Spatial and temporal changes of subchondral bone proceed to articular cartilage degeneration in rats subjected to knee immobilization

This study was aimed to investigate the spatial and temporal changes of subchondral bone and its overlying articular cartilage in rats following knee immobilization. A total of 36 male Wistar rats (11–13 months old) were assigned randomly and evenly into 3 groups. For each group, knee joints in 6 rats were immobilized unilaterally for 1, 4, or 8 weeks, respectively, while the remaining rats were allowed free activity and served as external control groups. For each animal, femurs at both sides were dissected after sacrificed. The distal part of femur was examined by micro‐CT. Subsequently, femoral condyles were collected for further histological observation and analysis. For articular cartilage, significant changes were observed only at 4 and 8 weeks of immobilization. The thickness of articular cartilage and chondrocytes numbers decreased with time. However, significant changes in subchondral bone were defined by micro‐CT following immobilization in a time‐dependent manner. Immobilization led to a thinner and more porous subchondral bone plate, as well as a reduction in trabecular thickness and separation with a more rod‐like architecture. Changes in subchondral bone occurred earlier than in articular cartilage. More importantly, immobilization‐induced changes in subchondral bone may contribute, at least partially, to changes in its overlying articular cartilage. Microsc. Res. Tech. 79:209–218, 2016. © 2016 Wiley Periodicals, Inc.     

Feasibility of detecting trabecular bone around percutaneous titanium implants in rabbits by in vivo microfocus computed tomography

Objectives: The goal of this study was to examine the feasibility of in vivo imaging of trabecular bone around titanium implants by means of microfocus computed tomography (micro‐CT) and the use of rabbits for this purpose. Materials and Methods: Ten male rabbits type Hollander, received a titanium implant (1.7 mm diameter and 10 mm length) in the trabecular bone of the left tibia. Seven weeks later a micro‐CT scan was taken. Four rabbits were used to monitor potential harmful effects from X‐ray absorption until 4 weeks after scanning. A second group of six rabbits was used for testing the hypothesis that a good correlation exists between in vivo micro‐CT images and histological images of trabecular bone around titanium implants. The six rabbits were scanned and sacrificed immediately. The tibias were extracted and submitted to standard histological procedures. This resulted in a total of 12 histological sections and their corresponding 12 micro‐CT images. Bone area measurements were performed at the left and right side of the implant in three regions: 0–500, 500–1000 and 1000–1500 μm distance from the implant interface. Intra‐class correlations (ICC) were calculated between both techniques. Results: The four rabbits did not show any sign of radiodermatitis 4 weeks after scanning. In the micro‐CT images of the group of six rabbits, trabeculae are visible, but not well defined, due to the presence of noise in the image. The ICC for the right implant side were 0.44 for zone 0–500 μm, 0.48 for zone 500–1000 μm and 0.40 for zone 1000–1500 μm. The ICC for the left implant side could not be calculated. Conclusion: A low agreement was found between the bone measurements from histology and in vivo micro‐CT images. The use of the in vivo micro‐CT for trabecular bone imaging around metallic implants should be restricted to track tendencies in follow‐up studies.     

Histochemical evidence of the initial chondrogenesis and osteogenesis in the periosteum of a rib fractured model: implications of osteocyte involvement in periosteal chondrogenesis

We have examined cellular events at the early stages of periosteal chondrogenesis and osteogenesis induced by bone fracture, using a well-standardized rib fracture model of the mouse. The initial cellular event was recognized as considerable proliferation in the deeper layer referred to as the "cambium layer" of the periosteum, as evidenced by numerous proliferating cell nuclear antigen-positive cells. The periosteal cartilage and bone were then regenerated directly from the region of the most-differentiated cell, i.e., mature osteoblasts of the cambium layer both close to and distant from the fracture site. Therefore, periosteal osteoblasts appeared to have the potential to differentiate into chondrogenic and osteoblastic lineages. CD31-positive blood vessels were uniformly localized along the periosteum that was regenerating cartilage and bone, being therefore indicative of less influence on the initiation of osteochondrogenesis. In contrast, however, the regenerated periosteal cartilage or bone extended from the cortical bones included dead or living osteocytes, respectively. Empty lacunae and lacunae embedded with amorphous materials were found close to the regenerated cartilage, while intact osteocytes persisted adjacent to the regenerated bone. The embedded lacunae with amorphous materials would render the tissue fluid, nutrients, oxygen, and several secretory factors such as dentin matrix protein-1 impossible to be delivered to the periosteal osteoblasts that interconnect osteocytes via gap junctions. Our study thus provides two major clues on initial cellular events in response to bone fracture: the potentiality of periosteal osteoblastic differentiation into a chondrogenic lineage, and a putative involvement of osteocytes in periosteal cartilage and bone regeneration.     

Osteocyte staining with rhodamine in osteonecrosis and osteoarthritis of the femoral head

Death of osteocytes is synonymous of bone death. Aseptic osteonecrosis of the femoral head is a lesion characterized by the death of osteocytes occurring after major vascular changes. The evolution may lead to hip osteoarthritis, which requires total hip arthroplasty in most cases. Evolution of aseptic osteonecrosis in four radiological stages is well known. We analyzed 24 femoral heads from patients with osteonecrosis or osteoarthritis, retrieved at the time of surgery for a hip arthroplasty. The aim of the study was to clearly identify the necrotic bone from the living bone in the histological samples. The femoral heads were sawed, and a large sample was harvested in the superior zone; it was stained en‐bloc with rhodamine dissolved in formalin to make the osteocytes fluorescent under UV light microscopy. Undecalcified sections, 7 μm thick, were obtained on a heavy‐duty microtome. A micrographic analysis using two UV excitation wavelengths visualized the living osteocytes (in green) and the bone matrix (in blue). A simple method to prepare combined images is described. In addition, the blocks can be analyzed by confocal microscopy to visualize more details. It is possible to identify at low magnification the osteocytes within the bone matrix and the osteonecrotic areas where osteocytes have disappeared. Identification of osteocytes showed that newly formed bone packets are laid on dead trabeculae in patients with aseptic osteonecrosis or with osteoarthritis. In the osteosclerotic areas, the enlarged trabeculae have a dead central core surrounded by recently apposed bone structure units.     

Quantitative zonal differentiation of articular cartilage by microscopic magnetic resonance imaging,polarized light microscopy,and Fourier‐transform infrared imaging

This study aimed to synchronize the zonal differentiation of the full‐thickness articular cartilage by three micro‐imaging techniques, namely microscopic magnetic resonance imaging (µMRI), polarized light microscopy (PLM), and Fourier‐transform infrared imaging (FTIRI). Eighteen cartilage‐bone blocks from three canine humeral joints were imaged by: (a) µMRI T₂ relaxation at 0° and 55° orientations in a 7 T magnetic field, (b) PLM optical retardation and azimuthal angle, and (c) FTIRI amide I and amide II anisotropies at 0° and 90° polarizations relative to the articular surface. In addition, µMRI T₁ relaxation was imaged before and after the tissue being immersed in gadolinium (contrast agent) solution, to calculate the proteoglycan concentration. A set of previously established criteria in cartilage imaging was revised. The new criteria could simultaneously correlate the thicknesses of the three consecutive subtissue zones in articular cartilage among these imaging techniques. Microsc. Res. Tech. 76:625–632, 2013. © 2013 Wiley Periodicals, Inc.     

Full supervised learning for osteoporosis diagnosis using micro‐CT images

Early osteoporosis diagnosis is of important significance for reducing fracture risk. Image analysis provides a new perspective for noninvasive diagnosis in recent years. In this article, we propose a novel method based on machine‐learning method performed on micro‐CT images todiagnose osteoporosis. The aim of this work is to find a way to more effectively and accurately diagnose osteoporosis on which many methods have been proposed and practiced. In this method, in contrast to the previously proposed methods in which features are analyzed individually, several features are combined to build a classifier for distinguishing osteoporosis group and normal group. Twelve features consisting of two groups are involved in our research, including bone volume/total volume (BV/TV), bone surface/bone volume (BS/BV), trabecular number (Tb.N), obtained from the software of micro‐CT, and other four features from volumetric topological analysis (VTA). Support vector machine (SVM) method and k‐nearest neighbor (kNN) method are introduced to create classifiers with these features due to their excellent performances on classification. In the experiment, 200 micro‐CT images are used in which half are from osteoporosis patients and the rest are from normal people. The performance of the obtained classifiers is evaluated by precision, recall, and F‐measure. The best performance with precision of 100%, recall of 100%, and F‐measure of 100% is acquired when all the features are included. The satisfying result demonstrates that SVM and kNN are effective for diagnosing osteoporosis with micro‐CT images. Microsc. Res. Tech. 76:333–341, 2013. © 2013 Wiley Periodicals, Inc.     

Effect of treatment with simvastatin on bone microarchitecture of the femoral head in an osteoporosis animal model

The objective of this study was to evaluate the microarchitecture and trabecular bone strength at the distal region of the femur, and its biomechanical properties with simvastatin administration with two different doses in ovariectomized (OVX) rats. Ninety rats were divided into six groups to evaluate treatment with the simvastatin drug (n = 15): SH (Sham surgery), SH‐5 (5 mg simvastatin), SH‐20 (20 mg simvastatin), OVX, OVX‐5, and OVX‐20. Euthanasia was performed at three different times, five animals per period: 7, 14, and 28 days. The effectiveness of the treatments was evaluated by mechanical testing and histomorphometric analysis of the femurs. The results of analysis by the linear model of mixed effects showed 20 mg of simvastatin results in increased trabecular bone after 14 days (P = 0.039) of ingestion in ovariectomized animals. However, ingestion of 5 mg of simvastatin is able to sensitize the trabecular bone only at 28 days (P = 0.005) of ingestion. In the mechanical tests stiffness improves within 28 days (P = 0.003). Regarding maximum strength, no statistical differences were observed. According to these results, it can be concluded that for a decrease in oral intake, longer treatment times are required. Microsc. Res. Tech. 79:684–690, 2016. © 2016 Wiley Periodicals, Inc.     

In vivo response to HA-polyhydroxybutyrate/polyhydroxyvalerate composite

This study examined the morphological and compositional structure of bone–implant interfaces after in vivo implantation into the tibias of rabbits. The implants were composed of biodegradable polyhydroxybutyrate/polyhydroxyvalerate copolymer reinforced with synthetic hydroxyapatite (HA) particles. Optical and scanning electron microscopy techniques were used, including energy‐dispersive X‐ray analysis. The interface was found to be morphologically, biologically and chemically active throughout the period of study. There was a strong tendency to rebuild the bone structure at the interface after implantation, independent of the composition of the implant, but direct bone bonding with the implant depended on the bioactive nature of the interface, as represented by the HA particles. At all implantation times, lamellar bone formed at the interface and replaced degrading polymer matrix, while engulfing HA filler particles. In regions about 50–100 from the interface, the bone region displayed an osteon organization. Osteoblasts and osteocytes were identified throughout the interface region. The thickness of the newly formed bone significantly increased over the period of the experiment from about 130 µm at 1 month to about 770 µm at 6 months. Materials that behave in this manner may be useful in some bone replacement therapies.     

Method for the study of the three-dimensional orientation of the nuclei of myocardial cells in fetal human heart by means of confocal scanning laser microscopy

A series of three-dimensional image analysis tools are used to measure the three-dimensional orientation of nuclei of myocardial cells. Confocal scanning laser microscopy makes it possible to acquire series of sections up to 100 μm inside thick tissue sections. A mean orientation vector of unit length is calculated for each segmented nucleus. The global orientation statistics are obtained by calculating the vectorial sum of the nuclear unit vectors. The final orientation is expressed by a mean azimuth angle, an elevation angle and a measure of the angular homogeneity. The method is illustrated for two different regions of the myocardium (interventricular septum and papillary muscle) of a normal human fetal heart. This quantitative method will be used to assess and calibrate the information provided by polarized light microscopy.     

Autologous resurfacing

Background: There is a discrepancy between the interest in joint‐reconstructions and the current knowledge about the healing‐processes involved. Major reconstructions are performed with osteosynthesized allografts and fresh allografts for cartilage. Objectives: The main question to be answered is: what do we know about metaphyseal and epiphyseal cancellous bone healing, contact healing of the subchondral bone and its influence on cartilage healing? Can we achieve healing of all four compartments in contact? Purpose: The purpose is to systematically investigate through animal testing the healing processes of metaphyseal and epiphyseal bone, including the subchondral bone and the healing of cartilage of press‐fit‐inserted grafts, considering nondemineralized high‐resolution histology. Material and Methods: Primary cancellous‐bone healing of osteosynthesized hemi‐osteotomies was studied in 13 canine tibial heads, the contact healing was investigated in 7 dogs and 18 giant‐rabbits comparing contact‐healing of press‐fit‐inserted autologs cylindrical grafts with empty defects applying the wet‐grinding diamond‐technology. Bench‐experiments on the epiphyseal bones of swine including pullout‐tests of cylindrical grafts formed the basis for validation of that press‐fit diamond technology. Results: Primary metaphyseal and epiphyseal contact healing, including hyaline cartilage, was found in all compartments of the meta‐and epiphysis in the precisely performed experiments. The press‐fit principle, which employs cylindrical grafts and diamond instrumentation featuring a difference of 15/100 mm between graft and recipient bed, achieved high loads between 73.48 and 178.95 N (mean value 118.16 and standard deviation 32.79) in the pullout tests. Conclusion: Autologous press‐fit grafting with alignment of the bony baseplate using wet‐grinding precision has attained promising histo‐morphological results. Microsc. Res. Tech., 78:40–51, 2015. © 2014 Wiley Periodicals, Inc.     

Modified enzymatic collagen digestion-mediated isolation of osteocytes

This study established a method for isolating large numbers of high-purity osteocytes from high-density bone. Bone fragments derived from mice tibia and femurs were alternately digested with type I collagenase and EDTA nine times, and the digested cells and bone chips (BC) were cultured, digested, and passaged when cells were fully grown. The types of cells obtained were identified by morphology, viable cell counts, alkaline phosphatase staining, and biochemical activity analyses, and specific osteocyte and osteoblast markers were evaluated by quantitative real-time polymerase chain reaction. Our results showed that among the cells obtained from the third digestion (fractions 7–9) of wild mice tibias and femurs and the remaining BCs, 85%–90% of the cells were osteocytes. Moreover, their morphology was approximately one-tenth to one-fifth the size of osteoblasts, star-shaped or polygonal, with a dendritic structure, negative for alkaline phosphatase staining, and showed a high expression of dmp1 and sclerostin. Ninety percent of the cells in fractions 1–3 were osteoblasts, and were fusiform or polygonal shape. The activity of osteoblast-specific alkaline phosphatase and mRNA expression were high in this fraction, while the expression of osteocyte-specific dmp1 and sclerostin was not detected. In the second portion (fractions 4–6), a large number were osteoblasts, mixed with a small number of osteocytes, and had high alkaline phosphatase activity and osteocyte mRNA levels, a specific level of the osteocyte marker dmp1, and no sclerostin was detected. Osteocytes in daβcat^ot mice were also successfully isolated by this method, and we found that Wnt signaling increased the proliferation of these osteocytes. The proposed method can be used to culture osteocytes and osteoblasts of high purity and can be used for isolation and culture of these two kinds of cells from high-density bone, which provides an avenue for the study of osteocyte function in vitro.     

A biomechanical study of osteoporotic vertebral trabecular bone: The use of micro-CT and high-resolution finite element analysis

Osteoporosis is one of the most dangerous skeletal diseases in relation to the highest fracture risk in vertebral bones. A considerable amount of work has been done to investigate the biomechanical characteristics of osteoporotic vertebral trabecular bone. Previous researchers studied the elastic characteristics using a micro-finite element (micro-FE) model, used to analyze realistic trabecular architectures in full detail, based on micro-computed tomography (μCT). Since osteoporotic compression fracture is closely associated with the mechanical characteristics of the vertebral trabecular bone and there were few micro-FE models to account for all of the elastic and plastic characteristics in vertebral trabecular bone, this study analyzed the effect of voxel resolution on the plastic characteristics as well as the elastic characteristics of three-dimensional (3D) osteoporotic lumbar trabecular bone models. Also, we evaluated the effect of specimen geometry on this problem. It has been reported that a cubic specimen with side length 6.5mm was suggested as standard specimens for the experimental test of trabecular bone. Current study examined whether or not the effect of the specimen geometry on the experimental test may be also applied to the simulated compression test of trabecular bone specimens. The experimental test employing the rapid prototyping (RP) technique and INSTRON test machine is performed to indirectly validate the results of the simulated compression test by micro-FE analysis. The review finished with the verification about the effects of the simulated compression test.     

Prediction of cement volume for vertebroplasty based on imaging and biomechanical results

Control of bone cement volume (PMMA) may be critical for preventing complications in vertebroplasty, the percutaneous injection of PMMA into vertebra. The purpose of this study was to predict the optimal volume of PMMA injection based on CT images. For this, correlation between PMMA volume and textural features of CT images was examined before and after surgery to evaluate the appropriate PMMA amount. The gray level run length analysis was used to determine the textural features of the trabecular bone. Estimation of PMMA volume was done using 3D visualization with semi-automatic segmentation on postoperative CT images. Then, finite element (FE) models were constructed based on the CT image data of patients and PMMA volume. Appropriate material properties for the trabecular bone were assigned by converting BMD to elastic modulus. Structural reinforcement due to the changes in PMMA volume and BMD was assessed in terms of axial displacement of the superior endplate. A strong correlation was found between the injected PMMA volume and the area of the intertrabecular space and that of trabecular bone calculated from the CT images (r-0.90 and −0.90, respectively). FE results suggested that vertebroplasty could effectively reinforce the osteoporotic vertebra regardless of BMD or PMMA volume. Effectiveness of additional PMMA injection tended to decrease. For patients with BMD well lower than 50mg/ml, injection of up to 30% volume of the vertebral body is recommended. However, less than 30% is recommended otherwise to avoid any complications from excessive PMMA because the strength has already reached the normal level.     

Mouse calvarial defect Model: An approach for the micro‐tomographic evaluation of polymer scaffolds

The ability of bone repair scaffolds to form bone is traditionally evaluated using cell culture and animal experiments. Mouse calvarial organ culture maintains the natural cell‐to‐cell and cell‐to‐matrix relationships as well as the anatomical order, and this model has been used to study the biological behavior of intramembranous bones. The aim of this study was to evaluate the potential of mouse calvarial organ culture to be used as an in vitro model to study the bone regenerative ability of bone repair polymer scaffolds. Critical size defects (CSD) were created in the parietal bones. Electrospun poly(ε‐caprolactone) scaffolds were placed into one group of defects. The remaining defects served as a control. The bones were cultured for 38 days and analyzed with μCT, phase‐contrast microscopy, dissecting microscopy, scanning electron microscopy, and energy‐dispersive X‐ray analyses. This organ culture technique is easily available and could permit researchers to quickly establish a valuable database of candidate bone repair scaffolds. Microsc. Res. Tech. 77:1037–1043, 2014. © 2014 Wiley Periodicals, Inc.     

Histological analysis of the alterations on cortical bone channels network after radiotherapy: A rabbit study

The aim of this study was to evaluate the effects of radiotherapy in cortical bone channels network. Fourteen rabbits were divided in two groups and test group received single dose of 15 Gy cobalt‐60 radiation in tibia, bilaterally. The animals were sacrificed and a segment of tibia was removed and histologically processed. Histological images were taken and had their bone channels segmented and called regions of interest (ROI). Images were analyzed through developed algorithms using the SCILAB mathematical environment, getting percentage of bone matrix, ROI areas, ROI perimeters, their standard deviations and Lacunarity. The osteocytes and empty lacunae were also counted. Data were evaluated using Kolmogorov‐Smirnov, Mann Whitney, and Student's t test (P < 0.05). Significant differences in bone matrix percentage, area and perimeters of the channels, their respective standard deviations and lacunarity were found between groups. In conclusion, the radiotherapy causes reduction of bone matrix and modifies the morphology of bone channels network. Microsc. Res. Tech. 73:1015–1018, 2010. © 2010 Wiley‐Liss, Inc.     

Application of autofluorescence robotic histology for quantitative evaluation of the 3‐dimensional morphology of murine articular cartilage

Murine models of osteoarthritis (OA) are increasingly important for understating pathogenesis and for testing new therapeutic approaches. Their translational potential is, however, limited by the reduced size of mouse limbs which requires a much higher resolution to evaluate their articular cartilage compared to clinical imaging tools. In experimental models, this tissue has been predominantly assessed by time‐consuming histopathology using standardized semi‐quantitative scoring systems. This study aimed to develop a novel imaging method for 3‐dimensional (3D) histology of mouse articular cartilage, using a robotic system—termed here “3D histocutter”—which automatically sections tissue samples and serially acquires fluorescence microscopy images of each section. Tibiae dissected from C57Bl/6 mice, either naïve or OA‐induced by surgical destabilization of the medial meniscus (DMM), were imaged using the 3D histocutter by exploiting tissue autofluorescence. Accuracy of 3D imaging was validated by ex vivo contrast‐enhanced micro‐CT and sensitivity to lesion detection compared with conventional histology. Reconstructions of tibiae obtained from 3D histocutter serial sections showed an excellent agreement with contrast‐enhanced micro‐CT reconstructions. Furthermore, osteoarthritic features, including articular cartilage loss and osteophytes, were also visualized. An in‐house developed software allowed to automatically evaluate articular cartilage morphology, eliminating the subjectivity associated to semi‐quantitative scoring and considerably increasing analysis throughput. The novelty of this methodology is, not only the increased throughput in imaging and evaluating mouse articular cartilage morphology starting from conventionally embedded samples, but also the ability to add the third dimension to conventional histomorphometry which might be useful to improve disease assessment in the model.     

Non‐Contact Evaluation of Osmosis‐Induced Shrinkage and Swelling Behavior of Articular Cartilage In‐Situ Using High‐Frequency Ultrasound

Abstract

The aim of the present study was to use high‐frequency ultrasound for the investigation of the transient osmosis‐induced free shrinkage‐swelling behaviors of normal articular cartilage in situ. Full‐thickness cartilage‐bone specimens were prepared from normal bovine patellae. The transient shrinkage and swelling strains of the cartilage induced by changing the bathing solution between physiological saline (0.15 M) and hypertonic saline (2 M) were monitored using a 50 MHz focused ultrasound beam. Both shrinkage and swelling strains showed temporary overshoots, followed by relaxation phases. The absolute peak value of the shrinkage strain (1.01%±0.62%) was significantly larger (p<0.05) than that of the swelling strain (0.40%±0.33%). It was found that the change of the mean ultrasound speed in cartilage could be approximately represented by an exponential function of time after the change of saline concentration.

This study successfully demonstrated that a high‐frequency focused ultrasound beam could be used to monitor the transient osmosis‐induced deformation of articular cartilage in a non‐contact way. Since the osmosis‐swelling behavior of cartilage relates to its compositional and structural characteristics and degeneration status, the reported ultrasound method may have potential for the characterization of cartilage degeneration, such as osteoarthritis.     

Scanning electron microscopy and histomorphology of the epiglottis in dromedaries: A study revealing unusual structure with the probable functional role

While epiglottis is essentially a mammalian structure, studying its microstructure in any placental model will add an important information to the field of comparative anatomy and the related branches of biology. The aim of this study was to describe the structure of the epiglottis in dromedary camels using light and scanning electron microscopy (SEM), with reference to the possible functions. A total of 11 epiglottis cartilages from 11 larynges were used. The study revealed unusual, deeply situated glands just beneath the cartilage plate. They have unusually, wide surface‐openings, while their ducts were partly located within the cartilage. This is presumed to be an adaptation to the need for rapid and efficient mucosal surface hydration in the arid conditions. The possible secretion transport mechanisms in these glands were also discussed. Furthermore, the SEM revealed for the first time, the presence of taste buds in camel epiglottis. However, in histological sections, visibility of taste buds was dependent upon the staining techniques. The taste buds were not seen with standard H& E stain, as they blended imperceptibly with the surrounding epithelium. Conversely, Mallory's trichrome showed contrasting colors, and taste buds were visible. In conclusion, camel epiglottis has an unusual structure, which may be correlated to environmental adaptation and important for the general health of upper respiratory tract in this species.     

The preparation of human articular cartilage for scanning electron microscopy

This paper compares sixteen preparative techniques thought to be of advantage in the study by scanning electron microscopy (SEM) of human articular cartilage surfaces. The adequacy of surface preservation obtained with the techniques, was judged subjectively, first, by the reproducibility of secondary electron images of normal cartilage, and second, by comparing the results with those obtained by reflected light microscopy of the fresh unfixed cartilage surface over a magnification range of × 20 – × 240. Adequate surface preservation was confirmed when cartilage surfaces had been dehydrated through ethanol to propylene oxide and vacuum dried; dehydrated through amyl acetate and quenched in Freon before freeze-drying; dehydrated and passed through amyl acetate at low temperature before freeze drying. Valuable information can be obtained from different specimens by varying the technique of preparation. At different ages, different surface features are best preserved. In a systematic study it has been found essential to adopt a uniform preparative method and to control the results by reflected light microscopy. Even with the most perfect preparation, the surface appearances cannot be identical with those that function under load in vivo.     

Stereological measures of trabecular bone structure: comparison of 3D micro computed tomography with 2D histological sections in human proximal tibial bone biopsies

Stereology applied on histological sections is the ‘gold standard’ for obtaining quantitative information on cancellous bone structure. Recent advances in micro computed tomography (µCT) have made it possible to acquire three-dimensional (3D) data non-destructively. However, before the 3D methods can be used as a substitute for the current ‘gold standard’ they have to be verified against the existing standard. The aim of this study was to compare bone structural measures obtained from 3D µCT data sets with those obtained by stereology performed on conventional histological sections using human tibial bone biopsies. Furthermore, this study forms the first step in introducing the proximal tibia as a potential bone examination location by peripheral quantitative CT and CT. Twenty-nine trabecular bone biopsies were obtained from autopsy material at the medial side of the proximal tibial metaphysis. The biopsies were embedded in methylmetacrylate before µCT scanning in a Scanco µCT 40 scanner at a resolution of 20 × 20 × 20 µm³, and the 3D data sets were analysed with a computer program. After µCT scanning, 16 sections were cut from the central 2 mm of each biopsy and analysed with a computerized method. Trabecular bone volume (BV/TV) and connectivity density (CD) were estimated in both modalities, whereas trabecular bone pattern factor (TBPf) was estimated on the histological sections only. Trabecular thickness (Tb.Th), number (Tb.N) and separation (Tb.Sp), and structure model index (SMI) were estimated with the µCT method only. Excellent correlations were found between the two techniques for BV/TV (r = 0.95) and CD (r = 0.95). Additionally, an excellent relationship (r = 0.95) was ascertained between TBPf and SMI. The study revealed high correlations between measures of bone structure obtained from conventional 2D sections and 3D µCT data. This indicates that 3D µCT data sets can be used as a substitute for conventional histological sections for bone structural evaluations.