JV Lobato1, 2, 3, N Sooraj Hussain4, 5, CM Botelho4,5, AC Maurício2, 3, A Afonso6, N Ali7, JD Santos4, 5
1
CHVNG – Serviço de Estomatologia, Centro Hospitalar de Vila Nova de Gaia, Portugal
2
ICBAS - Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Largo Professor Abel Salazar, 2, 4099-003 Porto, Portugal
3
CECA/ICETA - Centro de Estudos de Ciência Animal, Instituto de Ciências e Tecnologias Agrárias e Agro-Alimentares, Campus Agrário de Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, Portugal
4
INEB - Instituto de Engenharia Biomédica, Laboratório de Biomateriais, Rua Campo Alegre, 823, 4150 -180, Porto, Portugal
5
FEUP – Faculdade de Engenharia da Universidade do Porto, DEMM, Rua Dr. Roberto Frias, 4200 - 465 Porto, Portugal
6
FMDUP – Faculdade de Medicina Dentária da Universidade do Porto
7
Department of Mechanical Engineering, University of Aveiro, Aveiro, 3812-000 Portugal
Abstract
Synthetic bone grafts have been developed to provide an alternative to autografts and allografts. Bonelike® is a patented synthetic osteoconductive bone graft that mimics the mineral composition of natural bone. In the present preliminary animals studies a user- friendly version of synthetic bone graft Bonelike® have been developed by using a resorbable matrix, FloSeal®, as a vehicle and raloxifene hydrochloride as a therapeutic molecule, that is known to decrease osteoclast activity and therefore enhanced bone formation. From histological and scanning electron microscopy evaluations, the use of Bonelike® associated with FloSeal® and raloxifene hydrochloride showed that new bone was rapidly apposed on implanted granules and also that the presence of the matrix and therapeutic molecule does not alter the proven highly osteoconductivity properties of Bonelike®. Therefore, this association may be one step-forward for the clinical applications of Bonelike® scaffolds since it is much more easy-to-handle when compared to granular materials.
Keywords: Bonelike®, FloSeal®, Raloxifene Hydrochloride, Animal Model, Histological Analysis.
Introduction
Nowadays, the life expectancy is two times higher than in the beginning of the 20th century (e.g. in the EUA in 1900 the life expectancy was approximately 48 years and now its around 75-80 years). So, the human body is subjected to higher cumulative stress that results in degradation of the tissues and hence new therapies are required to overcome these problems1,2, such as bone grafts, that is the second most common transplantation tissue3. Recently, Murugan et al4 reported that, in Europe the number of bone grafting procedures was 287,300 in 2000 and it is expected to increase to approximately 479,079 in 2005. The worldwide use of bone grafts was estimated in 1 million, of which 15% were synthetic bone grafts and it was also suggested that future growth is mainly due the development of tissue-engineered composites, i.e. composites containing osteogenic cells and growth factors4.
A bone graft should have particular characteristics depending on its application, for example if a quick bond to bone is required then a highly bioactive implant material should be used. L. Hench defined a bioactive material as “a non-toxic, biologically active and that forms an interfacial bond with the host”1.
The ultimate goal of a synthetic bone graft field is to mimic the biological properties of natural bone. Therefore, the morphology and properties of natural bone should be use as a standard that have to be met by the ideal bone substitute5. According to its origin bone grafts can be classified as autografts, allografts, and xenografts6. All of them present an advantages and disadvantages7. Autograft, do not induce an immunological reaction and it has the ability to provide osteoinductive growth factors, osteogenic cells and structural scaffolds8, although they require an additional incision site and increased blood loss9,10. With the use of allografts there is the risk of transferring viral contaminants such as HIV, hepatitis B, hepatitis C and the promotion of immunological reactions. Due to adverse antigenic responses xenografts are not considered suitable for bone grafting. Hence, synthetic bone graft substitutes have been developed and clinically used to provide an alternative to autografts and allografts.
The bone grafts can be classified into three types depending upon their biological properties namely osteoconductive, osteoinductive and osteogenic grafts3. In literature wide varieties of bone grafts materials have been reported for use on de novo bone formation in vivo3, 4, 7,11-13.
Osteoconductive synthetic hydroxyapatite (HA), Ca10(PO4)6(OH)2 has been marketed in a variety of forms and used as a graft material due to its chemical composition, which has similarities with the mineral phase of bone. The natural apatite can be described as a multi-substituted calcium phosphate apatite14,15. Hence, one-way to improve HA bioactivity is by the incorporation of different ions into the HA lattice. Santos et al showed that the bioactivity of HA can also be enhanced by the incorporation of glass based on the P2O5-CaO system, a material patented and recently marketed as Bonelike®16-20. This system allows the incorporation of several ions, such as magnesium, sodium and fluoride resulting in a bone graft with a chemical composition similar to the mineral phase of bone. This novel biomaterial as a result of its controlled chemical phase composition of HA, α and β-tricalcium phosphate (TCP) and its microstructure showed to have better mechanical properties and enhanced bioactivity than the actual commercially HA21-22. The use of Bonelike® associated with a vehicle will facilitate the medical application of this bone graft and also allow the incorporation of biological molecules that can stimulate bone formation in vivo.
FloSeal® is a haemostatic sealant23 composed of collagen-derived particles and topical bovine-derived thrombin24-26 and it has been proven to control bleeding in several medical applications, like vascular surgery, adenoidectomy, laparoscopy and partial nephrectomy24,27,28. FloSeal® is easily used and it can be extruded from a syringe and applied topically to the bleeding area. This haemostatic agent has the ability to acquire
irregular shapes fitting the wounded site27,28. When the FloSeal® is in contact with blood the collagen particles are hydrated and swell, restricting the blood flow. The thrombin present converts the patient fibrinogen into a fibrin polymer, originating a clot around the granules27,28.
Estrogen is well known for its beneficial effect on osteoporosis29,30. The raloxifene hydrochloride is a known selective estrogen receptor modulator (SERM). This molecule acts as an estrogen agonist on bone and liver, also increasing bone mineral density31, being therefore used for prevention of osteoporosis in postmenopausal women. It has also the advantage of being an antagonist on estrogen receptors in the breast and uterus decreasing the risk of cancer. This SERM can be described as [6-hydroxy-2-(4- hydroxyphenyl)benzo-[b]thien-3-yl][4-[2-(1-piperidinyl)ethoxy] - phenyl]ethanone hydrochloride31. Raloxifene hydrochloride inhibits in vitro mammalian osteoclast differentiation and bone resorption in the presence of interleukina 6 (IL- 6). Also produces a similar activity of TGF-β3 (a cytokine associated with inhibition of osteoclast differentiation and activity) in ovariectomized rats32-35. Several studies shown that this molecule can prevent bone loss33,36,37.
Recently, the authors have reported the potential of Bonelike® graft for bone regeneration for a period of 12 weeks by using an animal model38 and in clinical applications39,40.
The aim of this work was to assess in vivo the osteoconductivity and biofunctionality of Bonelike® granules associated to FloSeal® and raloxifene hydrochloride and also to verify the effect of both FloSeal® and raloxifene on the bioactivity of Bonelike®.