Characterization of novel bioactive glass coated hydroxyapatite granules in correlation with in vitro and in vivo studies.
Human skeletal system that evolved over millions of years has the unique capacity to regenerate and repair damages that occur below the critical size. Management of defects in bone that are difficult to reunite was a major challenge to human minds over centuries. There are archeological records of humans using ivory, inert metals, gemstones and bone products to manage defects in bone (1). Local defects in bone arising as a result of trauma, tumor, and infection are frequently restored by bone graft substitutes and preferably used are autografts and allografts. Autografts being the most preferred remains the gold standard for osseous reconstruction in clinical practice but has problems like limited blood supply, donor site morbidity, increased operation time, blood loss and frequent insufficiency of bone graft (2).
Allografts on the other hand have the high risk of transmission of infections, develop adverse immunological reactions in the recipient's body, delay vascular penetration, slow bone formation and prevents incomplete graft incorporation when used as an accepted bone graft substitute. Some of the most significant advances in biomaterials over the last 20 years have been in the field of bone graft substitutes. Bioresorbable porous bone graft substitutes especially bioceramics may significantly reduce the disadvantages associated with autografts, allografts and other synthetic materials currently used in bone graft procedures. They share numerous advantages over autografts and allografts which include unlimited supply, non toxic and non allergic nature, easy sterilization, and storage. The use of ceramics as implantation materials originated from research in early 1970s, which lead to the introduction of variety of bioceramics. Owing to the close resemblance in physiochemical properties, calcium phosphates have received attention as a promising material to be used as bone substitutes. Hydroxyapatite, Bioglass and apatite wollastonite (AW) glass ceramic of different forms and sizes are widely used as substitutes for bone augmentation and restoration (3, 4, 5, 6, 7, 8) and in tissue engineering (9). Such materials favour osteointegration and are biocompatible (10, 11, 12). They form a viable alternative to autogenous bone grafting, either as osteoconductive bone void fillers or as bone graft extenders.
The aim of our study was to characterize the in-house synthesized hydroxyapatite (HA) and novel bioactive glass coated hydroxyapatite (BGHA) for their biocompatibility in vitro and compare their respective osseous regeneration in the healing of a critical size defect in vivo in a lapine femoral model. Thereby, this will enable to establish their potential ability for use as a non-load bearing bone substitute to replace bone lost due to trauma, diseased or any congenital abnormality.
Preparation of porous hydroxyapatite and bioactive glass coated porous hydroxyapatite granules:
In-house synthesized and developed bioactive glass coated porous hydroxyapatite granules (BGHA--test material) and porous Hydroxyapatite granules [Ca.sub.10][(P[O.sub.4]).sub.6][(OH).sub.2] (HA--control material) were used for the study. Hydroxyapatite powder was synthesized by the precipitation reaction between aqueous calcium and aqueous phosphate solution in the stoichiometric proportion, under basic medium. The precipitate was aged, washed, centrifuged, calcined and ball milled to get a fine powder. The powder was mixed with pore former compacted uniaxially into cylinders of porous HA and biscuit fired. The discs were crushed into granules. The granules were kept in a thin silica sol, removed the excess sol, dried and sintered at 1200[degrees]C. The sintered granules were thoroughly cleaned in acetone and deionized water in an ultrasonicator. Subsequently they were sterilized in an autoclave, prior to in vitro and in vivo studies. The control granules were prepared by sintering HA cylinders followed by crushing.
Physiochemical characterizations were done to establish the micro architecture, phase purity, crystallinity, composition and the functional groups of hydroxyapatite and bioactive glass coated hydroxyapatite.
Scanning Electron Microscope (SEM) and Energy Dispersive Analysis by X-ray (EDAX): The granules were gold coated in an ion sputter (E101-Hitachi) and examined for the microstructure (pore shape, size and interconnectivity) in SEM (S2400 Hitachi), and the EDAX spectra was plotted using the OXFORD X-ray microanalysis software.
X-ray diffraction analysis (XRD): Sintered HA and BGHA granules were powdered and the XRD spectrum was recorded in a diffractometer (SiemensD5005), performed at 40 kV and 30 mA, using step size of 0.02[degrees], scan rate of 2[degrees] per minute, and a scan range between 20[degrees] and 50[degrees], 2 [theta] in flat plane geometry with K-Alpha 1 radiation to check the phase purity and crystallinity.
Transmission Electron Microscope (TEM) analysis: The HA and BGHA granules were fine powdered in a pestle and mortar and the fine powders were dispersed in acetone by gentle mixing and a drop of the suspension was placed on a formvar carbon support film on copper grid of 200 mesh type and viewed under TEM (H-600) at an accelerating voltage of 75 kV, to view the ultrastructure of the fine crystals and its selected- area electron diffraction pattern.
Fourier transform infrared spectroscopy (FTIR): The IR spectrum of HA and BGHA granules were recorded using a Nicolet Impact 410 FT-IR spectroscopy, using the KBr pellet technique. Sintered HA and BGHA granules were powdered and mixed with KBr powder in a weight ratio of 1:100 and pressed into pellets and analyzed at a resolution of four wave numbers, operating from 400 [cm.sup.-1] to 4000 [cm.sup.-1].
In vitro cytotoxicity studies
An in vitro model comprising of Human Osteosarcoma Cell line (HOS) was used to check the toxicity of HA and BGHA granules.
Test on extract (Based on ISO 10993-5, 1999): Extracts of porous HA and BGHA, were prepared by incubating the materials in saline at 37[degrees]C for 24 h at an extraction ratio of 0.2 g/ml.100 % extracts were diluted to get concentrations of 50 % and 25 % with MEM supplemented with Foetal Bovine Serum. Different dilutions of extracts of Test sample, negative control (Ultra High Molecular Weight Poly Ethylene) and positive control (Dilute phenol) in triplicate were placed on sub confluent monolayer of Human Osteosarcoma Cell line (HOS) obtained from NCCS (National Centre for Cell Science, Pune, India) After incubation of cells with extracts of test sample and controls at 37 +/- 2[degrees]C for 24 +/ -1 h, cell culture was examined microscopically for cellular response. Cellular responses were scored as 0, 1, 2, 3 and 4 according to none, slight, mild, moderate and severe based on USP.
In vivo studies
Animal model: Twenty New Zealand white male/ female rabbits weighing 2 to 2.8 kg were randomly selected to five groups of four rabbits each. BGHA granules (test material) were implanted in distal and proximal critical size defects in the right femur of the animal. HA granules (control material) were implanted on the proximal part and an unfilled defect (sham) was made on the distal part of the left femur. The events occurring in a test defect, control defect and in the sham were compared at consecutive period of 1, 2, 4, 6 and 12 weeks.
Surgical procedure: The surgical procedure was carried out according to the standards of ASTM (American Society for Testing Materials, F 981-99 Standard). Management of animal husbandry and post operative care of the rabbits in the Vivarium are standardized in-house as per the guidelines of the Institutional Animal Ethics Committee (IAEC). The animals were sacrificed at 1, 2, 4, 6 and 12 weeks postimplantation, for assessing the material/ tissue interaction in bonding.
Histological analysis: Animals were euthanized at the specific study period. The bone was disarticulated form the anterior and posterior joints and immediately fixed in 10 % formalin and thereafter carefully cleaned to remove the attached muscles. The implant site was carefully exposed and a perpendicular cut was made through the implant site using a high precision diamond saw (Beuhler Isomet) and was immediately immersed in 10 % neutral buffered formalin.
Fixed samples were dehydrated in graded series of alcohol and embedded in PMMA. Thin sections (100--150 [micro]m) were cut using a diamond saw and the undecalcified sections were stained with Stevenel's blue and counter stained with Van Gieson picrofuschin (13, 14). The sections were examined using Nikon E600 Light Microscope.
Atomic force microscopy (AFM): The samples were fixed in 3 % gluteraldehyde, and dehydrated in ascending grades of acetone. The topographical scanning was done to determine any surface modifications of the bone and the implanted test material (1 wk), using Atomic force microscopy (Nanoscope V) in TappingMode[TM] at a scan size of 1 [micro]m to 10 [micro]m (data scale 600-700 nm, image data -height) and a scan rate of 1.489 Hz to 2 Hz.
Results and Discussion:
Understanding the events occurring at the tissue material interface is a challenge for material scientists in developing an ideal bone substitute. The mechanical stability of bone results from the incorporation of mineral into the osteoid matrix template and the factors that initiate the deposition of mineral in the calcification of bone are not well understood. This study was focused on synthesizing bioactive glass coated porous hydroxyapatite granule as a substitute for cortical bone grafts and to compare its efficacy with uncoated porous HA granules. Materials synthesized by sol-gel process are more bioactive than the materials of the same composition prepared by other methods (15). Apart from the physiochemical properties, the structural parameters like pore size, pore roughness and interconnections in the material play a vital role in the attraction, attachment and proliferation of osteoblast cells. An ideal material should mimic the micro architecture of the bone.
SEM (Fig.1.a & Fig.2.a) depicted BGHA and HA granules to be of size 300 to 500 [micro]m with well-defined pores of approximately 100 [micro]m with interconnections which is ideal for osteoblast invasion and proliferation. BGHA surface showed a glassy appearance. The surface physiochemical properties play a vital role in the bone bonding mechanism (16) because the physical properties and the crystal chemistry of the surface together with the chemical composition of the surrounding extracellular fluid determines the nature of the new surface to be formed on the material.
[FIGURES 1-2 OMITTED]
The EDAX spectrum of HA showed specific Ca and P peaks, which has an elemental distribution of 67.22 % and 32.78 % respectively in the material (Fig.1.b). The EDAX spectrum of BGHA showed silica peaks apart from Ca and P peak and the elemental composition in the material is Si = 6.27 %, P = 27.11 % and Ca = 66.61 % (Fig.2.b).
The XRD technique was employed to assess the phase purity and crystallographic changes. The properties of HA and BGHA differ with respect to the preparation route and reaction ingredients. Among many properties, crystallographic changes directly affect the bioactivity of HA and BGHA; thus, it is necessary to study its phase purity and crystallography before intending for in vitro and in vivo trials (17). XRD spectrum of HA defined the crystallinity of the material with major peaks assigned for hydroxyapatite (Fig.3.a) but BGHA showed characteristic major peaks for hydroxyapatite, and calcium silicate and minor peaks for tricalcium phosphate (Fig.3.b) .
[FIGURE 3 OMITTED]
At a higher magnification using TEM, submicron crystallites of HA and BGHA were seen confirming the materials crystallinity with respect to their unit crystal arrangement (hexagonal) with characteristic diffraction spots. Selected-area electron diffraction studies using TEM is considered as a more effective tool for identifying the phase formations in bioceramics (18).
The IR spectrum of HA showed characteristic functional groups of phosphate and hydroxyl moieties. Phosphate peaks were observed between 570 [cm.sup.-1] and 1050 [cm.sup.-1] (571.05 [cm.sup.-1], 601.52 [cm.sup.-1], 961.64 [cm.sup.-1], 1048 [cm.sup.-1]). The characteristic hydroxyl peak was observed at 3571.06 [cm.sup.-1]. In BGHA apart from phosphate(589.23 [cm.sup.-1],601.43 [cm.sup.-1], 1048 [cm.sup.-1]) and hydroxyl functional group (3570.32 [cm.sup.-1]) an additional silica peak was observed at 1089.42 [cm.sup.-1] which strongly supports a uniform coating of silica over the material which was absent in HA granules (Fig.4). It is very well understood that in the biological mineralization of bone tissue, hydroxyl ions and P[O.sub.4.sup.3-] is substituted in the apatite structure by C[O.sub.3.sup.2-] and HP[O.sub.4.sup.2-]. The formation of carbonate apatite crystals in HA and BGHA surface is a very complex process and this attributes to the establishment of strong bonding zone at the bone-material interface.
[FIGURE 4 OMITTED]
Non-cytotoxicity of HA and BGHA was proved by preparing extracts of the respective granules in saline at 37[degrees]C for 24 h and was diluted (100 %, 50 % and 25 %) with MEM supplemented with Fetal Bovine Serum and incubated along with a confluent growth of HOS cell line. The extracts of the materials were made in such a way that the maximum leaching of the constituents was achieved so as to compare the in vitro leaching of the materials due to the bioactive nature that becomes stable when the super saturation of the components is achieved (19). The extract of materials showed 'none cytotoxic' response to osteoblast cells at an extraction ratio of 0.1 gm/ ml. In the extract of BGHA, silica content proved non-toxic to the cell line.
Osteointegration of the material with the host bone is a prerequisite for the success of the implant. The rabbit as an experimental animal was chosen on the basis that it has a very well defined cortical bone Haversian system and the stimulation of remodeling after injury is more similar to that of other larger animals. Gross observation of the implant area did not show any inflammation or necrosis during the study period. Histologically, the osteogenesis was of intra membranous type and no cartilage cells were observed at any point of time. Formation of trabecular bone was observed as early as 1 wk in between the interstitial spaces of the BGHA granules while in HA granules the area of the newly formed bony trabecular was less, but there was a random distribution of numerous active osteoblast cells at the forefront of bone formation. A non-toxic surface is essential for the migration, attachment and proliferation of the osteoblasts (20). AFM revealed a rough surface architecture at the implanted site at 1 wk (Fig. 7). A small scanner size of 10[micro] x 10[micro] and roughness of the site was a hindrance for further study of the bone-material interface. After 4 weeks (Fig. 8. a), the empty spaces between the granules were almost occupied by de novo bone and small bone islands were observed on the granule particles. The empty gaps in the BGHA implanted site were less compared to the HA implanted site. The observaton of osteoid seams with active plump osteoblast cells lining the bone and numerous osteocytes within lacunae of the newly formed bone are all indicators of continuing bone formation. By 6 weeks, a fine network of woven bony architecture with good cellular infiltration was observed and the free spaces gradually decreased with time. BGHA and HA granules had almost completely bridged the defect by bone apposition without any fibrous tissue intervention. There was adequate material integration at the margins to the surrounding bone tissue, bridging between the proximal and distal ends of the defect. By 12 weeks, mature woven bone remodeled into mature lamellar bone embedded with osteocytes in the periosteal and endosteal regions and the defect was completely healed whereas the granules still persisted (Fig.8. b).
[FIGURES 5-8 OMITTED]
Morphologically, osteointegration is realized when there is direct contact between the viable bone and the material surface without an interposition of soft tissue at the light microscopical level (21). As time progressed BGHA granules started cracking paving way for bony trabeculae to delve into the crevices of the granule. Usually macrophages are the dominant infiltrating cells that respond rapidly to biomaterial implantation in soft and hard tissue and are implicated in the biodegradation of implanted biomaterials (22,23). However these cells were not observed here and osteogenesis appeared faster in the case of BGHA than with HA granules. This could be due to the formation of silica gel layer which eventually gets transformed into bone like apatite layer and it is well known that proteins bind to the apatite crystal surface of the ceramic favoring secondary nucleation, thereby affecting the dissolution and reprecipitation process on the ceramic (24). Studies have also showed that pores present in BGHA (25) are nucleation sites of apatite. So apatite formation is a feature in bone formation and bioactive glass coated granules were found to induce more apatite formation leading to osteogenesis and osteointegration. Kokubo et al., has reported in vitro formation of apatite layer on AW-glass ceramic as early as 7 days in pseudo extracellular fluid. (26). Among bioactive ceramics, glass ceramics containing apatite and wollastanite crystals have been found to have a high mechanical strength and showed newly formed apatite layer on their surface in the physiological environment (27). Shors and Holmes (28) have stated that an ideal bone graft substitute would mimic the natural bone and help in the stimulation of osteoblast cells to differentiate and release the mineralizing matrix factors.
Bone substitutes are in great demand in various degenerating bone disorders like osteoporosis, periodontal disease, juvenile periodontitis, dental periapical abscess, osteoarthritis, bone tumors, vascular necrosis and trauma. In this study, after twelve weeks of implantation, the defect was completely healed, but BGHA and HA granules still persisted. The rate of bone formation was faster in BGHA implanted sites. BGHA granules were fragmented with de novo bone tissue in between them when compared to the larger bits of HA granules, accelerating the process of osteogenesis and osteointegration. Interestingly, bone islands were also observed on the surface of BGHA granules. In comparison to HA, BGHA micron granules proved to be a promising material for orthopedic application in non-load bearing sites.
Authors gratefully acknowledge The Director, SCTIMST for the financial support and facilities to carry out this work. Authors are thankful to Dr.P.V.Mohanan, for the animal surgical procedures in Toxicology Laboratory; Dr. Mira Mohanty, for utilising the facilities in the Histopathology Laboratory; Mr. R .Sreekumar for SEM; Mr. R. Moses for FTIR; and Dr. George Thomas, RRL, Thiruvananthapuram, for AFM evaluation.
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Sandeep G, H. K. Varma, T.V. Kumary, Suresh Babu S and Annie John
Biomedical Technology Wing
Sree Chitra Institute for Medical Sciences & Technology
Poojapura, Trivandrum, India-695 012
Corresponding author: Dr.Annie John.Tel: +91-471-2520215;Fax: +91-471-2341814
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|Author:||G., Sandeep; Varma, H.K.; Kumary, T.V.; Babu S., Suresh; John, Annie|
|Publication:||Trends in Biomaterials and Artificial Organs|
|Date:||Jan 1, 2006|
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