on a different track, using ceramic inks and 3D printing technology. By blending a ceramic ink that mimics bone architecture, scientists have found a way to create composites to replace sections of diseased or missing bone and encouraging existing structures to bind with the new artificial composite. The method, if successful, could lead to reduced pain and speedier recoveries. “Our method has the potential to radically change current practice, reducing patient suffering and ultimately saving lives,” says Kristopher Kilian, scientia associate professor and co-director of the Australian Centre for Nanomedicine, University of New South Wales. “It paves the way for numerous opportunities that could prove transformational.”

The method he speaks of is called ceramic omnidirectional bioprinting in cell-suspensions (COBICS). In layman’s terms, that means printing bone-like structures directly into cavities in bone. The bioink is made of calcium phosphate and hardens when placed in water, meaning to replace a lost bone using COBICS would be much faster than relying on the natural process of osteogenesis. The ability to 3D print bone-like structures isn’t the novel aspect of Kilian’s research however, it’s the in-vivo 3D printing application.

Still in its infancy, 3D bone printing has depended on designing and building the structure material outside of a patient’s body, relying on laboratory-based printers which are unable to operate at room temperature and need toxic chemicals to sterilise the new bone material. But using COBICS, which Kilian claims is the first method that can integrate living cells and operate at room temperature, he foresees a lightweight, portable 3D printer that can be taken into the operating rooms. “This opens up the opportunity for us to directly print a patient’s bone within a cavity during surgery,” he explains. “If proven, the system could apply for patients if they have some bone resected for disease, or if they have a tumour taken out. We can take scans of that bone, digitalise it for output to a printer and directly print within the cavity of a patient.”

Kilian and his fellow researchers at the university have tested the COBICS technique, producing “excellent performance” in a rabbit model when compared with materials used in the current standard of care. The next stage is testing in larger animal models and eventually starting the regulatory approval process to move toward conducting in-human trials. There’s also the fact that the bioink is currently manufactured in-house, which limits the scale of production. “Currently, we make the ‘ink’ ourselves, but there is a lack of assistance on the manufacturing side. Support from investors would help us move forward more rapidly,” says Kilian.

With both Kilian and Salmeron-Sanchez reliant on funding to continue the journey with their technologies, the holy grail remains out of reach, at least for patients. Kilian remains optimistic, however, for a better standard of care: “I imagine a day soon where a patient needing a bone graft can walk into a clinic and the anatomical structure of their bone is imaged on the computer, translated to a 3D printer, and directly printed into the cavity with their own cells.”