Final Report Summary - NANOFACT (DEVELOPMENT OF BIOACTIVE NANOCOMPOSITES FOR BONE TISSUE ENGINEERING APPLICATIONS)
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 weeks . 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 year . 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 procedure . 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.
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 minutes , it is necessary to use supraphysiological doses to be effective. For instance, BMP-4 in human serum is 0.5-1.5 ng/ml however 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 in various combinations to facilitate fracture repair . Hence it clear that work is needed in the optimisation of the delivery device used as a carrier for these treatments.
In the current study, a novel technology has been developed which consists of a biomimetic composite scaffold which covalently binds growth factors within its structure has been developed. The main components of the scaffold are hydroxyapatite (HAp) which is similar to the mineral component of natural bone and chitosan (CS) a natural polymer. CS was used as a replacement for collagen which is the organic component of bone, as collagen is thought to affect the pharmacokinetics of growth factors 5. Additionally it has been reported that CS has antimicrobial properties and is osteoconductive. To this scaffold osteogenic and angiogenic growth factors have covalently linked using a photocrosslinking method 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 healing.
Using this method, 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. 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. Post mortem analysis of healing indicated that there was significantly more bone in the defect of scaffolds treated with growth factor containing scaffolds compared to scaffold alone. However, histological analysis indicated that the implanted scaffold remained intact for the duration of the in vivo experiment which prevented complete bone bridging inside the defect.
To overcome these issues poly-vinyl alcohol (PVA) and poly-acrylic acid (PAA) blends were utilised as a polymer matrix and were combined with a ceramic materials whose composition consists of βTCP, Wollastonite and Magnesium silicate. This ceramic material was dispersed in the polymer matrix which was solubilised with water and the final 3D construct was physically crosslinked using various freeze thawing (F/T) cycles. The resultant composite materials were characterization by FTIR, SEM, swelling studies, compression testing and rheometry. The antimicrobial activity of the scaffold containing ciprofloxacin was tested against a pathogen associated with osteomyelitis.
FTIR and SEM analysis illustrated that the ceramic was dispersed within the composite and improved the hydrogen bonds of these hydrogels. Morphologically, the structure of the composite was porous, with the porosity dependent on the ceramic content and number of freeze thaw cycles. Swelling studies in buffer solution pH 7.4 showed an increase in polymer swelling when ceramic was added. However, rheological testing demonstrated that the incorporation of the ceramic caused an increase in mechanical properties. These results indicate an increase in intramolecular bonding based on the concentration of the ceramics and number of F/T cycles. DSC thermograms showed increase in Tg values for the samples containing ceramics indicating a stiffening of the polymeric backbone chain. Antimicrobial activity tests indicated that ciprofloxacin released from the composite actively created a zone of inhibition surrounding the composite samples.
The combination of increased strength and ability to encapsulate a clinically relevant pharmaceutical ingredients, indicate that the composites developed in this work programme have potential for the treatment of osteomyelitis.
Contact: Dr. Declan Devine, email: ddevine@ait.ie
The treatment of choice for these ailments is bone grafting and an estimated 2.2 million such procedures occur worldwide each year . 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 procedure . 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.
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 minutes , it is necessary to use supraphysiological doses to be effective. For instance, BMP-4 in human serum is 0.5-1.5 ng/ml however 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 in various combinations to facilitate fracture repair . Hence it clear that work is needed in the optimisation of the delivery device used as a carrier for these treatments.
In the current study, a novel technology has been developed which consists of a biomimetic composite scaffold which covalently binds growth factors within its structure has been developed. The main components of the scaffold are hydroxyapatite (HAp) which is similar to the mineral component of natural bone and chitosan (CS) a natural polymer. CS was used as a replacement for collagen which is the organic component of bone, as collagen is thought to affect the pharmacokinetics of growth factors 5. Additionally it has been reported that CS has antimicrobial properties and is osteoconductive. To this scaffold osteogenic and angiogenic growth factors have covalently linked using a photocrosslinking method 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 healing.
Using this method, 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. 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. Post mortem analysis of healing indicated that there was significantly more bone in the defect of scaffolds treated with growth factor containing scaffolds compared to scaffold alone. However, histological analysis indicated that the implanted scaffold remained intact for the duration of the in vivo experiment which prevented complete bone bridging inside the defect.
To overcome these issues poly-vinyl alcohol (PVA) and poly-acrylic acid (PAA) blends were utilised as a polymer matrix and were combined with a ceramic materials whose composition consists of βTCP, Wollastonite and Magnesium silicate. This ceramic material was dispersed in the polymer matrix which was solubilised with water and the final 3D construct was physically crosslinked using various freeze thawing (F/T) cycles. The resultant composite materials were characterization by FTIR, SEM, swelling studies, compression testing and rheometry. The antimicrobial activity of the scaffold containing ciprofloxacin was tested against a pathogen associated with osteomyelitis.
FTIR and SEM analysis illustrated that the ceramic was dispersed within the composite and improved the hydrogen bonds of these hydrogels. Morphologically, the structure of the composite was porous, with the porosity dependent on the ceramic content and number of freeze thaw cycles. Swelling studies in buffer solution pH 7.4 showed an increase in polymer swelling when ceramic was added. However, rheological testing demonstrated that the incorporation of the ceramic caused an increase in mechanical properties. These results indicate an increase in intramolecular bonding based on the concentration of the ceramics and number of F/T cycles. DSC thermograms showed increase in Tg values for the samples containing ceramics indicating a stiffening of the polymeric backbone chain. Antimicrobial activity tests indicated that ciprofloxacin released from the composite actively created a zone of inhibition surrounding the composite samples.
The combination of increased strength and ability to encapsulate a clinically relevant pharmaceutical ingredients, indicate that the composites developed in this work programme have potential for the treatment of osteomyelitis.
Contact: Dr. Declan Devine, email: ddevine@ait.ie