Equine Bone Research Articles

Evaluation of Motion Correction Processing in Equine Bone Scintigraphy by Scheffé’s Method of Paired Comparisons

Equine bone scintigraphy is usually performed with horses in standing position under sedation. However, swaying motion often leads to poor-quality images. To examine the usefulness of motion correction (MC) processing, equine bone scintigrams were evaluated using Scheffé's method of paired comparisons. A significant difference in evaluation scores was detected by analysis of variance (F test, P<0.01). According to all observers, Yardstick analysis scores were higher for images use of MC processing than for those no use of MC processing, for all parts. Overall scores of 5 observers were as follows: without MC 100% acquisition time (AT, lowest), use of MC with 25% AT, MC 50% AT, MC 75% AT and MC 100% AT (highest). Thus, MC processing shortens AT in equine bone scintigraphy, and it contributes to a reduction in the external radiation exposure of nurses/technicians.

Equine bone marrow‐derived mesenchymal stromal cells (BMDMSCs) from the ilium and sternum: Are there differences?

The 2 sites of bone marrow harvest for isolation of mesenchymal stromal cells (MSC) in the horse are the sternum and ilium. The technical procedure is based on practitioner preference, but no studies have compared MSC concentrations and growth rates between the sites in horses aged 2-5 years. The objective of this study was to compare nucleated cell counts and growth rates between the sternum and ilium and between consecutive 5 ml bone marrow aspirates. We hypothesised that there would be a higher concentration of MSCs in the sternum than the ilium, and that the first sequential aspirate from either site would yield the greatest concentration of MSCs. We hypothesised that growth rates of cells from each site would not differ. Seven horses, aged 2 to 5 years, had 2 sequential 5 ml marrow aspirates taken from the sternum and ilium. Nucleated cell counts (NCCs) were obtained before and after marrow processing. Cells were expanded in culture for 3 passages and growth rate characteristics compared for all aspirates. The NCCs of the first 5 ml aspirate were higher than those of the second 5 ml aspirate for both sites (P<0.05). There was no difference between growth rates for any of the groups (P>0.05). The NCCs and growth rates of progenitor cells in the ilium and sternum are similar for horses in the 2-5 year age category. The first 5 ml bone marrow aspirate has a higher concentration of NCCs and resulting bone marrow-derived MSC population than subsequent aspirates. The first 5 ml aspirates from the sternum and ilium offer a rich supply of bone marrow-derived MSCs with similar growth rate characteristics. The harvesting procedure of only a 5 ml draw from either the sternum or ilium should result in adequate numbers of MSCs.

Avaliação da composição macroelementar do osso terceiro metacarpiano de equinos

The equine third metacarpal bone has been seen as a region of particular interest due to the large number of injuries to which it is subject. Thus, the detailed study of its anatomical structure, size and elemental composition is justified for the improvement of diagnostic techniques, treatment and prevention of injuries. In this work it has been studied the macro elemental composition of 30 pairs of equine third metacarpal bone and its relation with age, sex, weight, breed, nutrition and activity of animals. The average concentrations of calcium and phosphorus of the samples were 205 ± 62 mg/g and 97 ± 32 mg/g, respectively. The ratio [Ca] / [P] was found to be 2.12 ± 0.13, indicating that the ratio of calcium and phosphorus in bone tissue is constant and approximately equal to the ratio 2:1. The statistical analysis showed normal values for the entire set of measurements. In the evaluation of the animals with intense physical activity, and animals with light physical activity, it was found that the amount of mineralized material for the first and second sets respectively ([Ca] = 222 ± 57 mg/g [Ca] = 179 ± 47 mg/g), showing the existence of statistical correlation between the deposit of mineral material and the function performed by the animals. For the other characteristics were not found statistical evidence of correlations. These findings will be input for further studies of possible correlations with diseases of nutritional origin or physiological outside the series of fractures and other disease common to the equine locomotor system.

Estudo da correlação da densitometria óssea e do perímetro diafisário medial do terceiro metacarpiano em equinos

In this study, 14 post mortem equine metacarpal bones were evaluated to correlate the medial III metacarpus perimeter to dorsal bone density of the same region using Computed Tomography and Quantitative Ultrasound (QUS). According to Pearson's test, there were not correlations between these variables. Considering there are few bone densitometry studies in horses and there are many variables that can interfere with bone mineralization, new studies on equine bone density applying standard variables are recommended.

Expansion under hypoxic conditions enhances the chondrogenic potential of equine bone marrow-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are widely used in regenerative medicine in horses. Most of the molecular characterisations of BM-MSCs have been made at 20% O(2), a higher oxygen level than the one surrounding the cells inside the bone marrow. The present work compares the lifespan and the tri-lineage potential of equine BM-MSCs expanded in normoxia (20% O(2)) and hypoxia (5% O(2)). No significant differences were found in long-term cultures for osteogenesis and adipogenesis between normoxic and hypoxic expanded BM-MSCs. An up-regulation of the chondrogenesis-related genes (COL2A1, ACAN, LUM, BGL, and COMP) and an increase of the extracellular sulphated glycosaminoglycan content were found in cells that were expanded under hypoxia. These results suggest that the expansion of BM-MSCs in hypoxic conditions enhances chondrogenesis in equine BM-MSCs.

Treatment of mandibular atrophy by an equine bone substitute: an immunohistochemical study in man

ObjectiveTo perform an immunohistochemical evaluation of microvessel density (MVD), vascular endothelial growth factor (VEGF) and nitric oxide synthase (NOS) expression in mandibular ridge regeneration using an equine-derived bone substitute. Materials and methodsRidge augmentations were performed in six patients using equine bone. After a six-month healing period, four bone cores from no augmented (control) and four from augmented sites (test) were retrieved and processed for immunohistochemistry. ResultsStatistically, significant differences were found in the MVD expression between control and test sites (p<0.05). The high and low intensities of VEGF, NOS1 and NOS3 were mainly expressed in test samples with statistical differences with control (p<0.05). ConclusionsWithin the limits of the small sample size, the present study indicated that equine bone is able to support neovascularization when used as a grafting material.

Mesenchymal stromal cell cryopreservation.

The advent of stem cells and stem cell-based therapies for specific diseases requires particular knowledge of laboratory procedures, which not only guarantee the continuous production of cells, but also provide them an identity and integrity as close as possible to their origin. Their cryopreservation at temperatures below -80°C and typically below -140°C is of paramount importance. This target can be achieved by incorporating high molar concentrations of cryoprotectant mixtures that preserve cells from deleterious ice crystal formation. Usually, dimethyl sulfoxide (DMSO) and animal proteins are used as protectant reagents, but unexpected changes in stem cell fate and downstream toxicity effects have been reported, limiting their wide use in clinical settings. In scientific reviews, there are not much data regarding viability of mesenchymal stromal cells (MSCs) after the freezing/thawing process. During our routine analysis, a poor resistance to cryopreservation of these cells was observed, as well as their weak ability to replicate. This is an important point in the study of MSCs; moreover, it represents a limit for preservation and long-term storage. For this reason, MSCs isolated from equine, ovine, and rodent bone marrow and equine adipose tissue were compared using different cryopreservation solutions for this study of vitality. Our findings showed the best results regarding cell viability using a solution of fetal bovine serum with addition of 10% DMSO. In particular, we noted an increase in survival of equine bone marrow MSCs. This parameter has been evaluated by Trypan blue staining at fixed times (0, 24, and 48 hours post-thaw). This result highlights the fact that equine bone marrow MSCs are the frailest we analyzed. Therefore, it could be useful to delve further into this topic in order to improve the storage possibility for these cells and their potential use in cell-based therapies.

A method for proteomic analysis of equine subchondral bone and epiphyseal cartilage

Proteomic analyses of cartilage and, to a lesser extent, of bone have long been impaired because of technical challenges related to their structure and biochemical properties. We have developed a unified method based on phenol extraction, 2DE, silver staining, and subsequent LC-MS/MS. This method proved to be efficient to characterize the proteome of equine cartilage and bone samples collected in vivo. Since proteins from several cellular compartments could be recovered, our procedure is mainly suitable for in situ molecular physiology studies focused on the cellular content of chondrocytes, osteoblasts, and osteoclasts as well as that of the extracellular matrix, with the exception of proteoglycans. Our method alleviates some drawbacks of cell culture that can mask physiological differences, as well as reduced reproducibility due to fractionation. Proteomic comparative studies between cartilage and bone samples from healthy and affected animals were thus achieved successfully. This achievement will contribute to increasing knowledge on the molecular mechanisms underlying the physiopathology of numerous osteoarticular diseases in horses and in humans.

Lipids in biocalcification: contrasts and similarities between intimal and medial vascular calcification and bone by NMR

Pathomechanisms underlying vascular calcification biogenesis are still incompletely understood. Biomineral from human atherosclerotic intimal plaques; human, equine, and bovine medial vascular calcifications; and human and equine bone was released from collagenous organic matrix by sodium hydroxide/sodium hypochlorite digestion. Solid-state (13)C NMR of intimal plaque mineral shows signals from cholesterol/cholesteryl esters and fatty acids. In contrast, in mineral from pure medial calcifications and bone mineral, fatty acid signals predominate. Refluxing (chloroform/methanol) intimal plaque calcifications removes the cholesterylic but not the fatty acyl signals. The lipid composition of this refluxed mineral now closely resembles that of the medial and bone mineral, which is unchanged by reflux. Thus, intimal and medial vascular calcifications and bone mineral have in common a pool of occluded mineral-entrained fatty acyl-rich lipids. This population of fatty acid may contain methyl-branched fatty acids, possibly representing lipoprotein particle remnants. Cell signaling and mechanistic parallels between physiological (orthotopic) and pathological (ectopic) calcification are also reflected thus in the NMR spectroscopic fingerprints of mineral-associated and mineral-entrained lipids. Additionally the atherosclerotic plaque mineral alone shows a significant independent pool of cholesterylic lipids. Colocalization of mineral and lipid may be coincidental, but it could also reflect an essential mechanistic component of biomineralization.

Comparison of in vitro adherence, proliferation and osteogenesis of equine bone marrow‐derived mesenchymal stem cells to identify an allogeneic donor

Equine mesenchymal stem cells (eMSCs) are promising candidates to repair and to regenerate damaged tissue in horses. To avoid immune reactions, the use of autologous eMSCs is desired, but the number of cells obtained from biopsies is generally too low for direct implantation. As a result, it takes several weeks for in vitro expansion of eMSC cultures to generate enough numbers for injection into the same horse. The only way to satisfy this high demand for eMSCs is the use of allogeneic eMSCs. eMSCs lack major histocompatibility complex II antigens and are able to avoid immune recognition allowing for allogeneic administration of these cells. Similar to humans, equine bone marrow‐derived MSCs exhibit variability in their rates of proliferation and differentiation in vitro. Experiments were carried out to identify a “reliable” donor and establish MSC cultures in order to generate off‐the‐shelf allogeneic cell stocks for immediate administration into client cases. Results of experiments to compare the cell viability (live/dead staining), proliferation (MTS assay), in vitro differentiation into osteoblasts (alkaline phosphatase and alizarin red staining), and gene expression from 7 horses are presented. Only one out of seven horses demonstrated a high rate of proliferation, and potential for in vitro osteogenesis, hence, was found to be superior to the others. This research is sponsored by a grant from the Center of Excellence in Livestock Diseases and Human Health.

Proliferation of equine bone marrow-derived mesenchymal stem cells in gelatin/β-tricalcium phosphate sponges

A three dimensional scaffold is essential in mesenchymal stem cells (MSCs) delivery in cell-based therapy for facilitating cell adherence, migration, proliferation, and differentiation. The objectives of this study were to evaluate the possibility of β-tricalcium phosphate incorporated gelatin sponges (Gelatin/β-TCP sponge) as scaffolds for equine MSCs and to examine the effects of seeding density and seeding method on the proliferation of equine MSCs in the Gelatin/β-TCP sponges. Mononuclear cells and MSCs isolated from bone marrow were seeded into Gelatin/β-TCP sponges at different densities by different seeding methods—static or agitated methods. Proliferation of the MSCs in Gelatin/β-TCP was assessed using the Cell Counting Kit-8 assay and histological examination. Distribution and proliferation of MSCs in the Gelatin/β-TCP sponge were observed, and the Gelatin/β-TCP sponge supported limited growth when seeded at high density. We also found that the agitated seeding method enhanced the proliferation of MSCs. This study demonstrated the suitability of Gelatin/β-TCP sponges for the proliferation and maintenance of equine MSCs. These results contribute to the application of MSC-seeded Gelatin/β-TCP sponges in equine medicine.

Accuracy and precision of computer-assisted analysis of bone density via conventional and digital radiography in relation to dual-energy x-ray absorptiometry

To evaluate the precision and accuracy of assessing bone mineral density (BMD) by use of mean gray value (MGV) on digitalized and digital images of conventional and digital radiographs, respectively, of ex vivo bovine and equine bone specimens in relation to the gold-standard technique of dual-energy x-ray absorptiometry (DEXA). Left and right metatarsal bones from 11 beef cattle and right femurs from 2 horses. Bovine specimens were imaged by use of conventional radiography, whereas equine specimens were imaged by use of computed radiography (digital radiography). Each specimen was subsequently scanned by use of the same DEXA equipment. The BMD values resulting from each DEXA scan were paired with the MGVs obtained by use of software on the corresponding digitalized or digital radiographic image. The MGV analysis of digitalized and digital x-ray images was a precise (coefficient of variation, 0.1 and 0.09, respectively) and highly accurate method for assessing BMD, compared with DEXA (correlation coefficient, 0.910 and 0.937 for conventional and digital radiography, respectively). The high correlation between MGV and BMD indicated that MGV analysis may be a reliable alternative to DEXA in assessing radiographic bone density. This may provide a new, inexpensive, and readily available estimate of BMD.

A Fresh Look at the Process of Arriving at a Clinical Prognosis Part 4: Fractures

Equine bone and biomechanics research continues to provide us with sound and practical concepts for preventing fractures in equine athletes, accelerating a safe return to training and competition, and avoiding recurrence. Bone is a living, dynamic tissue that is constantly in a state of turnover as part of its ongoing maintenance, and is constantly responding to the various forces it experiences, from within and without. Even the seemingly inert, acellular components of bone are in a perpetual state of flux under both biomechanical and metabolic/nutritional influences. Homeostasis in bone involves both modeling and remodeling; modeling is bone's adaptive response to increases or decreases in load that are of insufficient magnitude to damage the bone. It involves the addition or removal of existing bone, changing the shape and loading capacity of the bone as needed. Remodeling is bone's reparative response to injury; it involves the removal and replacement of bone in the area of damage. Fractured bone needs the following conditions for best repair: protection—remove the primary cause of bone injury and prevention of further such injury; stability—depends on the type and site of fracture; blood flow—bone is dependent on good blood supply for repair; competent immune response—a robust white cell response is necessary both for tissue repair and for prevention/resolution of bacterial infection at the site; nutrients—all living tissues need adequate calories, and protein, minerals (including trace minerals), and vitamin D are of primary importance for optimal bone repair; and load—after fracture healing, increasing the loading capacity of the bone directs further bone repair and modeling. The prognosis may be adversely affected when any of these conditions are not met adequately. Generally, fractures in horses are not fatal injuries by themselves but most often prompted by a poor prognosis for return to soundness because of the fracture type, the development of serious complications, and the economic investment required for adequate repair. Factors that determine the prognosis for survival are degree of structural failure, ease of fixation and potential for complications, adequacy of stabilization, concurrent and complicating conditions, owner's wishes, and prognosis for return to athletic performance. Fractures of the metacarpus and metatarsus are used to illustrate the aforementioned principles.

Effects of mineral content on the fracture properties of equine cortical bone in double-notched beams

We recently developed a method to measure cortical bone fracture initiation toughness using a double-notched beam in four-point bending. This method was used to test the hypothesis that mineralization around the two notch roots is correlated with fracture toughness and crack extension (physical damage). Total energy absorbed to failure negatively correlated with average mineralization of the beam (r2=0.62), but not with notch root mineralization. Fracture initiation toughness was positively correlated to mineralization at the broken notch root (r2=0.34). Crack length extension at the unbroken notch was strongly negatively correlated with the average mineralization of the notch roots (r2=0.81) whereas crack length extension at the broken notch did not correlate with any of the mineralization measurements. Mineralization at the notch roots and the average mineralization contributed independently to the mechanical and damage properties. The data are consistent with a hypothesis that a) high notch root mineralization results in less stable crack length extension but high force to initiate unstable crack propagation while b) higher average mineralization leads to low post-yield (and total) energy absorption to failure.

In vivo osteoinductivity of gelatin β-tri-calcium phosphate sponge and bone morphogenetic protein-2 on an equine third metacarpal bone defect

This study evaluated the therapeutic effects of a gelatin-β-TCP sponge (sponge) incorporating BMP-2 (BMP-2/sponge) on bone regeneration in equines. Six bone defects were created in third metacarpals of five thoroughbred horses, and a total of six treatments were applied in a randomized manner. The treatments were BMP-2/sponge, BMP-2/gelatin hydrogel sheet (sheet), free BMP-2, bFGF/sheet, plain sponge, and plain sheet. The defects were monitored for 16weeks by radiography and then examined by histological analysis. Radiographic evaluation scores of bone regeneration revealed significantly greater bone regeneration of defects treated with BMP-2/sponge than defects treated with plain sponge or BMP-2 sheet (P<0.05). In histological analysis, compact bone was observed over a wide area in the BMP-2/sponge treatment. We concluded that the treatment with BMP-2/sponge accelerated bone regeneration in the equines of this study.

Stem Cell and Tissue Engineering Applications in Orthopaedics and Musculoskeletal Medicine

Stem Cell and Tissue Engineering Applications in Orthopaedics and Musculoskeletal Medicine

In VivoHealing of Meniscal Lacerations Using Bone Marrow-Derived Mesenchymal Stem Cells and Fibrin Glue

Fibrin glue created from a patient's own blood can be used as a carrier to deliver cells to the specific site of an injury. An experimental model for optimizing various permutations of this delivery system in vivo was tested in this study. Harvested equine meniscal sections were reapposed with fibrin glue or fibrin glue and equine bone marrow-derived mesenchymal stem cells (BMSCs). These constructs were then implanted subcutaneously in nude mice. After harvesting of the constructs, BMSC containing constructs showed significantly increased vascularization, and histology showed subjectively decreased thickness of repair tissue and increased total bonding compared to fibrin alone constructs. This model allowed direct comparison of different meniscal treatment groups while using a small number of animals. This in vivo model could be valuable in the future to optimize fibrin and cellular treatments for meniscal lesions in the horse and potentially humans as well.

Effects of a novel hydrogel on equine bone healing: A pilot study

To examine the efficacy and biocompatibility of a thiolated gelatin-thiolated carboxymethyl hyaluronan (CMHA-SGX) sponge as an osteoconductive device in an equine second and fourth metacarpal bone defect model. Seven millimetre segmental ostectomies were created bilaterally in the second and fourth metacarpal bones of four horses. The left and right metacarpal defects were randomly assigned to (1) be filled with a CMHA-SGX sponge (treated) or (2) were left unfilled (control). The duration of the study was nine weeks. Bone healing was evaluated using serial radiology, as well as histologically and histomorphometrically. Data were analyzed using an analysis of variance (ANOVA). The level of significance was p<0.05. Serial radiographic evaluation revealed improved healing in the treated compared to the control defects at weeks eight and nine (p = 0.02). This finding was not corroborated histologically. Histomorphometry did not reveal any significant differences in healing between experimental groups. The CMHA-SGX sponge did not inhibit bone formation, induce local inflammation or lead to surgical site infection. While further optimization to improve osteoconductive properties should be considered, the CMHA-SGX sponge appears to be a biocompatible orthopaedic implant and its use as a carrier for osteogenic proteins warrants further investigation.

Comparison of Gene-Specific DNA Methylation Patterns in Equine Induced Pluripotent Stem Cell Lines with Cells Derived From Equine Adult and Fetal Tissues

Cellular pluripotency is associated with expression of the homeobox transcription factor genes NANOG, SOX2, and POU5F1 (OCT3/4 protein). Some reports suggest that mesenchymal progenitor cells (MPCs) may express increased quantities of these genes, creating the possibility that MPCs are more "pluripotent" than other adult cell types. The objective of this study was to determine whether equine bone marrow-derived MPCs had gene expression or DNA methylation patterns that differed from either early fetal-derived or terminally differentiated adult cells. Specifically, this study compared DNA methylation of the NANOG and SOX2 promoter regions and concurrent gene expression of NANOG, SOX2, and POU5F1 in equine induced pluripotent stem (iPS) cells, fetal fibroblasts, fetal brain cells, adult chondrocytes, and MPCs. Results indicate that NANOG and POU5F1 were not detectable in appreciable quantities in tissues other than the equine iPS cell lines. Equine iPS cells expressed large quantities of all three genes examined. Significantly increased quantities of SOX2 were noted in iPS cells and both fetal-derived cell types compared with adult cells. MPCs and adult chondrocytes expressed equivalent, low quantities of SOX2. Further, NANOG and SOX2 expression inversely correlated with the DNA methylation pattern in the promoter region, such that as gene expression increased, DNA methylation decreased. The equine iPS cell lines examined demonstrated DNA methylation and gene expression patterns that were consistent with pluripotency features described in other species. Results do not support previous reports that NANOG, SOX2, and POU5F1 are poised for increased activity in MPCs compared with other adult cells.

Processing of equine bone marrow using the automated MarrowXpress System: RBC depletion, volume reduction, and mononuclear cell recovery

The therapeutic use of bone marrow-derived mononuclear cells (MNCs) and mesenchymal stem cells for the treatment of soft tissue and orthopedic injuries in equine patients is expanding. After collection, bone marrow must be reduced in volume and depleted of RBCs for immediate therapeutic use or to prepare cells for culture or cryopreservation and storage. The MarrowXpress (MXP) System is an automated, closed, sterile system designed to process human bone marrow samples. The purpose of this study was to evaluate the capacity of the MXP System to process equine bone marrow to reduce volume, deplete RBCs, and enhance recovery of MNCs. Bone marrow was collected from 47 horses into 2 60-mL syringes containing heparin and processed using the MXP System. HCT, total nucleated cell (TNC) count, and MNC count were obtained for each sample before and after processing using an Advia 120 hematology analyzer. Volume reduction, RBC depletion, and recovery of TNCs and MNCs were calculated. For equine bone marrow samples, mean values were 73.2% for RBC depletion and 78.0% for volume reduction. TNC count before processing was 2.5 ± 1.2 × 10(7) and after processing was significantly higher at 7.8 ± 3.3 × 10(7) (P < .0001), with a recovery of 68.5 ± 24.5% (mean ± SD). MNC count before processing was 1.1 ± 0.9 × 10(7) and after processing was significantly higher at 3.8 ± 1.9 × 10(7) (P < .0001), with a recovery 73.0 ± 31.5%. The MXP System can reliably reduce volume and deplete RBCs from aspirates of equine bone marrow aspirates. MNCs can be recovered in a reproducible and sterile manner. Further studies evaluating the effects of the MXP System on cell viability, identification of mesenchymal stem cells (MSCs), and the efficacy of MSC expansion are warranted.