Bone healers

Calcium phosphates are the major class of compounds for synthetic bone substitution materials, not surprisingly as they are the principal component of bone. Matthias Epple’s team in the department of inorganic chemistry at the University of Duisburg-Essen, Germany, for example, has developed a nanoparticle paste that mimics the bone-forming action of calcium phosphate.

Genes for two growth factors are encapsulated within the synthetic calcium phosphate nanoparticles: bone morphogenetic protein 7 (BMP-7), which stimulates bone-forming cells; and vascular endothelial growth factor (VEGF), which induces the growth of blood vessels for bone-cell nutrition. The idea is that, once injected, surrounding cells take up the nanoparticles, and acidic conditions in the cells dissolve the calcium phosphate, releasing the genes and inducing the synthesis of proteins that accelerate bone growth.

In laboratory tests on three different cell types, Epple reports that the growth factors BMP-7 and VEGF-A were expressed in cells.² In further studies, the team studied the paste in bone defects in rabbit models. Although results have not yet been published, Epple says: ‘The results are promising. We can deduce that the paste promotes bone in-growth to a large extent. After 12 weeks, the defect was partially filled. I would assume that it will take three-12 months for the defect to be filled, depending on the size of the defect, the age of the patient and the location of the bone defect.’

Epple believes the nanoparticle paste could work well for bone defects after trauma or tumour extraction. It might also be used to build up bone around an endoprosthesis, a device used to replace a missing body part that is placed inside the body.

The success of any bone graft depends on the ability of a surrounding scaffold to assist the natural healing process. Researchers often treat the scaffold with growth factors. Two of the most important bone growth factors are platelet-derived growth factor (PDGF) and bone morphogenetic protein 2 (BMP-2). PDGF is released immediately following a bone injury, such as a fracture. Then other factors, including BMP-2, help to create the right environment for bone regeneration by recruiting cells that can produce bone and forming a supportive structure, including blood vessels.

However, if very large quantities of growth factors are delivered too quickly, they are rapidly cleared from the treatment site so they have reduced impact on tissue repair, and can also induce unwanted side effects. ‘You want to recruit the native adult stem cells we have in our bone marrow to go to the site of injury and then generate bone around the scaffold, and you want to generate a vascular system to go with it,’ says Paula Hammond of Massachusetts Institute of Technology’s (MIT) in Boston, US. This process, she explains, takes time, so ideally the growth factors should be released slowly  in nano or microgram quantities, over several days or weeks.

To achieve this, the MIT team created a 0.1mm thick, porous scaffold sheet made from biodegradable poly(lactide-co-glycolide) (PLGA), a polymer that is widely used in medical treatment and can be tuned to disintegrate at a specific rate. The researchers coated the sheet with about 40 layers of BMP-2, followed by 40 layers of PDGF. This allowed PDGF to be released more quickly, along with a more sustained BMP-2 release, mimicking aspects of natural healing.

The researchers tested the scaffold in rats with a skull defect 8mm in diameter.³ Once the scaffold was implanted, growth factors were released at different rates. PDGF, released during the first few days after implantation, helped initiate the wound-healing cascade and mobilise different precursor cells to the site of the wound. BMP, released more slowly, then induced some of these immature cells to become osteoblasts, which produce bone. When both growth factors were used together, these cells generated a layer of bone, just two weeks after surgery, that was indistinguishable from natural bone in its appearance and mechanical properties, the researchers say.

‘Using this combination allows us to not only have accelerated proliferation first, but also facilitates laying down some vascular tissue, which provides a route for both the stem cells and the precursor osteoblasts and other players to get in and do their jobs,’ says Hammond. ‘You end up with a very uniform healed system.’ She suggests that patients with severe bone injuries, such as soldiers wounded in battle, or patients suffering from congenital bone defects, could benefit from the new tissue scaffold.

Richard Oreffo, professor of musculoskeletal science at the University of Southampton, UK, also works with plastic scaffold materials in the drive to generate what he calls ‘living bone composites’ for patient application. In collaboration with Mark Bradley, professor of chemistry at the University of Edinburgh, UK, he has developed a lightweight material with a honeycomb structure that allows blood to flow through it, enabling stem cells from the patient’s bone marrow to attach to the material and grow new bone. Over time, the plastic slowly degrades as the implant is replaced by newly grown bone.

Bradley’s lab made and tested hundreds of candidate materials – mixtures of natural and synthetic polymers – and selected those able to support cell and bone growth.⁴ The team found that several of the blends provided excellent support for human bone marrow-derived skeletal cells and foetal skeletal cells. The best blend – poly (L-lactic acid/poly (vinyl acetate)/ poly(ε-caprolactone) – had an architecture like bone and produced promising results in mice, which was very exciting, says Oreffo.

In collaboration with professors Matt Dalby and Nikolaj Gadegaard at the University of Glasgow, UK, Oreffo has recently shown how two types of stem cells – cultured human embryonic stem cells and mesenchymal stem cells from the bone marrow of adults – grown on plastic surfaces can be manipulated to turn into bone cells, without any chemical intervention.⁵ Disorder in the embossed square patterns on the plastic – the holes were about 100nm deep and 120nm in diameter – helped mesenchymal stem cells to change into bone cells, he says, while ordered square patterns helped retain the stem cell properties.

‘Our research may offer a whole new approach to skeletal regenerative medicine,’ Oreffo comments. ‘The use of nanotopographical patterns offers approaches in new cell culture designs, innovative device designs, and could herald the development of new bone repair therapies as well as further human stem cell research.’

Oreffo is now thinking about how to incorporate nanotopography into titanium bone implants. In May 2014, he was involved in Southampton General Hospital’s first hip surgery with a 3D printed implant, made from titanium, and a bone stem cell graft. The patient is showing very good bone in-growth after 12 months, he reports. Four other patients have since received similar implants.

The implant provides a new socket for the ball of the femur bone to enter. In between the implant and the pelvis, the surgeons inserted a graft or bone scaffold containing bone stem cells; the graft acts as a filler for the loss of bone. The patient’s own bone marrow cells are in the graft to provide a source of bone stem cells to encourage bone regeneration behind and around the implant. Stem cells from the bone marrow should attach to the scaffold material and grow new bone to support the implant, creating a living bone composite, Oreffo explains.

‘The 3D printing of the implant in titanium, from CT scans of the patient and stem cell graft offers the possibility of improved outcomes for patients,’ says Oreffo. ‘Growing bone at the point of injury together with a hip implant designed to the exact fit of the patient offers real opportunities for improved patient recovery and quality of life. In essence, we have a bone graft material with excellent biocompatibility and strength and that will fill the defect behind the bone well. The stem cells allow growth and the construct to fuse together.’ Doctors at Southampton General Hospital believe this is a game changer. Douglas Dunlop, consultant orthopaedic surgeon,  says: ‘The benefits to the patient through this pioneering procedure are numerous. The titanium used to make the hip is durable and has been printed to match the patient’s exact measurements – this should improve fit and could reduce the risk of having to have further surgery.’

The next step, says Oreffo, is to use 3D printing to generate patient stem cells within the scaffolds, thus providing greater control over where cells are placed and located. Oreffo comments: ‘3D printing has the potential to significantly change some of our approaches to skeletal tissue repair. We seek to harness stem cells and appropriate smart biocompatible materials to create living bone composites for orthopaedic application. A key factor in these materials is how well they incorporate into the body.’

However, working with stem cells can be challenging. When they are used to regenerate bone tissue, many migrate away from the repair site, which disrupts the healing process. A research team at the University of Rochester, US, has, however, found a way to keep the stem cells in place by encasing them in hydrogels that are designed to degrade and disappear when their work is done.

The team, led by Danielle Benoit, assistant professor of biomedical engineering, coated the donor bone fragments  with the hydrogels, which contained fluorescently labelled stem cells, and implanted them into defects in mouse bone, both in vivo and in vitro.⁶ The researchers monitored the repair process using the fluorescence signals and found that there was no measurable difference between the concentrations of stem cells in the various samples, despite the fact that the in vivo sample was part of a dynamic environment – which included enzymes and blood flow – making it easier for the stem cells to migrate away from the target site. Thus virtually all the stem cells stayed in place to complete their work in generating new bone tissue.

Benoit’s team was able to manipulate the time taken for the hydrogels to dissolve by introducing groups of atoms within the polymer molecules ‘Some types of tissue repair take more time to heal than do others,’ says Benoit. ‘Our success opens the door for many, and more complicated, types of bone repair.’

1 http://corpcom.frost.com/forms/EU-PR_AZanch_D68D_TI_10Jul15

2 M. Epple et al, RSC Advances, 2013; doi: 10.1039/C3RA23450A

3 P. T. Hammond et al, PNAS, 2014; doi: 10.1073/pnas. 1408035111

4 M. Bradley et al, Advanced Functional Materials, 2013; doi: 10.1002/adfm.201202710

5 R. O. Oreffo et al, Small, 2013; doi: 10-1002/smll.201202340

6 D. S. W. Benoit et al, Acta Biomaterialia, 2014; DOI: 10.1016/j.actbio.2014.04.012