1. Publishable summary
Bone is often considered to be a solid inert material, but in fact it is a dynamic tissue which is
constantly undergoing microfracturing and repair during every day loading. It is this innate ability to
undergo repair that allows bone to heal following fracture without the formation of scar tissue.
However, a Clinical problem arises when the size of the injury exceeds the healing capacity of the
bone. A normal fracture heals in 6-8 weeks1. However, in 5-13% of cases the bone does not heal
properly and the incidence of this increases if an open fracture occurs. When bone healing is delayed
it is called a delayed union, if however the bone does not heal it is considered a non-union which in
severe cases can lead to amputation. Another clinical problem occurs with critical sized bone defects
which result from the loss of a bone segment which exceeds the natural healing capacity of the
bone. These circumstances can result from a variety of incidents, such as: high speed trauma;
debridement of tissue following infection; or for pathological reasons, such as osteosarcoma
resections.
The treatment of choice for these ailments is bone grafting and an estimated 2.2 million such
procedures occur worldwide each year2. The gold standard bone graft uses autologous bone taken
from the patient’s own non-essential bone stock, typically the iliac crest. However, despite its
success rate and widespread use it has inherent disadvantages such as: the need for a second
surgery to harvest the bone, which has a high incidence of morbidity and increases the risk of
infection; limited bone stock, especially in patients suffering with osteoporosis or patients that have
already underwent a similar procedure3. To circumvent these issues allogeneic grafts are sometimes
used. However, they are less osteogenic than autologous bone grafts and they have an increased risk
of infection, or worse, disease transmission. Furthermore, any measure taken to reduce these risks
adversely affects the osteogenicity of the bone graft. Therefore, there is a clinical need to develop
off-the-shelf alternatives to bone grafts which tissue engineering aims to address 4.
Tissue engineering in its traditional sense involves harvesting the patient’s cells, combining these
cells with signalling molecules and adding this to a 3D matrix which is subsequently cultured for 1-2
weeks prior to re-implantation. However, if the need to use cells is removed by using combinations
of growth factors, the patient could be treated immediately in a single surgery, without leaving them
with a space filling implant needed while the cells grow to a sufficient number. Growth factor
delivery systems, should be designed such that the growth factor reaches the site of injury without
degradation or denaturing and remains there until it has fulfilled its function 4.
There are currently growth factor treatments available in the form of Medtronic’s Infuse® (Bone
morphogenetic protein (BMP)-2_and Stryker’s OP-1® (BMP-7). Both products come as a two part
system where the growth factor is reconstituted and mixed with the carrier collagen sponge
(Infuse®) or powder (OP-1®). This results in the growth factor being only loosely bound to the
scaffold from which it can then wash away intraoperatively. As the plasma half-life of BMP-2 is
approximately 10 minutes5, it is necessary to use supraphysiological doses to be effective. For
instance, BMP-4 in human serum is 0.5-1.5 ng/ml 6however OP-1 contains 3.3mg per treatment a
dose one millions times higher. It is also known that the BMPs are present in normal fracture repairs
throughout all phases of bone regeneration and remodelling and that growth factors are produced
1
Mckibbin, Biology of Fracture Healing in Long Bones. Journal of Bone and Joint Surgery-British Volume, 1978. 60(2): p. 150-162.
Calori, et al., The use of bone-graft substitutes in large bone defects: Any specific needs? Injury International Journal of the Care of the
Injured, 2011. 42: p. S56-S63.
3 Cancedda, R., P. Giannoni, and M. Mastrogiacomo, A tissue engineering approach to bone repair in large animal models and in clinical
practice. Biomaterials, 2007. 28(29): p. 4240-4250.
4
Evans. Advances in Regenerative Orthopedics. Mayo Clin Proc. 2013;88(11):1323-1339
5 Lieberman et al. The role of growth factors in the repair of bone. Biology and clinical applications. J Bone Joint Surg Am. 2002 Jun;84A(6):1032-44
6 Herrera B, Inman GJ. A rapid and sensitive bioassay for the simultaneous measurement of multiple bone morphogenetic proteins.
Identification and quantification of BMP4, BMP6 and BMP9 in bovine and human serum. BMC Cell Biol. 2009 Mar 19;10:20.
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in various combinations to facilitate fracture repair7. Hence it clear that work is needed in the
optimisation of the delivery device used as a carrier for these treatments.
There are numerous publications outlining techniques for the delivery of growth factors. However
the main limitations of these systems are the inability of the scaffold to contribute to load bearing,
which itself can lead to non-unions 8. Additionally, if these scaffolds are based on biodegradable
polymers like polylactic acid, they release acidic by-products during degradation which can adversely
affect surrounding tissue. Moveover, the degradation rate is often too slow to complement bone
repair.
In the current study, the load bearing of the scaffold has been enhanced utilising nanocomposite
technology which mimics natural bone. A composite is a material with at least two phases, which
when combined produce a material with characteristics different from the individual components.
The individual components, namely hydroxyapatite (HAp) and chitosan, remain separate and distinct
within the finished structure and loads are distributed to the HAp by the chitosan matrix that holds
the HAp particles together. Chitosan was used as a replacement for collagen, which is the polymer
phase of natural bone, as collagen is thought to affect the pharmacokinetics of growth factors 5.
Chitosan is a deacylated form of chitin which itself is an abundant naturally occurring polymer,
derived from the exoskeleton of crustaceans (and insects) and a waste product of the food industry.
Additionally it also has reported antimicrobial properties and is osteoconductive. To this scaffold
osteogenic and angiogenic growth factors have been bound to signal host cells to regenerate bone.
BMP-4 was selected as the osteogenic growth factor as it is expressed in all stages of bone healing
including remodelling. Vascular endothelial growth factor-A (VEGF-A) was selected as the angiogenic
growth factor, as it increases vascularity in bone, improves the healing of experimental non unions
and bone has an absolute requirement for avascular supply. Additionally, it has been shown that
BMP-4 and VEGF-A have a synergistic effect on bone healing9.
To create the novel osteogenic scaffold these components were mixed together with an initiator and
a crosslinking agent to give a solid scaffold ‘after curing’, which retained the growth factors within its
structure. Hence, the growth factors are retained in the scaffold and are released progressively as
the scaffold itself degrades. This in turn is controlled by the in vivo degradation of the chitosan. This
has reported to take from 2 to 8 weeks depending on the level of crosslinking. Once the crosslinking
had been optimised, the strength of the composite was maximised to yield Young’s modulus values
for the composite which were double those of native chitosan. Degradation studies illustrated that
samples retained their shape for over 10 weeks under static physiological conditions. The
cytocompatibility of the scaffold was assessed in vitro by co-culture with a pre-osteoblasic cell line
MC3T3-E1. From this analysis it was found that the scaffold did not have a cytotoxic effect on the
cells tested. When bovine serum albumin was added to the crosslinking reaction as a model protein
at a concentration of 400ug per 200mg of scaffold, it was found that between 25% and 100% of the
protein was released over a ten day period depending on the structure of the scaffold. However,
when low doses of BMP-4 and VEGF-A were added alone or in combination, it was found that the
scaffold retained almost all of the protein within its structure after a ten day release period.
The osteogenic potential of the scaffolds were tested in vivo using a rat defect model. In this model a
custom made HDPE plate was fixed in position using four threaded k-wires and a 5mm defect was
created using a 0.22mm Gligi saw. Initial results obtained from X-ray analysis of healing indicating
that the scaffold containing growth factors healed considerably better than those without.
Contact: Dr. Declan Devine, email: ddevine@ait.ie
7
Marsell & Einhorn. The role of endogenous bone morphogenetic proteins in normal skeletal repair. Injury. 2009; 40 Suppl 3:S4-7
King & Krebsbach. Growth factor delivery: How surface interactions modulate release in vitro
and in vivo. Advanced Drug Delivery Reviews 2012; 64: 1239–1256
9 Peng et al. Synergistic enhancement of bone formation and healing by stem cell-expressed VEGF and bone morphogenetic protein-4. J
Clin Invest. 2002 Sep; 110(6):751-9
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