Repair of rabbit radial bone defects using bone morphogenetic protein-2 combined with 3D porous silk fibroin/ß-tricalcium phosphate hybrid scaffolds.

Our study aimed to investigate the effect of bone morphogenetic protein-2 (BMP-2) bound to silk fibroin and ß-tricalcium phosphate (SF/ß-TCP) hybrid on the healing of critical-size radial defects in rabbits. A 15-mm critical-size defect was induced at mid-diaphysis in the left radius of 20 New Zealand white rabbits (average age, 3.5 months; weight, 2.5-3.0 kg). The animals were randomized into Group 1 (SF/ß-TCP combined with BMP-2), Group 2 (SF/ß-TCP alone), and Group 3 (nothing implanted). Radiographs were obtained every 2 weeks and euthanasia was performed after 8 weeks for visual, radiological, micro-computed tomography (micro-CT), and histological studies. Eight weeks after implantation (SF/ß-TCP combined with BMP-2), radiographs showed that new bone formed on the surface of the implant and had bridged the defect in Group 1. Micro-CT imaging also confirmed the formation of new bone around the implant, and the newly formed bone was quantified. Histological examination revealed newly formed bone in the implanted area. Meanwhile, there was no formation of new bone in Group 3. Among the groups, most active formation of new bones was found in Group 1, while there was no difference between Group 2 and Group 3. Based on these results, we concluded that BMP-2-SF/ß-TCP showed significant improvement in healing of critical-size defects. Therefore, the combination of BMP-2 and SF/ß-TCP would be useful in the field of bone tissue engineering.

Autogenous bone particle/titanium fiber composites for bone regeneration in a rabbit radius critical-size defect model.

PURPOSE: To explore the effects of autogenous bone particle/titanium fiber composites on repairing segmental bone defects in rabbits. MATERIALS AND METHODS: A model of bilateral radial bone defect was established in 36 New Zealand white rabbits which were randomly divided into 3 groups according to filling materials used for bilaterally defect treatment: in group C, 9 animal bone defect areas were prepared into simple bilateral radius bone defect (empty sham) as the control group; 27 rabbits were used in groups ABP and ABP-Ti. In group ABP, left defects were simply implanted with autogenous bone particles; meanwhile, group ABP-Ti animals had right defects implanted with autogenous bone particle/titanium fiber composites. Animals were sacrificed at 4, 8, and 12 weeks, respectively, after operation. RESULTS: Micro-CT showed that group C could not complete bone regeneration. Bone volume to tissue volume values in group ABP-Ti were better than group ABP. From histology and histomorphometry Groups ABP and ABP-Ti achieved bone repair, the bone formation of group ABP-Ti was better. The mechanical strength of group ABP-Ti was superior to that of other groups. CONCLUSIONS: These results confirmed the effectiveness of autologous bone particle/titanium fiber composites for promoting bone regeneration and mechanical strength.

7T MRI of distal radius trabecular bone microarchitecture: How trabecular bone quality varies depending on distance from end-of-bone.

PURPOSE: To use 7T magnetic resonance imaging (MRI) to determine how trabecular bone microarchitecture varies at the epiphysis, metaphysis, and diaphysis of the distal radius. MATERIALS AND METHODS: The distal radius of 24 females (mean age = 56 years, range = 24-78 years) was scanned on a 7T MRI using a 3D fast low-angle shot sequence (0.169 × 0.169 × 1 mm). Digital topological analysis was applied at the epiphysis, metaphysis, and diaphysis to compute: total trabecular bone volume; trabecular thickness, number, connectivity, and erosion index (a measure of network resorption). Differences and correlations were assessed using standard statistical methods. RESULTS: The metaphysis and epiphysis had 83-123% greater total bone volume and 14-16% greater trabecular number than the diaphysis (both P < 0.0001). The erosion index was significantly higher at the diaphysis than the metaphysis and epiphysis (both P < 0.01). The most elderly volunteers had lower trabecular number (<66 years mean 0.29 ± 0.01; ≥66 years, 0.27 ± 0.02, P < 0.05) and higher erosion index (<66 years mean 1.18 ± 0.17; age ≥66 years, mean 1.42 ± 0.46, P < 0.05) at the epiphysis; differences not detected by total trabecular bone volume. CONCLUSION: 7T MRI reveals trabecular bone microarchitecture varies depending on scan location at the end-of-bone, being of overall higher quality distally (epiphysis) than proximally (diaphysis). Age-related differences in trabecular microarchitecture can be detected by 7T MRI. The results highlight the potential sensitivity of 7T MRI to microarchitectural differences and the potential importance of standardizing scan location for future clinical studies of fracture risk or treatment response. LEVEL OF EVIDENCE: 3 J. Magn. Reson. Imaging 2017;45:872-878.

A Study of BMP-2-Loaded Bipotential Electrolytic Complex around a Biphasic Calcium Phosphate-Derived (BCP) Scaffold for Repair of Large Segmental Bone Defect.

A bipotential polyelectrolyte complex with biphasic calcium phosphate (BCP) powder dispersion provides an excellent option for protein adsorption and cell attachment and can facilitate enhanced bone regeneration. Application of the bipotential polyelectrolyte complex embedded in a spongy scaffold for faster healing of large segmental bone defects (LSBD) can be a promising endeavor in tissue engineering application. In the present study, a hollow scaffold suitable for segmental long bone replacement was fabricated by the sponge replica method applying the microwave sintering process. The fabricated scaffold was coated with calcium alginate at the shell surface, and genipin-crosslinked chitosan with biphasic calcium phosphate (BCP) dispersion was loaded at the central hollow core. The chitosan core was subsequently loaded with BMP-2. The electrolytic complex was characterized using SEM, porosity measurement, FTIR spectroscopy and BMP-2 release for 30 days. In vitro studies such as MTT, live/dead, cell proliferation and cell differentiation were performed. The scaffold was implanted into a 12 mm critical size defect of a rabbit radius. The efficacy of this complex is evaluated through an in vivo study, one and two month post implantation. BV/TV ratio for BMP-2 loaded sample was (42±1.76) higher compared with hollow BCP scaffold (32±0.225).

Cell-free scaffolds with different stiffness but same microstructure promote bone regeneration in rabbit large bone defect model.

To promote bone healing, bone repair biomaterials are increasingly designed to incorporate growth factors. However, the impact of matrix mechanics of cell-free scaffold independent of microstructure on the osteogenic differentiation of endogenous osteoprogenitor cells orchestrating bone repair and regeneration remains not to be fully understood. In our recent study, three-dimensional (3D) scaffolds with different stiffness but same microstructure have been successfully fabricated by coating decellularized bone with collagen/hydroxyapatite (HA) mixture with different collagen rations. It has been demonstrated that the scaffold with optimal stiffness can induce the osteogenic differentiation of MSCs in vitro and in the subcutaneous tissue. The present in vivo study further investigated the repair efficiency of these scaffolds in a rabbit radius with a critical-sized segmental defect model and its potential mechanism. Micro-computed tomography (µ-CT), X-ray and histological analysis were carried out to evaluate the repair capacity of these scaffolds. The results demonstrated that the cell-free scaffold with optimal stiffness incorporation of endogenous osteoprogenitor cells significantly promoted the repair and reconstruction quality of mass bone defect. One of the crucial mechanisms was that hypoxia and stromal cell-derived factor-1α (SDF-1α) mediated mesenchymal stem cells (MSCs) migration by which matrix mechanics exerted influence on bone fracture healing. These findings suggested that only modulating the matrix stiffness of cell-free scaffold can be one of the most attractive strategies for promoting the progression of bone healing.

Repairing rabbit radial defects by combining bone marrow stroma stem cells with bone scaffold material comprising a core-cladding structure.

We prepared a bone scaffold material comprising a PLGA/ß-TCP core and a Type I collagen cladding, and recombined it with bone marrow stroma stem cells (BMSCs) to evaluate its potential for use in bone tissue engineering by in vivo and in vitro experiments. PLGA/ß-TCP without a cladding was used for comparison. The adherence rate of the BMSCs to the scaffold was determined by cell counting. Cell proliferation rate was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The osteogenic capability was evaluated by alkaline phosphatase activity. The scaffold materials were recombined with the BMSCs and implanted into a large segmental rabbit radial defect model to evaluate defect repair. Osteogenesis was assessed in the scaffold materials by histological and double immunofluorescence labeling, etc. The adherence number, proliferation number, and alkaline phosphatase expression of the cells on the bone scaffold material with core-cladding structure were significantly higher than the corresponding values in the PLGA/ß-TCP composite scaffold material (P < 0.05). An in vivo test indicated that the bone scaffold material with core-cladding structure completely degraded at the bone defect site and bone formation was completed. The rabbit large sentimental radial defect was successfully repaired. The degradation and osteogenesis rates matched well. The bone scaffold with core-cladding structure exhibited better osteogenic activity and capacity to repair a large segmental bone defect compared to the PLGA/ß-TCP composite scaffold. The bone scaffold with core-cladding structure has excellent physical properties and biocompatibility. It is an ideal scaffold material for bone tissue engineering.

Structural analysis of cortical porosity applied to HR-pQCT data.

PURPOSE: The investigation of cortical porosity is an important aspect of understanding biological, pathoetiological, and biomechanical processes occurring within the skeleton. With the emergence of HR-pQCT as a noninvasive tool suitable for clinical use, cortical porosity at appendicular sites can be directly visualized in vivo. The aim of this study was to introduce a novel topological analysis of the cortical pore network for HR-pQCT data and determine the influence of resolution on measures of cortical pore network microstructure and topology. METHODS: Cadaveric radii were scanned using HR-pQCT at two different voxel sizes (41 and 82 µm) and also using µCT at a voxel size of 18 µm. HR-pQCT and µCT image sets were spatially coregistered. Segmentation and quantification of cortical porosity (Ct.Po) and mean pore diameter (Ct.Po.Dm) were achieved using an established extended cortical analysis technique. Topological classification of individual pores was performed using topology-preserving skeletonization and multicolor dilation algorithms. Based on the pore skeleton topological classification, the following parameters were quantified: total number of planar surface-skeleton canals (N.Slabs), tubular curve-skeleton canals (N.Tubes), and junction elements (N.Junctions), mean slab volume (Slab.Vol), mean tube volume (Tube.Vol), mean slab orientation (Slab.θ), mean tube orientation (Tube.θ), N.Slabs/N.Tubes, and integral (total) slab volume/integral tube volume (iSlab.Vol/iTube.Vol). An in vivo reproducibility study was also conducted to assess short-term precision of the topology parameters. Precision error was characterized using root mean square coefficient of variation (RMSCV%). RESULTS: Correlations to µCT values for Ct.Po were significant for both the 41 and 82 µm HR-pQCT data (41: r(2) = 0.82, p < 0.001, 82: r(2) = 0.75, p < 0.001). For Ct.Po.Dm, only the 41 µm data were significantly predictive of µCT values (r(2) = 0.72, p < 0.01) Data at both HR-pQCT voxel sizes were strongly predictive of the µCT values for N.Slabs (41: r(2) = 0.93, p < 0.001; 82: r(2) = 0.84, p < 0.001), N.Tubes (41: r(2) = 0.94, p < 0.001; 82: r(2) = 0.84, p < 0.001), and N.Junctions (41: r(2) = 0.93, p < 0.001; 82: r(2) = 0.78, p < 0.001), though proportional bias was evident in these correlations. Weak correlations were seen for iSlab.Vol/iTube.Vol at both voxel sizes (41: r(2) = 0.52, p < 0.01; 82: r(2) = 0.39, p < 0.05). Slab.Vol was significantly correlated to µCT data at 41 µm (r(2) = 0.60, p < 0.01) but not at 82 µm, while Tube.Vol was significantly correlated at both voxel sizes (41: r(2) = 0.79, p < 0.001; 82: r(2) = 0.68, p < 0.01). In vivo precision error for these parameters ranged from 2.31 to 9.68 RMSCV%. CONCLUSIONS: Strong correlations between µCT- and HR-pQCT-derived measurements were found, particularly in HR-pQCT images obtained at 41 µm. These data are in agreement with our previous study investigating the effect of voxel size on standard HR-pQCT metrics of trabecular and cortical microstructure, and extend our previous findings to include topological descriptors of the cortical pore network.

Bone microarchitecture and estimated strength in 499 adult Danish women and men: a cross-sectional, population-based high-resolution peripheral quantitative computed tomographic study on peak bone structure.

High-resolution peripheral quantitative computed tomography (HR-pQCT) allows in vivo assessment of cortical and trabecular bone mineral density (BMD), geometry, and microarchitecture at the distal radius and tibia in unprecedented detail. In this cross-sectional study, we provide normative and descriptive HR-pQCT data from a large population-based sample of Danish Caucasian women and men (n = 499) aged 20-80 years. In young adults (<35 years), women (n = 100) compared to men (n = 64) had smaller total and cortical areas, inferior metric trabecular indices, higher network inhomogeneity, lower cortical porosity, and lower finite element estimated bone strength. The changes in parameters with age were estimated from multiple regression analyses. In men, with age the greatest changes (from parameter minimum or maximum) until 80 years were found for cortical porosity (1.91 IQR), BV/TV (-1.09 IQR), and trabecular thickness (-0.87 IQR) in the radius and BV/TV (-1.55 IQR), cortical BMD (-1.25 IQR), and cortical porosity (1.25 IQR) in the tibia. In women changes were most pronounced for cortical porosity (4.76 IQR), trabecular inhomogeneity (3.84 IQR), and cortical BMD (-2.86 IQR) in the radius and cortical BMD (-5.06 IQR), cortical porosity (3.86 IQR), and cortical area (-1.64 IQR) in the tibia. These findings emphasize the age- and sex-related differences in bone morphology, with men having a structural advantage over women from early adult life translating into superior indices of bone strength. With age women are further disadvantaged compared to men by greater decrements in cortical and trabecular architecture in the radius and cortical architecture in the tibia.

Prevascularisation with endothelial progenitor cells improved restoration of the architectural and functional properties of newly formed bone for bone reconstruction.

PURPOSE: The aim of this study was to examine whether the addition of endothelial progenitor cells (EPCs) contributes to restoring the architectural and functional properties of newly formed bone for reconstruction of bone defects. METHODS: Bone marrow-derived EPCs and mesenchymal stem cells (MSCs) were co-seeded onto demineralized bone matrix (DBM) as a prevascularized tissue-engineered bone (TEB) for the repair of segmental bone defects to evaluate the effects of prevascularization of TEB on ameliorating morphological, haemodynamic and mechanical characteristics. RESULTS: The restoration of the intraosseous vasculature and medullary cavity was improved markedly compared to the non-prevascularized groups. The blood supply, biomechanical strength, and bone mineral density of the prevascularized group were significantly higher than those of the non-prevascularized groups during bone reconstruction. CONCLUSIONS: The present study indicates that EPC-dependent prevascularization contributes to bone healing with structural reconstruction and functional recovery and may improve the understanding of correlation between angiogenesis and osteogenesis.

Equivalence of mean intercept length and gradient fabric tensors - 3D study.

In this study the relationship between the mean intercept length (MIL) method - the current standard histomorphometric method of assessing structural anisotropy and an alternative method of the gray-level structure tensor (GST) is investigated. Both methods are applied to a set of 25 three-dimensional binary µCT images of trabecular bone. It is shown that there is a very strong correlation between the logarithms of the principal values of the MIL and the GST fabric tensors (Pearson's coefficient of correlation higher than 0.98) and between the logarithms of the invariants of the MIL and the GST fabric tensors (Pearson's coefficient of correlation higher than 0.999). There is also a good correlation between the degree of anisotropy calculated from the MIL and from the GST tensors (Pearson's coefficient of correlation equal to 0.90). The principal anisotropy directions of the MIL and the GST fabric tensors coincide at the 5% significance level. Additionally, the performance of both methods is tested, based on a set of artificial structures with prescribed orientations.

[Effects of surface roughness of bone cements on histological characteristics of induced membranes].

OBJECTIVE: To explore surface roughness of bone cement and surround tissue on histological characteristic of induced membranes. METHODS: Bone cements with smooth and rough surface were implanted in radius bone defect, intramuscular and subcutaneous sites of rabbits, and formed induced membranes. Membranes were obtained and stained (HE) 6 weeks later. Images of membrane tissue were obtained and analyzed with an automated image analysis system. Five histological parameters of membranes were measured with thickness,area,cell density,ECM density and microvessel density. Double factor variance analysis was used to evaluate the effect of the two factors on histological characteristics of induced membranes. RESULTS: Membranes can be induced by each kind of bone cement and at all the three tissue sites. In histological parameters of thickness,area and micro vessel,there were significant differences among the membranes induced at different tissue sites (P = 0.000, P = 0.000, P = 0.000); whereas, there were no significant differences in histological parameters of cell density and ECM density (P = 0.734, P = 0.638). In all five histological parameters of membranes, there were no significant differences between the membranes induced by bone cements with different surface roughness (P = 0.506, P = 0.185, P = 0.883, P = 0.093, P = 0.918). CONCLUSION: Surround tissue rather than surface roughness of bone cements can affect the histological characteristics of induced membranes. The fibrocystic number, vascularity, mechanical tension and micro motion of the surround tissue may be closely correlated with the histological characteristics of induced membranes.

A novel cell-based therapy in segmental bone defect: using adipose derived stromal cells.

BACKGROUND: Cell-based therapy is currently focusing on bone marrow as an ideal source. However, harvesting of bone marrow is always associated with some potential morbidity. It was recently revealed that adipose derived stromal cells contain a cell population with multipotency. In addition to an adequate supply, these cells do not appear to decline with age. The purpose of our study was to assess osteogenic capability of these cells in vitro and in vivo. MATERIALS AND METHODS: Adipose derived stromal cells were isolated from New Zealand white rabbit fat pads. After primary culture in basic medium and expanded to two passages, the cells were cultured in an osteogenic medium for 2-4 wk to induce osteogenesis. In vivo, segmental bone defects were created at left radius in 30 rabbits. The cultured cells were implanted into the defects through open operation. RESULTS: Adipose derived stromal cells were able to be induced to osteogenesis confirmed by ALP and von Kossa staining. Some specific markers, such as ALP, osteopontin, osteocalcin, were also detected by PCR. The segmental defects had complete union, judged by radiographic analysis and histologic analysis, in the group transplanted with these cells. CONCLUSION: Adipose derived stromal cells have the potential to differentiate into osteogenic lineage both in vitro and in vivo. It would be a promising novel cell-based therapy for healing bone defects in the clinical setting.

A weighted osteon morphotype score outperforms regional osteon percent prevalence calculations for interpreting cortical bone adaptation.

Using circularly polarized light microscopy,we described a weighted-scoring method for quantifying regional distributions of six secondary osteon morphotypes(Skedros et al.: Bone 44 (2009) 392-403). This osteon morphotype score (MTS) strongly correlated with "tension" and "compression" cortices produced by habitual bending. In the present study, we hypothesized that the osteon MTS is superior to a relatively simpler method based on the percent prevalence (PP) of these osteon morphotypes. This was tested in proximal femoral diaphyses of adult chimpanzees and habitually bent bones: calcanei from sheep, deer, and horses, radii from sheep and horses, and third metacarpals (MC3s) from horses. Sheep tibiae were examined because their comparatively greater torsion/shear would not require regional variations in osteon morphotypes. Predominant collagen fiber orientation (CFO), a predictor of regionally prevalent/predominant strain mode, was quantified as image gray levels (birefringence). Ten PP calculations were conducted. Although PP calculations were similar to the osteon MTS in corroborating CFO differences between "tension" and "compression" cortices of the chimpanzee femora and most of the habitually bent bones, PP calculations failed to show a compression/tension difference in equine MC3s and sheep radii. With the exception of the prevalence of the "distributed" osteon morphotype, correlations of PP calculations with CFO were weak and/or negative. By contrast, the osteon MTS consistently showed positive correlations with predominant CFO. Compared with the osteon MTS and predominant CFO, regional variations in PP of osteon morpho types are not stronger predictors of nonuniform strain distributions produced by bending.

Interpreting cortical bone adaptation and load history by quantifying osteon morphotypes in circularly polarized light images.

Birefringence variations in circularly polarized light (CPL) images of thin plane-parallel sections of cortical bone can be used to quantify regional differences in predominant collagen fiber orientation (CFO). Using CPL images of equine third metacarpals (MC3s), R.B. Martin, V.A. Gibson, S.M. Stover, J.C. Gibeling, and L.V. Griffin. (40) described six secondary osteon variants ('morphotypes') and suggested that differences in their regional prevalence affect fatigue resistance and toughness. They devised a numerical osteon morphotype score (MTS) for quantifying regional differences in osteon morphotypes. We have observed that a modification of this score could significantly improve its use for interpreting load history. We hypothesized that our modified osteon MTS would more accurately reveal differences in osteon MTSs between opposing "tension" and "compression" cortices of diaphyses of habitually bent bones. This was tested using CPL images in transverse sections of calcanei from sheep, deer, and horses, and radii from sheep and horses. Equine MC3s and sheep tibiae were examined as controls because they experience comparatively greater load complexity that, because of increased prevalence of torsion/shear, would not require regional mechanical enhancements provided by different osteon morphotypes. Predominant CFO, which can reliably reflect adaptation for a regionally prevalent strain mode, was quantified as mean gray levels from birefringence of entire images (excluding pore spaces) in anterior, posterior, medial, and lateral cortices. Results showed that, in contrast to the original scoring scheme of Martin et al., the modified scheme revealed significant anterior/posterior differences in osteon MTSs in nearly all "tension/compression" bones (p<0.0001), but not in equine MC3s (p=0.30) and sheep tibiae (p=0.35). Among habitually bent bones, sheep radii were the exception; relatively lower osteon populations and the birefringence of the primary bone contributed to this result. Correlations between osteon MTSs using the scoring scheme of Martin et al. with CFO data from all regions of each bone invariably demonstrated weak-to-moderate negative correlations. This contrasts with typically high positive correlations between modified osteon MTSs and regional CFO. These results show that the modified osteon MTS can be a strong correlate of predominant CFO and of the non-uniform strain distribution produced by habitual bending.

Periostin-like-factor and Periostin in an animal model of work-related musculoskeletal disorder.

Work-related musculoskeletal disorders (WMSDs), also known as overuse injuries, account for a substantial proportion of work injuries and workers' compensation claims in the United States. However, the pathophysiological mechanisms underlying WMSDs are not well understood, especially the early events in their development. In this study we used an animal model of upper extremity WMSD, in which rats perform a voluntary repetitive reaching and pulling task for a food reward. This innovative model provides us an opportunity to investigate the role of molecules which may be used either as markers of early diagnosis of these disorders, and/or could be targeted for therapeutic purposes in the future. Periostin-like-factor (PLF), and Periostin were examined in this study. Both belong to a family of vitamin K-dependent gamma carboxylated proteins characterized by the presence of conserved Fasciclin domains and not detected in adult tissues except under conditions of chronic overload, injury, stress or pathology. The spatial and temporal pattern of PLF and Periostin localization was examined by immunohistochemistry and western blot analysis in the radius and ulna of animals performing a high repetition, high force task for up to 12 weeks and in controls. We found that PLF was present primarily in the cellular periosteum, articular cartilage, osteoblasts, osteocytes and osteoclasts at weeks 3 and 6 in all distal bone sites examined. This increase coincided with a transient increase in serum osteocalcin in week 6, indicative of adaptive bone formation at this time point. PLF immunoexpression decreased in the distal periosteum and metaphysis by week 12, coincided temporally with an increase in serum Trap5b, thinning of the growth plate and reduced cortical thickness. In contrast to PLF, once Periostin was induced by task performance, it continued to be present at a uniformly high level between 3 and 12 weeks in the trabeculae, fibrous and cellular periosteum, osteoblasts and osteocytes. In general, the data suggest that PLF is located in tissues during the early adaptive stage of remodeling but not during the pathological phase and therefore might be a marker of early adaptive remodeling.

In vitro heterogeneity of osteogenic cell populations at various equine skeletal sites.

Bone cell cultures were evaluated to determine if osteogenic cell populations at different skeletal sites in the horse are heterogeneous. Osteogenic cells were isolated from cortical and cancellous bone in vitro by an explant culture method. Subcultured cells were induced to differentiate into bone-forming osteoblasts. The osteoblast phenotype was confirmed by immunohistochemical testing for osteocalcin and substantiated by positive staining of cells for alkaline phosphatase and the matrix materials collagen and glycosaminoglycans. Bone nodules were stained by the von Kossa method and counted. The numbers of nodules produced from osteogenic cells harvested from different skeletal sites were compared with the use of a mixed linear model. On average, cortical bone sites yielded significantly greater numbers of nodules than did cancellous bone sites. Between cortical bone sites, there was no significant difference in nodule numbers. Among cancellous sites, the radial cancellous bone yielded significantly more nodules than did the tibial cancellous bone. Among appendicular skeletal sites, tibial metaphyseal bone yielded significantly fewer nodules than did all other long bone sites. This study detected evidence of heterogeneity of equine osteogenic cell populations at various skeletal sites. Further characterization of the dissimilarities is warranted to determine the potential role heterogeneity plays in differential rates of fracture healing between skeletal sites.

Spatial distribution of osteocyte lacunae in equine radii and third metacarpals: considerations for cellular communication, microdamage detection and metabolism.

Osteocytes, which are embedded in bone matrix, are the most abundant cells in bone. Despite the ideal location of osteocytes to sense the local environment and influence bone remodeling, their functions, and the relative importance of these functions, remain controversial. In this study, we tested several hypotheses that address the possibilities that population densities of osteocyte lacunae (Ot.Lc.N/B.Ar) correlate with strain-, remodeling- or metabolism-related aspects of the local biomechanical environments of mid-third diaphyseal equine radii and third metacarpals from skeletally mature animals. Ot.Lc.N/B.Ar data, quantified in multiple cortical locations, were analyzed for possible correlations with (1) structural and material characteristics (e.g., cortical thickness, percent ash, secondary osteon population density, mean osteon cross-sectional area, and predominant collagen fiber orientation), (2) strain characteristics, including prevalent/predominant strain magnitude and mode (tension, compression, shear), (3) hypothesized strain-mode-related microdamage characteristics, which might be perceived by osteocyte 'operational' networks, and (4) variations in remodeling dynamics and/or metabolism (i.e. presumably higher in endocortical regions than in other transcortical locations). Results showed relatively uniform Ot.Lc.N/B.Ar between regions with highly non-uniform strain and strain-related environments and markedly heterogeneous structural and material organization. These results suggest that population densities of these cells are poorly correlated with mechanobiological characteristics, including local variations in metabolic rate and strain magnitude/mode. Although osteocytes hypothetically evolved both as strain sensors and fatigue damage sensors able to direct the removal of damage as needed, the mechanisms that govern the distribution of these cells remain unclear. The results of this study provide little or no evidence that the number of osteocyte lacunae has a functional role in mechanotransduction pathways that are typically considered in bone adaptation.

Methods: a comparative analysis of radiography, microcomputed tomography, and histology for bone tissue engineering.

This study focused on the assessment of radiography, microcomputed tomography, and histology for the evaluation of bone formation in a 15.0-mm defect in the rabbit radius after the implantation of a tissue-engineered construct. Radiography was found to be useful as a noninvasive method for obtaining images of calcified tissue throughout the time course of the experiment. With this method, however, image quality was low, making it difficult to obtain precise information about the location and quantity of the bone formed. Microcomputed tomography was used to create three-dimensional reconstructions of the bone (25-microm resolution). These reconstructions allowed for greater spatial resolution than the radiography, but did not allow for imaging of the implanted scaffold material or the surrounding, nonmineralized tissue. To visualize all materials within the defect area at the cellular level, histology was used. Histological analysis, however, is a destructive technique that did not allow for any further analysis of the samples. Each technique examined here has its own advantages and limitations, but each yields unique information regarding bone regeneration. It is only through the use of all three techniques that complete characterization of the bone growth and tissue/construct responses after implantation in vivo.

Osteocyte apoptosis and osteoclast presence in chicken radii 0-4 days following osteotomy.

Osteocyte apoptosis caused by load-induced microdamage is followed by osteoclastic bone remodeling, and a causal link between apoptosis and repair has been suggested. The objectives of the present study were to use a chick model to examine the incidence of osteocyte apoptosis and the presence of osteoclasts during the first 96 hours following an osteotomy, prior to extensive callus mineralization. Osteotomies were performed on the right radii of 24 chicks at 23-24 days of age. The left radii served as controls. Radii were collected and processed at six time points following surgery (0, 12, 24, 48, 72, and 96 hours). Decalcified bone tissue sections were stained either for apoptosis using a modified TUNEL procedure or for tartrate-resistant acid phosphatase to identify osteoclasts in the intracortical and periosteal envelopes. The percentage of apoptotic osteocytes, as well as osteoclast counts (n/mm or n/mm2) were quantified in four regions (0-1, 1-2, 2-4, and 4-8 mm from the site of the osteotomy; regions 1-4, respectively) in the osteotomized radii and in the same measured areas in the control radii. Data for osteocyte apoptosis and osteoclasts in the control limb were subtracted from the osteotomized limb data to identify differences due to surgical influence. The incidence of osteocyte apoptosis was significantly higher at 12, 24, 48, and 72 hours versus 0 hours following osteotomy, and the response was highest in region 1; however, there was no interaction between time and region. Intracortical osteoclast counts (n/mm2) were elevated after 48 hours, and the response was similar in all regions. The data demonstrate that osteocyte apoptosis occurs within 24 hours in response to an osteotomy and temporally precedes an increase in osteoclast presence. Hence, osteocyte apoptosis may play a role in signaling during the bone healing process.

Analysis of avian bone response to mechanical loading-Part one: Distribution of bone fluid shear stress induced by bending and axial loading.

Mechanical loading-induced signals are hypothesized to be transmitted and integrated by a bone-connected cellular network (CCN) before reaching the bone surfaces where adaptation occurs. Our objective is to establish a computational model to explore how bone cells transmit the signals through intercellular communication. In this first part of the study the bone fluid shear stress acting on every bone cell in a CCN is acquired as the excitation signal for the computational model. Bending and axial loading-induced fluid shear stress is computed in transverse sections of avian long bones for two adaptation experiments (Gross et al. in J Bone Miner Res 12:982-988, 1997 and Judex et al. in J Bone Miner Res 12:1737-1745, 1997). The computed fluid shear stress is found to be correlated with the radial strain gradient but not with bone formation. These results suggest that the radial strain gradient is the driving force for bone fluid flow in the radially distributed lacunar-canalicular system and that bone formation is not linearly related to the loading-induced local stimulus.