Daniel CHAPPARD, Hélène LIBOUBAN, Erick LEGRAND, Michel Félix BASLE and Maurice AUDRAN

GEROM-LHEA, CHU Angers                     Updated: 01 septembre 2017

MicroCT microcomputed tomography microscanner bone microarchitecture architecture biomaterials, bone biomaterial,osteoporosis, osteoporose, bone metastasis, bone cancer, tooth, dental surgery, endodontics, pulp chamber, dental root bone subsitute, bone graft, bone grafting, tissu osseux, scanner, microscanner, skyscan, bone structure, porous material.

Introduction 

Microcomputed tomography (microCT) is a miniaturized version of computerized axial tomography co mmonly 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 calcified tissues in various research fields.

 

The Skyscan microtomographic systems

We  used the Skyscan computed microtomographs (Bruker - Aartselaar, Belgium) in the cone beam acquisition mode. Our firs 1072 model has been changed for the 1172 microtomograph in 2010 and then by the most recent Skyscan 1272 in 2017. We also have the 1076 in vivo system. These systems are composed of a sealed microfocus X-ray tube, air cooled with a spot size less and a CCD camera. Images are usuallly obtained at 80V and 100µA with an aluminum filter each time calcified material is present in the specimen. Specimens can be studied either in the wet or dry form: biopsies or fragile bones are placed in an Eppendorf test tube containing the fixative and the vial is fixed to a stub with plasticine ; large and dry specimens are directly fixed with plasticine. For each specimen, a series of projection images are obtained with a rotation of between each image (form 0.45° to 0.1°). 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). With the 1172 system, a better resolution can be obtained and large bone biopsies are imaged with a pixel size of 3-4µm. Given a series of projection images, a stack of 2D sections is 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 First Skyscan 1072 microCT at GEROM Angers (France)

The second Skyscan 1172 microCT at GEROM Angers (France)

The third Skyscan 1272 microCT at GEROM Angers (France)

 

The Skyscan 1076 microCT at GEROM Angers (France)

nanotomograph

The nanotomograph NANOTOM at GEROM Angers (France)

 

Three dimensional (3D) modeling 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. However, the sofwares provided by the different manufacturers for measuring the parameters of bone mass and microarchitecture are not fully validated and differences exist when comparing the values of bone volume (BV/TV), trabecular bone thickness (Tb.Th) and trabecular bone separation (Tb.Sp) with values obtained by histomorphometry (10). So, clearly microCT is still actually not the "gold standard" for quantitative evaluation of bone mass and microarchitecture but is an incomparable tools for the viewing bone.


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]. A recent review reports all the 2D and 3D histological methods to evaluate bone microarchitecture in human bone biopsies [26].

 

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.

Click on the link below to see a movie of a human osteoporotic bone (from a lumbar vertebra).

MicroCT of a human osteoporotic bone from a human vertebra Video

 

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 can also be used as a rapid tool in the pre-diagnosis of malignancies (30 - 34). Breast and prostate cancers are known to be specially metastasizing to bone. Metastases from breast cancer usually exhibit a mixed osteolytic/osteosclerotic aspect, with osteolysis predominating. Osteosclerosis is a common finding in prostatic cancer although osteolysis occurs within the sclerotic lesions. B-cell malignancies (lymphoma, myeloma) are also associated with marked osteolysis. in a study on 247 patients with a malignant bone disease (metastasis, myeloma or bone lymphoma) microCT identified excess bone resorption on trabecular surfaces when eroded surfaces were >10.5% of bone surfaces as determined by histomorphometry. MicroCT failed to identify some patients with smoldering myeloma or some lymphomas with microresorption. MicroCT data are obtained within 4 hr and suggest the malignant invasion of bone marrow when excess of bone resorption/formation is obtained. MicroCT can be used in the immediate postbiopsy period making possible a fast identification of malignancy. However these signs are not specific and must be confirmed by histopathological analysis.

Transiliac bone biopsy from a patient with an osteosclerotic bone metastasis from a prostate adenocarcinoma.Bone volume is increased. Numerous foci of newly apposed bone are evidenced at the surface of the trabeculae.

Transiliac bone biopsy from a patient with a Multiple Myeloma. Note the focal disorganization of the trabecular network with an area where all the trabeculae have been resorbed.

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].

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.

In the botulinum toxin model of bone loss, microCT was used to see if changes occured during growth of small rats. The 2D sections were used to determine the gravity center of the bone and a 3D model of all gravity centers was used to determine the mean curvature of the bones (40).

3D models obtained with microCT of the tibia of Wistar rats showing (left) the bone model, (right) the line of centers of gravity -in yellow- with the bone model rendered semi-transparent in a blue pseudo-color.

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]. The possibility to treat lethally irradiated mice by grafting with allogeneic stem cells was investigated using bone marrow from transgenic mice expressing the green fluorescent protein  GFP+ C57B6. Transplanted GFPB6 presented a dramatic bone loss compared to B6 and did not restore their trabecular bone mass with time, despite a cortical thickening 6-months after BMT.   With aging, GFP mice have less trabeculae, thicker cortices but a narrower femoral shaft than B6. From 3-months after bone marrow transplantation, cortical characteristics of TransplantedGFPB6 differed statistically from B6 and were identical to those of GFP. GFP+ cells were located along trabecular surfaces and in periosteal and endosteal envelopes but no osteocyte expressed GFP. Engrafted cells did not restore the irradiation-induced trabecular bone loss, but reconstituted a marrow microenvironment and bone remodeling similar to those of the donor. The effects of irradiation and graft on bone remodeling differed between the cortical and trabecular bones [23].

3D models obtained with microCT of the distal femur at 6 months. (left) control B6 mouse, (center) control transgenic GFP mouse, see the marked reduction in trabecular bone and the increase in cortical thickness and (right) a B6 mouse transplanted with  bone marrow cells from a GFP+ B6 mice. Cortical bone is in light grey and trabeculae are darker.

Click on the link below to see a movie of a rabbit tibia.

MicroCT of a rabbit tibia Video

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 i mmunodeficient 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]. 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].

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)

Bisphosphonates (zoledronic acid or pamidronate) are used in preventing osteolysis in myltiple myeloma. However, loss of cortical bone in not possible to quantify by histomorphometry on histological sections or on microCT images. Osteolysis was studied in mice grafted with a malignant myeloma cell subline (5THL) to see if one drug was more active after 10 weeks. Mice were distributed into 4 groups: control, untreated, treated with pamidronate or with zoledronic acid. The right tibias and femurs were analyzed by microCT and trabecular morphometric parameters were obtained. Cortical bone osteolysis was analyzed by developing a new algorithm to unwrap microCT sections of the cortices, allowing measurement of the number of perforations, porosity and mean perforation area. The algorithm convert the round 2D sections into flat section in the same way that a world map is a flat representation of the earth. MicroCT was used to quantify the trabecular bone: a bone loss was evidenced in the untreated myeloma group and both bisphosphonates appeared equal to preserve trabecular mass. However, the number and size of cortical perforations cannot be determined on 3D models. Unwrapping microCT images provided flat images allowing a precise determination of cortical perforations. Pamidronate did not reduce the number and size of cortical perforations but significantly reduced porosity. Zoledronic acid appeared significantly superior and considerably reduced all parameters. Unwrapping microCT image is a new method allowing the measurement of cortical perforations in bone malignancies, a parameter that cannot be measured correctly on 2D histological sections [48]. The algorithm described is patented by APP Agency for Programs Protection IDDN.FR.001.310043.000.S.P.2013.000.31230.

myeloma

A: microCT reconstruction of a bone from a C57BL/KaLwRij mice with the 5T2 myeloma. Note the cortical perforations from the marrow cavity to the periosteum
B: a digitized 2D section from the bone, the section appears round with marked perforations.
C:
2D unwrapped image of the same section.
D: 3D unwrapped image of the same bone than in A. the perforations appear as hole on this flatten image.

In animals with metastasis, vascular injection of a radio-opaque material can identify the neo-angiogenesis due to the tumor growth. However, since the injection products (usually Microfill) have the same radioopacity than bone, it is necessary to perform 2 scans of the same bone: one without decalcification, and a second one after decalcification. On the 1st one, both injected vessels AND bone are seen; on the 2nd scan, only the vessels can be seen. The 3D models must be overimposed in 3D. The technique is described in ref 36.

MicroCT scan of the tibia of a rat with an osteolytic bone metastasis, vascular injection with Microfill. Note the area of neo-angiogenesis in the metaphyseal area

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.

 

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)

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.

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 allowed us to study the effects of glucocorticoids on alveolar bone. Glucocorticoids are highy deleterous for trabecular bone of the axial skeleton but their effects on the alveolar bone remained underevaluated. in 5-month-old male Swiss-Webster mice, the implantation of pellets with a control release of glucocorticoids was done. Animals were euthanized after 28 days. The right tibia and the right hemimandible of each mouse were analyzed by histomorphometry and microcomputed tomography. In the mouse, alveolar bone consists of a thin slab between the incisor and the molar roots connected with the alveolar processes. A 2D-frontal section was done through the pulp chamber of the first molar and was used to measure the thickness of the alveolar bone slab. A 2D-sagittal section was done through the pulp chamber of the three molars and was used to measure bone volume in the alveolar processes. Thickness and bone volume of alveolar bone were significantly decreased. At the tibia, glucocorticoids decreased bone formation with a reduced mineral apposition rate and bone formation rate. Although the amount of alveolar bone is very low in the mouse, glucocorticoids can induce an alveolar bone loss in long-term treated animals which is measurable by microCT (39).

The mandibula from a Swiss Webster mice; (A) control animal; alveolar bone is arrowed, (B and C) mice treated with glucocorticosteroids, note the thinning or the perforations occuring in the thin slab of 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.

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 to1600 µm). 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].

left: polystyrene microbeads examined by scanning electron microscopy.

middle: porous poly 2-hydroxymethacrylate with porosity created by 1200 mm polystyrene microbeads examined by scanning electron microscopy.

right:  the same porous block analyzed by microCT.

 

 MicroCT can be used to observe the porosity of biomaterials. β-TCP (tricalcium phosphate) can be prepared by the foam technology (e.g., Kasios™). The whole process was investigated using microCT and a special algorithm was proposed to separate the inner microporosity (created by the foam rods after sintering) from the macroporosity (created by the open pores of the foam) [25].

 

 

A) microCT of the polyurethane foam used to prepare the β-TCP granules. The foam is made of interconnected rods.

B) microCT of a particle of β-TCP prepared by the foam technique; in blue, the biomaterial with interconnected macroporosity and in yellow, the inner microporosity created by the rods of the foam that have disappeared during the sintering steps.

 

Macrophysical properties of granular biomaterials usable to fill bone defects have been rarely considered. Granules of a given biomaterial occupy the 3D space when packed together and create a new type of macroporosity (inter-granule porosity) suitable for the invasion of vascular and bone cells. Granules of β-TCP were prepared by the polyurethane foam technology [] with increasing the amount of material powder in the slurry (10, 11, 15, 18, 21 and 25g). After sintering, granules of 1000-2000µm were selected by sieving. They were analyzed morphologically by SEM and placed in polyethylene test tubes to produce 3D scaffolds. MicroCT was used to image the scaffolds and to determine the inter-granule porosity and fractal dimension in 3D. 2D sections of the microCT models were binarized and used to compute classical morphometric parameters describing porosity (ICI, strut analysis, star volumes) and fractal dimensions. In addition, two newly important fractal parameters (lacunarity and succolarity) were measured. Compression analysis of the stacks of granules was done. Porosity decreased as the amount of material in the slurry increases but non-linear relationships were observed between microarchitectural parameters describing the pores and porosity. Lacunarity increased in the series of granules but succolarity (reflecting the penetration of a fluid) was maximal in the 15-18g groups and decreased noticeably in the 25g group. The 3D arrangement of biomaterial granules studied by these new fractal techniques allows deriving the optimal formulation based on the lowest amount of material, suitable mechanical resistance during crushing and the creation of large interconnected pores [47].

b-tcp 3D

 

Left) MicroCT analysis of a 3D arrangement of β-TCP granules prepared with the 25g formulation.

Right) Relationship between the fractal parameters and the amount of β-TCP used to prepare the granules, succolarity is exponentially related the groups of granule and lacunarity is maximal in the 15 group.

We have used microCT to explore the evolution of an injectable bone substitute in aged male rats with a massive bone loss in the femur provoked by the combined effect of castration and disuse (obtained after a botulinum toxin injection in the quadriceps). MicroCT was first used to measure the loss of bone mass and architecture in this model [18]. MicroCT was used in a second step to show that the injectable calcium/phosphate biomaterial failed to restore bone mass at long term [14].

The lower part of a femur from a Wistar rat; (left) a marked bone loss is obtained within one month after castration and disuses cause by clostridium botulinum toxin (Botox) Cortical bone is in grey, trabecular bone in green.  (right) injection of a calcium/phosphate biomaterial by a small hole done on the diaphysis. The material appears with a red pseudocolor.

Biomaterials, and especially polymers, can be used as carriers to deliver growth factors directely in bone defects. We have used poly (2-hydroxy ethyl methacrylate) cylinders to immobilize basic fibroblast growth factor (FGF-2). Cylinders were grafted in the supra-critical defects created in the distal femoral epiphysis of rabbits and healing was followed at 2 and 3 months after surgery. MicroCT was used to measure and observe the bone formation around the grafted area: bone trabeculae appeared as a thin lace formed at the surface of the polymer [22].

The lower part of a femur from a rabbit grafted with a cylinder of polymer allowing a controlled release of a growth factor (FGF-2). Note the thin lace of trabeculae that has developped onto the surface of the cylinder (the polymer is radiolucent and cannot be seen here)

When a biomaterial is used inside the body (temporarily or permanently), it is often necessary to visualize it through X-ray radiation. The well-known solution, especially in the case of dental or bone cements, is the incorporation of inorganic additives such as barium sulphate or zirconium dioxide particles. In the case of methacrylic bone cements, it has been reported that these particles diminish the mechanical properties (especially fatigue life) due to the creation of interfaces between the polymeric matrix and the inorganic radio-opacifying particles. We have developped several copolymer that are radioopaque due to the incorporation of iodinated monomers. MicroCT appeared an interesting tool to measure the radio opacity of polymer cylinders and to compare it with bone's one [25].

Cylinder of polymer containing a iodinated monomer (TIBOM). 5 concentrations of the iodinated monomer were used and the X-ray adsorption was measured on the cross sections of the cylinders. A piece of bone is figured.

 

Conclusion

MicroCT is a recent technology available in bone laboratories. Even if image acquisition and reconstructions are still time consuming (resp. ~ 1.30 h and ~ 2-3h) it has nothing to do with the 2-3 weeks necessary for bone histomorphometry after plastic embedding. However, histological studies are still necessary to evaluate the bone remodelling level and to identify and quantify bone or marrow cells. MicroCT also provides parameters that are not available in 2D (i.e., degree of anisotropy). The recent development of microtomographs for in vivo studies will also help to clarify the morphological changes occurring in the same animals upon time instead of sacrifying groups of animals at given time intervals.

 

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  22. Ruhe, P.Q. et al. rhBMP-2 release from injectable poly(DL-lactic-co-glycolic acid)/calcium-phosphate cement composites. J Bone Joint Surg Am 85-A S3, 75-81, 2003.

Studies performed by our group with microCT

  1. Libouban, H. et al. Osteolytic bone lesions in the 5T2 multiple myeloma model: radiographic, scanning electron microscopic and microtomographic studies. J. Histotechnol. 24, 81-86, 2001.

  2. Audran, M. et al. Bone microarchitecture and bone fragility in men: DXA and histomorphometry in humans and in the orchidectomized rat model. Calcif Tissue Int 69, 214-217., 2001.

  3. Libouban, H. et al. Increased bone remodeling due to ovariectomy dramatically increases tumoral growth in the 5T2 multiple myeloma mouse model. Bone 33, 283-292, 2003.

  4. Filmon, R. et al. Non-connected versus interconnected macroporosity in poly(2-hydroxyethyl methacrylate) polymers. An X-ray microtomographic and histomorphometric study. J Biomater Sci Polym Ed 13, 1105-1117, 2002.  

  5. Libouban H., et al. Intérêt des modèles animaux dans l’étude du myélome. Oncoscopie, 7, 4-9, 2002.

  6. Chappard D., et al.  Aluminium osteodystrophy and celiac disease. Calcif. Tissue Int. 74, 212-213, 2004.

  7. Chappard D., et al.  Ex-vivo bone mineral density of the wrist: influence of medullar fat. Bone, 34, 1023-1028, 2004

  8. Libouban H., et al.  Selection of an highly agressive myeloma cell line by an altered bone microenvironment in the C57BL/KaLwRij mouse. BBRC Biochem Biophys. Res. Commun. 316, 859-866, 2004.

  9. Bodic F., et al. Bone loss and teeth. Joint Bone Spine, 72, 215-221, 2005. Chappard D., et al. Microcomputed tomography for the study of hard tissues and bone biomaterials. Microscopy & Analysis, 19, 17-19, 2005.

  10. Chappard D., et al. Comparison insight bone measurements by histomorphometry and µCT. J. Bone Miner Res., 20, 1177-1184, 2005.

  11. Guillaume B, et al. Microcomputed tomography used in the analysis of the morphology of root canals in extracted wisdom teeth Brit. J. Oral Maxillofac. Surg. 44, 240-244, 2006.

  12. Guggenbuhl P., et al.  Texture analysis of X-ray radiographs is correlated with bone micro-computed tomography. Osteoporosis Int. 17, 447-454, 2006.

  13. Blouin S., et al.  An injectable calcium-phosphate substitute as a tentative to restore bone mass in severely osteopenic and aged rats. J. Biomed. Mater. Res. A 78, 570-580, 2006.

  14. Blouin S., et al., Rat models of bone metastases.  Clin. Exp. Metastasis. 22, 605-614, 2005.

  15. Schmidt et al., Bone changes in myelofibrosis with myeloid metaplasia: a histomorphometic and microcomputed tomographic study. Europ. J. Haematol., 78, 500-509, 2007.

  16. Chappard D. et al., Bone microarchitecture in males with corticosteroid induced osteoporosis. Osteoporosis Int. 18, 487 - 494, 2007.

  17. Blouin S., et al., Disuse and orchidectomy have additional effects on bone loss in the aged male rat. Osteoporosis Int. 18, 85-92, 2007

  18. Legrand E, et al., Trabecular bone microarchitecture is related to the number of risk factors and etiology in osteoporotic men. Microsc. Res. Tech., 70, 952-959, 2007

  19. Stancu I.C., et al., Preparation of macroporous poly (2-hydroxyethyl) methacrylate with interconnected porosity. J. Optoelectr. Adv. Mater. 9, 2125-2129, 2007.

  20. Libouban H., et al.,  Early preventive effects of risedronate in a rat model of osteopenia due to orchidectomy and localized disuse. Micron, 39, 998-1007, 2008.

  21. Mabilleau G., et al., Effects of FGF-2 release from a hydrogel polymer on bone mass and microarchitecture. Biomaterials, 29,1593-600, 2008

  22. Dumas A., Brigitte M., Moreau M.F., Chretien F., Baslé M. F. Chappard D. Bone mass and microarchitecture of irradiated and bone marrow-transplanted mice: influences of the donor strain. Osteoporosis Int. 20, 435-443, 2009.

  23. Zaharia C., Zecheru T., Marculescu B., Filmon R., Mabilleau G., Cincu C., Staikos G., Chappard D. Chemical structure of methylmethacrylate-2-[20,30,50-triiodobenzoyl]oxoethyl methacrylate copolymer, radio-opacity, in vitro and in vivo biocompatibility. Acta Biomater., 4, 1762-1769, 2008.

  24. Filmon R., Retailleau-Gaborit N., Brossard G., Grizon-Pascaretti F., Baslé M.F., Chappard D.  Preparation of β-TCP granular material by polyurethane foam technology. Image Anal. Stereol.  28:1-10, 2009.

  25. Chappard D., Baslé M.F., Legrand E., Audran M. Trabecular bone microarchitecture: a review. Morphologie 92, 162-170, 2008.

  26. Chappard D. Technical aspects: how do we best prepare bone samples for proper histological analysis? In: Heymann D, editor. "Bone cancer: progression and therapeutic approaches". London: Acad. Press. Elsevier Inc. p 203-210, 2009.

  27. Dumas A., Brigitte M., Moreau M.F., Chretien F., Baslé M. F. Chappard D. Bone mass and microarchitecture of irradiated and bone marrow-transplanted mice: influences of the donor strain. Osteoporosis Int. 20, 435-443, 2009

  28. Filmon R., Retailleau-Gaborit N., Brossard G., Grizon-Pascaretti F., Baslé M.F., Chappard D. Preparation of b-TCP granular material by polyurethane foam technology. Image Anal. Stereol. 28, 1-10, 2009.

  29. Libouban H., Massin P., Gaudin C, Mercier P., Basle, M.F., Chappard D. Migration of wear debris of polyethylene depends of bone microarchitecture. J. Biomed. Mater. Res –Appl. Biomat. B 90, 730-737, 2009.

  30. Chappard D., Libouban H., Legrand E., Ifrah, N., Masson C., Baslé M.F., Audran M. Computed microtomography of bone specimens for rapid analysis of bone changes associated with malignancy. Anat. Rec. 293, 1125-1133, 2010.
  31. Guillaume B., Libouban H., Baslé M.F., Chappard D. Comblement sinusien par β-TCP avant pose d’implants chez l’homme : Étude clinique et histologique. Titane, 7, 201-210, 2010.
  32. Nyangoga H., Blouin S., Libouban H., Baslé M.F., Chappard D. A single pretreatment by zoledronic acid converts metastases from osteolytic to osteoblastic in the rat. Microsc. Res. Techn. 73, 733-740, 2010.
  33. Nyangoga H., Aguado E., Goyenvalle E., Baslé M.F., Chappard D. A Non Steroidal Anti Inflammatory Drug (Ketoprofen) Does Not Delay b-Tcp Bone Graft Healing. Acta Biomater. 6, 3310–3317, 2010.
  34. Chappard D., Bouvard B., Basle M.F., Legrand E., Audran M. Bone metastasis: histological changes and pathophysiological mechanisms in osteolytic or osteosclerotic localizations. A review. Morphologie 95, 65-75, 2011
  35. Chappard D., Baslé M.F., Legrand E., Audran M. New laboratory tools in the assessment of bone quality. Osteoporosis Int. 22, 2225–2240, 2011.
  36. Nyangoga H., Mercier P., Libouban H., Baslé M.F., Chappard D. Three-dimensional characterization of the vascular bed in metastatic bone of rat. PlosOne 28, 6, e17336, 2011.
  37. Audran M., Maury E., Bouvard B., Legrand E., Baslé M.F., Chappard D. Is trans-iliac bone biopsy a painful procedure? Clin. Nephrol. 77, 97-104, 2012.
  38. Bodic F., Amouriq Y., Gayet-Delacroix M., Maugars Y., Hamel L., Baslé M.F., Chappard D. Relationships between mandibular and iliac bone mass and microarchitecture in endentulated subjects: a DXA, CT and microCT study. Gerodontology, 29, e585-e594, 2012.
  39. Bouvard B., Gallois Y., Legrand E., Baslé M.F. Audran M., Chappard D. Glucocorticoids reduce alveolar bone at the mandible in mice. Joint Bone Spine on line DOI 10.1016/j.jbspin.2012.01.009, 2012.
  40. Bouvard B., Mabilleau G., Legrand E., Audran M., Chappard D. Micro and macroarchitectural changes at the tibia after botulinum toxin injection in the growing rat. Bone. 50, 858-864, 2012.
  41. Deprez P., Nichane M., Rousseaux P., Devogelaer J.P., Chappard D., Lengelé B., Rezsöhazy R., Nyssen-Behets C. Postnatal growth defect in mice upon persistent Hoxa2 expression in the chondrogenic cell lineage. Differentiation 83, 158-67, 2012.
  42. Mallard F., Bouvard B., Mercier P., Bizot P., Cronier P., Chappard D. Trabecular microarchitecture in established osteoporosis: relationship between vertebrae, distal radius and calcaneus by texture analysis of X-ray images.Orthop.Traumatol. Surg. Res. 99, 52-59, 2013.
  43. Bouvard B., Gallois Y., Legrand E., Baslé M.F. Audran M., Chappard D. Glucocorticoids reduce alveolar bone at the mandible in mice. Joint Bone Spine 80, 77-81, 2013.
  44. Koufany M., Moulin D., Maire T., Netter P., Weryha G., Chappard D., Jouzeau J.Y. The Peroxisome Proliferator-Activated Receptor gamma (PPARγ) agonist pioglitazone preserves bone micro-architecture in experimental arthritis. Arthritis & Rheumatism 65, 3084-3095, 2013.
  45. N’Diaye M., Degeratu C., Bouler J.M., Chappard D. Biomaterials porosity determined by fractal dimensions, succolarity and lacunarity on microcomputed tomographic images. Mater. Sci. Engin. C Mater Biol Appl. 33, 2025-2030, 2013.
  46. Aguado E., Gaudin-Audrain C.,Goyenvalle E., Pascaretti-Grizon F., Chappard D. β-TCP granules mixed with reticulated hyaluronic acid induce an increase bone apposition. Biomed. Mater. 9, 015001.doi:10.1088/1748-6041/9/1/015001, 2014.
  47. N’Diaye M.,Terranova L., Mallet R., Mabilleau G., Chappard D. Three-dimensional arrangement of β-tricalcium phosphate granules evaluated by microcomputed tomography and fractal analysis. Biomed. Mater. online DOI: 10.1016/j.actbio.2014.09.015, 2014.
  48. N’Diaye M., Libouban H., Aguado E., Audran M., Chappard D. Unwrapping microcomputed tomographic images for measuring cortical osteolytic lesions in the 5T2 murine model of myeloma treated by bisphosphonate. Micron, online, DOI: 10.1016/j.micron.2014.10.001, 2014.

 

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