Present and future applications of biomaterials in bone disease –a 3d microcomputed tomography study
PRESENT AND FUTURE APPLICATIONS OF BIOMATERIALS IN BONE DISEASE –A 3D MICROCOMPUTED TOMOGRAPHY STUDY
Conf Dr Adrian Barbilian, Prof. Univ. Dr Ioan Sarbu, Dr Marinel Dignei, Dr Dragos Cuzino, Dr Trifu Marian
University Clinical CentralMilitaryHospital “ Dr Carol Davila”- Bucharest, Romania
1
The Romanian Review Precision Mechanics, Optics & Mecatronics, 2008 (18), No. 34
Present and future applications of biomaterials in bone disease –a 3d microcomputed tomography study
Abstract – Microcomputed tomography (microCT) is a miniaturized version of computerized axial tomography commonly used by radiologists but the systems have a resolution of the order of a few micrometers. These systems often makes use of laptop computers or working stations and provide images that are very close to those provided by synchrotrons (with a resolution in the order of the micrometer.)
Key words –microcomputed tomography (microCT), bone disease, biomaterials
Introduction
Microcomputed tomography (microCT) is a miniaturized version of computerized axial tomography commonly used by radiologists but the systems have a resolution of the order of a few micrometers. These systems often makes use of laptop computers or working stations and provide images that are very close to those provided by synchrotrons (with a resolution in the order of the micrometer.) Up to now, the use of microcomputed tomography has been successfully used in different branches of science for the study of porous or cavity-containing objects: metallic foams, electronics, stones, wood and composite polymers. In biology, the technique is well adapted to the study of hard tissues because of the high linear attenuation coefficient of the calcified bone and dental matrices [1]. The technique is now favored in the study of trabecular bone loss in osteoporotic patients or in animal models of osteoporosis [2-4]. In bone biology, a great body of literature is concerned with the measurement of characteristics of the trabecular network. Histomorphometry was developed in the ‘70ies as a method to quantify bone loss in osteoporosis on 2D histological sections. During decades, osteoporosis was considered as a disease associated with a low bone mass. However, it was only in the ‘80ies that the 3D alterations of trabecular bone were taken into account although bone, being a “living biomaterial”, adapts to strains by a redistribution of trabeculae by the remodeling process [5]. The importance of microarchitecture in the pathogenesis of bone fragility is now fully recognized and is part of the WHO definition of the disease: “…characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to enhanced bone fragility and a consequent increase in fracture risk”. Several attempts were done recently to measure the architectural characteristics of trabecular bone on 2D histological sections and on 3D models prepared from microCT analyses. We review hereafter the potent interest of microCT applied to calcium phosphate materials in various research fields.
Calcium Phosphate materials are used like adjuvant to dental implants, alveolar bridge augmentation, bioreactor (to grow cells), cements (dental and orthopedic) ,chewing gum, coating for implants (plasma-spraying; ion sputtering; laser deposition; electrodeposition), dentrifices, drug delivery, ear implants, fillers for composites (polymeric or non-polymeric), fillers for glass ionomer cements, maxillor-facial surgery, pulp-capping materials, repair of bony defects, repair of periodontal defects, repair of failing implants, research materials (standards or reagents), spine fusion . Since calcium phosphate cements were proposed, several formulations have been developed, some of them commercialised, and they have proven to be very efficient bone substitutes in different applications. Some of their properties, such as the injectability, or the low-temperature setting, which allows the incorporation of different drugs, make them very attractive candidates as drug carriers. In this article, the performance of calcium phosphate cements as carriers of different types of drugs, such as antibiotics, analgesics, anticancer, anti-inflammatory, as well as growth factors is reviewed.
Weuse the Skyscan 1072 X-ray computed microtomograph in the cone beam acquisition mode. The system is composed of a sealed microfocus X-ray tube, air cooled with a spot size less than 8 µm and a CCD camera. Images were obtained at 80V and 100µA with a 1 mm aluminum filter each time calcified material was present in the specimen. Specimens were studied either in the wet or dry form: biopsies or fragile bones were placed in an Eppendorf test tube containing the fixative and the vial was fixed to a stub with plasticine ; large and dry specimens were directly fixed with plasticine. For each specimen, a series of 400 projection images were obtained with a rotation of 0.45° between each image. The magnification used depends on the size of the objects: large human bone biopsies and rat femurs were scanned at x21 (pixel size = 11.4 µm), mice bones at x58 (pixel=5.26 µm) while human teeth, blocks of biomaterials and large blocks of human bone (vertebra, radius) were analysed at the lowest magnification (x14; pixel size=19.74 µm). Given a series of projection images, a stack of 2D sections was reconstructed for each specimen (the number of sections depending of the desired height) and stored in the .bmp format with indexed grey levels ranging from 0 (black) to 255 (white).
The 3D softwares
Three dimensional (3D) modelling and analysis reconstruction of the specimens were obtained with the ANT software (Skyscan - Aartselaar, Belgium). The program allows reconstruction of objects from the stack of 2D sections, after interactive thresholding. The reconstructed 3D models were obtained by a surface-rendering algorithm. Four different 3D models can be reconstructed and made visible on the computer screen simultaneously, thus offering the possibility to combine several images. In addition, the program offers facilities for 360° model rotations in all directions, displacements, lightening effects and colouring of the desired structures. A very interesting facility for the study of porous structures was the possibility to make the virtual models semi-transparent. Another interesting possibility was to obtained 2D reslices of the objects across a plane, positioned in a specified direction. Morphometric measurements can be done on 2D images and 3D models with software.
MicroCT in human bone diseases
MicroCT offers the unique possibility to visualize in 3D the microarchitectural changes occurring in the various types of osteoporosis. Thinning of trabeculae with rather well preserved architecture is associated with glucocorticoid therapy. On the other hand, focal disorganization of the network are observed in postmenopausal osteoporosis and hypogonadism osteoporosis in males [6]. In these cases, the increased osteoclastic activity has led to the complete removal of trabeculae. Several reports have shown that 3D measurements were highly correlated with 2D obtained by histomorphometry although microCT seems to provide slightly increased values for bone volume [7]. The method has also provided interesting results in the survey of anti-osteoporotic treatments such as bisphosphonates which can preserve bone architecture. Recently, microCT was used to characterize the microarchitecture of trabecular bone in glucocorticoid-induced osteoporosis and a very particular aspect was observed on plates that become thinner in their center with appearance of minute perforations[17].
left: anchorage of the 3D trabecular network (composed of plates and pilars on the endosteal surface of cortical bone.
right: reconstruction of the cortical bone (blue pseudocolor with a 50% transparency) with a reconstruction of the Haversian canals (yellow) to illustrate the complex branching of these structures.
left: transiliac bone biopsy from a patient with alcoholic osteoporosis.
right: transiliac bone biopsy from a patient with glucocorticosteroid induced osteoporosis; note the very thin trabeculae and plates with numerous perforations.
MicroCT is also very interesting in the understanding of bone changes in haematological disorders. In MMM (myelofibrosis with myeloid metaplasia), the mechanisms responsible for bone sclerosis appear underlined by a complex network of interacting cytokines.MicroCT found that newly-formed bone packets (lamellar or woven) are anchored at the surface of the trabeculae and the bone volume progressively can increase dramatically [16].
left: transiliac bone biopsy from a patient with MMM.Bone volume is increased. Numerous foci of newly apposed bone are evidenced at the surface of enlarged trabeculae and endosteum.
right: transiliac bone biopsy from a patient with a late stage of MMM. Bone volume is considerably increased and osteosclerosis has filled most of the marrow spaces.
MicroCT in animal models of osteoporosis
The development of animal models of osteoporosis is a very interesting approach in understanding the pathophysiological mechanisms of bone loss; in addition, models can be used to test the efficiency of new therapeutic strategies. The ovariectomized rat (OVX) is considered by WHO and FDA as the most suitable model of post menopausal osteoporosis [8]. Several studies have shown that microCT changes in ovariectomized models are very similar to human osteoporosis; they associate a reduction of trabecular number and a conversion of trabecular plates into rods. In the same way, orchidectomy in the male rat has similar effects and can be used as a model for hypogonadic osteoporosis.
left: tibial metaphysis of a control Wistar male rat.
right: tibial metaphysis of an orchidectomised (ORX) rat 16 week after surgery, note the importance reduction in bone mass and the marked conversion of trabeculae into pilars.
Disuse osteoporosis can be obtained by bandaging or local injection of botulinum toxin [9]; combination of factors induces a considerable loss of trabecular bone. Similar effects have been found in humans when several risks factors are associated in the same subject: the number of fractures dramatically increases, parallel to the trabecular bone disorganization [19]. Trangenic animals offer the possibility to study the consequences of the deletion of a protein on the bone skeleton.
left: femoral metaphysis of a control Wistar male rat.
right: femoral metaphysis of a rat with a severe bone loss obtained by combining disused (provoked by botulinum toxin injection) and orchidectomy (ORX)
MicroCT analyses of mice of various strains have revealed enormous variations in the trabecular bone mass and architecture [10, 11]. The technique is specially useful since it permits the visualization of the external and inner part of the same bone without altering the specimen, making it usable for other histological techniques [12]. Here again, the effects of pharmaceutical compounds on bone mass and architecture can be explored [13, 14]. In the orchidectomized rat with disuse obtained on one hindlimb by botulinum toxin, we have used testosterone or risedronate (a 3rd generation aminobisphosphonate). Testosterone had limited effects on the bone loss; on the other hand, risedronate preserved bone mass and architecture in the trabecular bone but was found less active on the cortical bone loss [21].
1
The Romanian Review Precision Mechanics, Optics & Mecatronics, 2008 (18), No. 34
Present and future applications of biomaterials in bone disease –a 3d microcomputed tomography study
A: Tibial metaphysis of a control Wistar male rat.
B: Tibial metaphysis of a male rat having had orchidectomy + unilateral paralysis of the quadriceps with botulinum toxin.
C: Similar to B, but with risedronate as a countermeasure.
D: Similar to B, but with testosterone as a countermeasure.
1
The Romanian Review Precision Mechanics, Optics & Mecatronics, 2008 (18), No. 34
Present and future applications of biomaterials in bone disease –a 3d microcomputed tomography study
The 2D sections obtained by the microCT can also be used to model the bony structure by finite element analysis and to obtain multiple replica by stereolithography [15, 16].
MicroCT in animal models of cancer diseases
The relationships between metastatic cancer cells and bone remodelling are now well identified in some types of malignancies. Breast cancer cells have a propensity to metastasize in bone marrow and stimulate bone remodelling, leading to osteolytic or osteolytic/osteocondensing metastases. Prostate cancer cells stimulate the osteoblastic cells and induce osteosclerotic metastases. Multiple myeloma (a hematologic malignancy of the plasma cell) is associated with osteolytic foci in 95% of patients. Numerous models are described in the literature in small laboratory animals: they include the injection of allogenic cells in mice or rats or the use of immunodeficient strains (nude or SCID) for the evaluation of human neoplastic cells. These models offer the possibility to study bone changes that mimic human metastases: osteolytic lesions obtained with 13762 ma mmary carcinoma in the rat have been explored by microCT [17]. One of the best model human myeloma is the 5T2 MM described in C57BL/KaLwRij mice. MicroCT offered the interesting possibility to quantify trabecular bone resorption but also to determine the exact number of cortical perforations (a parameter that cannot be obtained by 2D histomorphometry) [4], and the needed volume to be charged with biomaterials. In this model we were able to show the protective effect of the bisphosphonic compound pamidronate on the dramatic bone loss induced by 5T2 MM cells [18].
1
The Romanian Review Precision Mechanics, Optics & Mecatronics, 2008 (18), No. 34
Present and future applications of biomaterials in bone disease –a 3d microcomputed tomography study
1
The Romanian Review Precision Mechanics, Optics & Mecatronics, 2008 (18), No. 34
Present and future applications of biomaterials in bone disease –a 3d microcomputed tomography study
first 3 images: different views of the femur of a C57BL/KaLwRij mice with the 5T2 myeloma. Note the complete disappearance of the trabecular bone and cortical perforations from the marrow cavity to the periosteum
middle: tibia from a C57BL/KaLwRij mice with the 5THL myeloma. Note the marked perforations and the removal of trabecular bone.
right: 2D microCT scan of the tibia of a Copenhagen rat wearing the MatLyLu tumor (arrow indicates a tumor nodule with osteolysis)
MicroCT and dental research
MicroCT can be used to study the ex-vivo micro-anatomy of teeth. The possibility to used semi transparent models allows a perfect identification of the pulpar chamber and root canals through the heavily calcified dentin and enamel. On 2D sections, the different mineralization degrees of enamel >dentin >cement can be easily identified [19]. MicroCT has been used to calculate the volume of root canals in endodontal research [20]. In animals, some studies have stressed the interest of the technique to measure the alveolar bone loss induced by sex hormone deprivation.
1
The Romanian Review Precision Mechanics, Optics & Mecatronics, 2008 (18), No. 34
Present and future applications of biomaterials in bone disease –a 3d microcomputed tomography study
1
The Romanian Review Precision Mechanics, Optics & Mecatronics, 2008 (18), No. 34
Present and future applications of biomaterials in bone disease –a 3d microcomputed tomography study
A human wisdom tooth with 3D reconstruction (left), semi-transparent imaging (middle) to see the pulp chamber in yellow and the roots, after having "sectionned" the crown to look at the pulp chamber by the top (right)
1
The Romanian Review Precision Mechanics, Optics & Mecatronics, 2008 (18), No. 34
Present and future applications of biomaterials in bone disease –a 3d microcomputed tomography study
1
The Romanian Review Precision Mechanics, Optics & Mecatronics, 2008 (18), No. 34
Present and future applications of biomaterials in bone disease –a 3d microcomputed tomography study
A human wisdom tooth with 3D reconstruction and semi-transparent imaging (left and middle) to see the pulp chamber and the root canals -in blue, On the right, the pulp chamber and the canals have been reconstructed and appear as a solid volume.
1
The Romanian Review Precision Mechanics, Optics & Mecatronics, 2008 (18), No. 34
Present and future applications of biomaterials in bone disease –a 3d microcomputed tomography study
1
The Romanian Review Precision Mechanics, Optics & Mecatronics, 2008 (18), No. 34
Present and future applications of biomaterials in bone disease –a 3d microcomputed tomography study
The mandibula from a Wistar rat; (left) photo showing the incisor and the 3 molars; (middle and right) 3D reconstruction with a cuting plane on the incisor region, in yellow and throught the molars, in green.
The mandibula from a Wistar rat; (left) photo showing the 2D section along the yellow cutting plane on the incisor region, and through the molars, green cutting plane. The highly calcified enamel is in black, note the pulp chamber of the incisor and molar and the alveolar bone.
MicroCT and biomaterials
Biomaterials are used to replace a given function of the human body. For bone, prostheses can supply the loss of articular function and are mainly composed of metals (Cr Ni, Ti….) that preclude the use of microCT. Metals induce reconstruction artefacts related to X-ray absorption; however, the use of microfocus beam could help to appreciate the titanium/bone interface in some cases [21]. On the other hand, bone substitutes offers to fully explore the possibility of microtomography. The design of new porous materials can be controlled with microCT and the interconnected ness can be examined and measured [22]. The method is also well adapted to phosphocalcic or polymer bone substitutes and can be used to follow their bioerosion and their osteoconducting properties [23]. Biomimetic materials such as glass ionomers or self calcifying polymers can also be explored with microCT.
1
The Romanian Review Precision Mechanics, Optics & Mecatronics, 2008 (18), No. 34
Present and future applications of biomaterials in bone disease –a 3d microcomputed tomography study
1
The Romanian Review Precision Mechanics, Optics & Mecatronics, 2008 (18), No. 34
Present and future applications of biomaterials in bone disease –a 3d microcomputed tomography study
left: a titanium surgical screw implanted in trabecular bone
middle: synthetic biomaterial composed of poly 2-hydroxymethacrylate with an interconnected porosity, the material is viewed in semi-transparency.
right: overimposition of the porosity in green through the material.
The design of a macromolecular bone graft with interconnected macroporosity represents a major challenge in the field of orthopaedic biomaterials. Such a synthetic graft would combine the biocompatibility and the biomechanical behavior with a micro-architecture allowing the osteoconduction through the colonization of the implant by bony cells and blood vessels. Macroporous blocks of poly (2-hydroxy ethyl) methacrylate (pHEMA) were obtained by the monomer around polystyrene beads of various diameters (up to1600µm). MicroCT and scanning electron microscopy were used to evaluate the porosity and the interconnectivity. Porosity did not differ whatever the size of the beads used as porogen and was close to that of human trabecular bone [20].