Optimisation of bone marrow aspiration from the equine sternum for the safe recovery of mesenchymal stem cells

Reasons for performing study: Mesenchymal stem cell (MSC) therapy for orthopaedic disease is being used with increasing frequency; there is a need to define a safe, reliable and effective technique for the recovery of MSCs from the sternum of the horse. Objectives: To describe an optimised safe technique for obtaining bone marrow‐derived MSCs from the sternum of the Thoroughbred horse. Methods: The anatomical relationship of the sternum with the heart and internal anatomy was demonstrated in cadavers. Sternal anatomy was evaluated ultrasonographically and after midline sectioning. Sternebrae were examined histologically after aspiration to determine the effect of needle insertion. The quality of the aspirate was evaluated as the number of colony‐forming units from sequential and separately aspirated 5 ml aliquots and assessed for their multipotency using trilineage differentiation. Results: The optimal safe location for the needle was the 5th sternebra because it had a safe dorsoventral thickness and was cranial to the apex of the heart. This sternebra could be reliably identified ultrasonographically. Aspirates could also be obtained from the 4th and 6th sternebrae, although the former is between the front limbs and the latter closer to the heart. Minimal disruption of the internal bony architecture was seen after needle insertion through the thin outer cortex and the first 5 ml aliquot contained the greatest number of colony‐forming units of mesenchymal stem cells with trilineage capabilities. Conclusions: Accurate placement of a Jamshidi needle into the medullary cavity of the 4th–6th individual sternebrae is facilitated by the use of ultrasonography and enables aspiration of bone marrow reliably with minimal damage to the sternum and risk to the horse. Potential clinical relevance: Sternal marrow aspiration as described is a safe and reliable technique to obtain MSCs for orthopaedic cell‐based therapies.     

Isolation and characterization of bone marrow-derived equine mesenchymal stem cells

OBJECTIVE: To isolate and characterize bone marrow-derived equine mesenchymal stem cells (MSCs) for possible future therapeutic applications in horses. SAMPLE POPULATION: Equine MSCs were isolated from bone marrow aspirates obtained from the sternum of 30 donor horses. PROCEDURES: Cells were cultured in medium (alpha-minimum essential medium) with a fetal calf serum content of 20%. Equine MSC features were analyzed to determine selfrenewing and differentiation capacity. For potential therapeutic applications, the migratory potential of equine MSCs was determined. An adenoviral vector was used to determine the transduction rate of equine MSCs. RESULTS: Equine MSCs can be culture-expanded. Equine MSCs undergo cryopreservation in liquid nitrogen without altering morphologic characteristics. Furthermore, equine MSCs maintain their ability to proliferate and differentiate after thawing. Immunocytochemically, the expression of the stem cell marker CD90 can be detected on equine MSCs. The multilineage differentiation potential of equine MSCs was revealed by their ability to undergo adipogenic, osteogenic, and chondrogenic differentiation. CONCLUSIONS AND CLINICAL RELEVANCE: Our data indicate that bone marrow-derived stromal cells of horses can be characterized as MSCs. Equine MSCs have a high transduction rate and migratory potential and adapt to scaffold material in culture. As an autologous cell population, equine MSCs can be regarded as a promising cell population for tissue engineering in lesions of the musculoskeletal system in horses.     

Comparison of autogenous cancellous bone grafts obtained from the sternum and proximal portion of the tibia of llamas.

OBJECTIVE: To describe the anatomy of the sternum in llamas, define the surgical approach to the sternum for collection of cancellous bone graft tissue, and compare the histologic appearance of graft tissue obtained from the sternum with that obtained from the proximal portion of the tibia. DESIGN: Prospective study. ANIMALS: 12 llamas, 3 to 19 years old, that had been submitted for necropsy. PROCEDURE: Radiographs were taken of the sternum and left tibia of the llamas. Measurements of the sternum were determined from the radiographs and adjusted for magnification. Sternebrae volumes were estimated from these measurements. Anatomic dissections to the center of the fourth sternebra and the proximal portion of the tibia were made, and a surgical approach to the sternum was developed. Cancellous graft tissue was obtained from each site and submitted for histologic evaluation. RESULTS: Sternebrae 3, 4, and 5 were significantly larger in volume than the other sternebrae. The ventral aspect of the fourth sternebra was readily accessed for removal of graft tissue by making a 6-cm-long ventral midline incision centered 17 cm craniad to the xipnoid. Mean soft tissue thickness overlying the ventral aspect of the fourth sternebra was 3.1 cm. More tissue was obtained from the sternal (mean, 9.11 g) than from the tibial (mean, 5.16 g) sites. Sternal graft tissue consisted of trabecular bone spicules with predominantly hematopoietic marrow, whereas tibial tissue consisted of trabecular bone spicules with only fatty marrow. CONCLUSIONS AND CLINICAL RELEVANCE: The fourth sternebra in llamas is readily accessible for obtaining autogenous cancellous bone graft tissue that consists of predominantly hematopoietic marrow.     

Application of a novel sorting system for equine mesenchymal stem cells (MSCs)

The objective of this study was to validate non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs) into subpopulations, for use with MSCs derived from equine muscle tissue, periosteal tissue, bone marrow, and adipose tissue. Cells were collected from 6 young, adult horses, postmortem. Cells were isolated from left semitendinosus muscle tissue, periosteal tissue from the distomedial aspect of the right tibia, bone marrow aspirates from the fourth and fifth sternebrae, and left supragluteal subcutaneous adipose tissue. Aliquots of 800 × 10³ MSCs from each tissue source were separated and injected into a ribbon-like capillary device by continuous flow (GrFFF proprietary system). Cells were sorted into 6 fractions and absorbencies [optical density (OD)] were read. Six fractions from each of the 6 aliquots were then combined to provide pooled fractions that had adequate cell numbers to seed at equal concentrations into assays. Equine muscle tissue-derived, periosteal tissue-derived, bone marrow-derived, and adipose tissue-derived mesenchymal stem cells were consistently sorted into 6 fractions that remained viable for use in further assays. Fraction 1 had more cuboidal morphology in culture when compared to the other fractions. Statistical analysis of the fraction absorbencies (OD) revealed a P-value of < 0.05 when fractions 2 and 3 were compared to fractions 1, 4, 5, and 6. It was concluded that non-equilibrium GrFFF is a valid method for sorting equine muscle tissue-derived, periosteal tissue-derived, bone marrow-derived, and adipose tissue-derived mesenchymal stem cells into subpopulations that remain viable, thus securing its potential for use in equine stem cell applications and veterinary medicine.     

Bone marrow response to large volume blood collection in the horse

Evaluation of erythropoietic regeneration in horses is difficult unless serial bone marrow aspirates are performed. To investigate the acute and chronic erythropoietic regenerative response of equine bone marrow following acute removal or loss of blood, sequential bone marrow aspirates over 4 weeks were taken from the sternum of five horses from which 20 ml kg(-1)of blood had been removed. We found that the total number of erythroid cells counted (expressed as a percentage of the total number of erythroid and myeloid cells counted) expanded initially by 13.7 per cent within 3 days after blood removal, the erythroid response peaking by 9 days with a further 13.5 per cent increase. This peak coincided with the lowest M:E ratio. Concomitantly, a shift from proliferative phase cells to maturing phase cells occurred, which appeared to persist beyond 31 days post collection. Thus, we found that the equine bone marrow mounted a regenerative erythropoietic response more slowly than previously determined and, also, regeneration of the erythroid compartment was incomplete 31 days after blood removal of this magnitude.     

92匹马骨髓细胞正常值的测定

通过对92匹三河马（其中成年马67匹，育成马25匹）骨髓内各种细胞的形态学观察，计算出成年马和育成马骨髓细胞的正常值，为马血液疾病的诊断提供了参考依据。     

Comparison of bone marrow aspiration at the sternum and the tuber coxae in middle-aged horses

The objective of this study was to compare bone marrow (BM) aspirates from the sternum and the tuber coxae of middle-aged horses. Bone marrow was obtained from the sternum and both tubera coxae of 12 healthy, 13-year-old geldings. Two different puncture techniques were used for the tuber coxae. The 2 syringes used for sternal sampling were evaluated separately. The mononuclear cell (MNC) fraction of the BM was isolated and the mesenchymal stem cells (MSCs) were culture-expanded. At the sternum, BM aspiration was always possible. Bone marrow aspiration at the tuber coxae required straight and deep needle penetration combined with high negative pressure. With this technique a median sample amount of 11.0 mL with large individual variation was obtained. A median of 3.06 × 10(6) MNC/mL BM (1st syringe) and 2.46 × 10(6) MNC/mL BM (2nd syringe) was isolated from sternal samples. In contrast, the tuber coxae yielded a median of 0.27 × 10(6) MNC/mL BM. The first passage yielded a median of 2.19 × 10(6) MSC (1st syringe) and 1.13 × 10(6) MSC (2nd syringe) from sternal samples, compared to a significantly lower median number of MSC from tuber coxae BM (0.06 × 10(6) MSC). The number of MNC and MSC obtainable from the BM aspirates taken from the tuber coxae is significantly lower than that obtained from the sternal BM aspirates. Autologous BM for the equine athlete is particularly clinically relevant at an advanced age. Based on our findings, the tuber coxae cannot be recommended for BM aspiration in middle-aged horses.     

Osteogenic potential of sorted equine mesenchymal stem cell subpopulations

The objectives of this study were to use non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs), to sort equine muscle tissue-derived mesenchymal stem cells (MMSCs) and bone marrow-derived mesenchymal stem cells (BMSC) into subpopulations and to carry out assays in order to compare their osteogenic capabilities. Cells from 1 young adult horse were isolated from left semitendinosus muscle tissue and from bone marrow aspirates of the fourth and fifth sternebrae. Aliquots of 800 × 10³ MSCs from each tissue source were sorted into 5 fractions using non-equilibrium GrFFF (GrFFF proprietary system). Pooled fractions were cultured and expanded for use in osteogenic assays, including flow cytometry, histochemistry, bone nodule assays, and real-time quantitative polymerase chain reaction (qPCR) for gene expression of osteocalcin (OCN), RUNX2, and osterix. Equine MMSCs and BMSCs were consistently sorted into 5 fractions that remained viable for use in further osteogenic assays. Statistical analysis confirmed strongly significant upregulation of OCN, RUNX2, and osterix for the BMSC fraction 4 with P < 0.00001. Flow cytometry revealed different cell size and granularity for BMSC fraction 4 and MMSC fraction 2 compared to unsorted controls and other fractions. Histochemisty and bone nodule assays revealed positive staining nodules without differences in average nodule area, perimeter, or stain intensity between tissues or fractions. As there are different subpopulations of MSCs with different osteogenic capacities within equine muscle- and bone marrow-derived sources, these differences must be taken into account when using equine stem cell therapy to induce bone healing in veterinary medicine.     

Superficial Osteitis and Sequestrum Formation as a Result of Skin Avulsion in the Horse

Metatarsal injuries with exposed bone are described in seven horses. Superficial osteitis and sequestrum formation occurred in six of the seven horses. The basic pathophysiology includes local vascular stasis leading to ischemic necrosis of osteocytes and establishment of bacterial infection within dead bone. Radiographic features of superficial osteitis and sequestrum formation include reactive periosteal new bone formation, an area of radiodense necrotic bone (sequestrum) surrounded by a radiolucent zone of granulation tissue, which in turn, is surrounded by an area of increased radiodensity (involucrum). The presence of a sequestrum may usually be confirmed radiographically by 12–14 days after injury. Surgical management included wound debridement, wound irrigation, bandaging or casting, sequestrectomy and skin grafting. Five of the seven patients required sequestrectomy, in one the sequestrum disappeared without removal. Mixed bacterial populations were commonly found in the wounds. No specific etiologic agent was associated with sequestrum formation.     

Computed tomographic anatomy of the temporomandibular joint in the young horse

Reasons for performing study: The equine temporomandibular joint (TMJ) and its surrounding structures can be difficult to investigate in cases with a clinical problem related to the region. Little previous attention has been given either to a computed tomographic (CT) imaging protocol for the joint or an interpretation of the structures displayed in CT images of the normal joint. Objectives: To provide a CT atlas of the normal cross‐sectional anatomy of the equine TMJ using frozen and plastinated sections as anatomical reference. Methods: Eight TMJs from 4 immature pure‐bred Spanish horses were examined by helical CT. Scans were processed with a detailed algorithm to enhance bony and soft tissue. Transverse CT images were reformatted into sagittal and dorsal planes. Transverse, sagittal and dorsal cryosections were then obtained, photographed and plastinated. Relevant anatomic structures were identified in the CT images and corresponding anatomical sections. Results: In the CT images, a bone window provided excellent bone detail, however, the soft tissue components of the TMJ were not as well visualised using a soft tissue window. The articular cartilage was observed as a hyperattenuating stripe over the low attenuated subchondral bone and good delineation was obtained between cortex and medulla. The tympanic and petrous part of the temporal bone (middle and inner ear) and the temporohyoid joint were seen in close proximity to the TMJ. Conclusions: Helical CT provided excellent images of the TMJ bone components to characterise the CT anatomy of the normal joint. Potential relevance: Detailed information is provided that may be used as a reference by equine veterinarians for the CT investigation of the equine TMJ and serve to assist them in the diagnosis of disorders of the TMJ and related structures (middle and inner ear). The study was performed at an immature stage and further studies of mature individuals are required in order to confirm that the clinical interpretation is not affected by changes occurring with age.     

Sternal bone biopsy in standing horses

OBJECTIVE: To develop a technique for sternal bone biopsy in standing horses. STUDY DESIGN: Experimental study. ANIMALS: Five adult horses. METHODS: Horses were restrained in a standing stocks and sedated. The sternal biopsy site, identified by ultrasonographic examination, was clipped and prepared for aseptic surgery and infiltrated with local anesthetic. An electric bone biopsy drill (Osteocore; Institut Straumann AG, CH-4437, Waldenburg, Switzerland)), which yielded 4-mm-diameter bone specimens, was used to obtain sternal biopsies through a small skin incision. Sections (7 microm) of the bone specimens were stained with toluidine blue and Goldner's green trichrome and observed microscopically to determine suitability for histological and histomorphometric evaluation. RESULTS: The most suitable sternal biopsy site was at the 4th or 5th sternebra. The surgical procedure was easy to perform and well tolerated by the horses, and adequate samples were obtained on the first attempt. The only complications were incisional edema in all horses and wound drainage in 1 horse. CONCLUSIONS: Sternal bone biopsy may be successfully performed in standing horses, and the technique described in this report yields architecturally intact bone specimens. CLINICAL RELEVANCE: The sternum is an accessible site for cancellous bone biopsy specimens in standing horses.     

Comparison of Chondrogenic Potential in Equine Mesenchymal Stromal Cells Derived from Adipose Tissue and Bone Marrow

Objective— To compare the chondrogenic potential of adult equine mesenchymal stem cells derived from bone marrow (MSCs) or adipose tissue (ASCs). Study Design— In vitro experimental study. Animals— Adult Thoroughbred horses (n=11). Methods— BM (5 horses; mean [±SD] age, 4±1.4 years) or adipose tissue (6 horses; mean age, 3.5±1.1 years) samples were obtained. Cryopreserved MSCs and ASCs were used for pellet cultures in stromal medium (C) or induced into chondrogenesis±transforming growth factor‐3 (TGFβ₃) and bone morphogenic factor‐6 (BMP‐6). Pellets harvested after 3, 7, 14, and 21 days were examined for cross‐sectional size and tissue composition (hematoxylin and eosin), glycosaminoglycan (GAG) staining (Alcian blue), collagen type II immunohistochemistry, and by transmission electron microscopy. Pellet GAG and total DNA content were measured using dimethylmethylene blue and Hoechst DNA assays. Results— Collagen type II synthesis was predominantly observed in MSC pellets from Day 7 onward. Unlike ASC cultures, MSC pellets had hyaline‐like matrix by Day 14. GAG deposition occurred earlier in MSC cultures compared with ASC cultures and growth factors enhanced both MSC GAG concentrations (P<.0001) and MSC pellet size (P<.004) after 2 weeks in culture. Conclusion— Equine MSCs have superior chondrogenic potential compared with ASCs and the equine ASC growth factor response suggests possible differences compared with other species. Clinical Relevance— Elucidation of equine ASC and MSC receptor profiles will enhance the use of these cells in regenerative cartilage repair.     

Estudio Radiológico del Esternón del Ovino en Crecimiento

Radiological investigation of the sternum of the sheep A radiological investigation of the sternum of the sheep demonstrated distinct growth phases. Forty Merino sheep of mixed breeds were distributed into 4 age groups of 0, 45, 105 and 270 days. Each animal was radiographed in sternal and lateral recumbency. Measurements were made from the radiographs. The sternebra are rectangular in newborn and become concave in adult. The curvature of the sternum is 16 to 17° at day 0 and 27–28° at day 270. The nucleus of ossification was seen in all sternebrae at 45 days of age, they were present only in the most caudal sternebrae at 105 days.     

Musculoskeletal responses of 2-year-old Thoroughbred horses to early training. 8. Quantitative back-scattered electron scanning electron microscopy and confocal fluorescence microscopy of the epiphysis of the third metacarpal bone

AIM: To characterise and explain the increase in density evident by computerised tomography (CT) and radiography in companion studies as a response to training, in bone in the palmar and dorsal regions of the condyles of the third metacarpal bone (Mc3) of 2-year-old Thoroughbred horses.

METHODS: Compositional back-scattered electron (BSE) imaging in scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM) were conducted on polymethyl methacrylate (PMMA)-embedded mediolateral slices of the right distal Mc3 from seven 2-year-old Thoroughbred horses trained on a racetrack and seven untrained horses kept at pasture. One left Mc3 from each group was studied in transverse section planes. This study focussed on regions of Mc3 found to differ in density between the trained and untrained horses in companion studies using CT and radiography.

RESULTS: The increase of bone density in the condyles of Mc3 in trained horses compared with untrained horses occurred, without prior osteoclastic resorption, via the deposition of new bone on pre-existing internal surfaces. Within prior marrow spaces of cancellous bone, there was also rapid formation of immature strands and fronds of bone which were more cellular and mineralised, and more lamellar bone tissue was deposited on these new scaffolding elements in the trained horses. Both resulted in increased bone volume fraction (BVF). The microscopic mineralisation density of the bulk of the new tissue was lower than in pre-existing bone, and CT and radiography underestimated the increase in BVF. The new tissue was thus probably less stiff at the microscopic scale than pre-existing bone, though its addition would stiffen the global structure.

CONCLUSIONS: In Mc3 of all the trained horses, there were obvious differences in microscopic structure compared with those from the untrained horses. Moderate, industry-standard levels of exercise used to prepare young horses for racing induced the formation of new bone in non-bone spaces in bone tissue, such that the bone organ should better withstand later increased levels of exercise.     

Immunohistochemical localization of alpha 2-beta 1-glycoprotein in horses

Alpha 2-beta 1-glycoprotein may be found free in horse serum or complexed with alpha-1-proteinase inhibitor to form pre-alpha 2-elastase inhibitor. There has been little information published concerning alpha 2-beta 1-glycoprotein and its possible tissue sources in horses. A peroxidase-antiperoxidase technique was used to identify alpha 2-beta 1-glycoprotein in buffy coat and bone marrow neutrophils of healthy horses. Macrophages and neutrophils in bronchoalveolar lavage samples from clinically normal horses and from horses with chronic pulmonary disease also were positive for alpha 2-beta 1-glycoprotein. Alpha 2-beta 1-glycoprotein was identified in some instances in normal equine hepatocytes of formalin-fixed liver sections. In formalin-fixed lung sections from horses with chronic, small-airway disease and chronic bronchointerstitial pneumonia, alpha 2-beta 1-glycoprotein was observed in some airway secretions and in macrophages.     

Standing computed tomography of the equine limb using a multi-slice helical scanner: Technique and feasibility study

Computed tomography (CT) is an important cross-sectional diagnostic modality for lameness localised to the equine distal limb. The necessity of general anaesthesia to perform CT scans has limited its use in the equine orthopaedic field. Therefore, many attempts have been made to perform CT of the distal limb in standing horses. This retrospective report aims to describe the technical set-up and the feasibility of using a multi-slice helical CT unit recently introduced into the equine market. The medical records of the patients undergoing a standing CT in the period between March 2019 and January 2020 were reviewed. The imaged anatomical region and the image quality were assessed. Thirty-two horses met the inclusion criteria, and the following anatomical areas have been imaged: front foot/pastern (n = 14), metacarpophalangeal joint (n = 11), front proximal suspensory ligament (n = 2), carpus (n = 2), metatarsophalangeal joint (n = 2) and tarsus (n = 1). In 97% of the cases, excellent imaging quality was obtained. Motion artefact is the main cause of poor image quality. The feet and the metacarpophalangeal region can be easily imaged. Imaging the proximal anatomical regions of the limb is more challenging but achievable.     

Computed tomography of normal cranioencephalic structures in two horses

The purpose of this investigation was to define the anatomy of the cranioencephalic structures in horses using computed tomography (CT). Transverse images of two isolated equine cadaver heads were obtained using a Toshiba 600 HQ (third-generation equipment TCT). CT images were compared to corresponding frozen cross-sections of the cadaver head. Relevant anatomical structures were identified and labelled at each level. The resulting images provided excellent anatomic detail of the structures of the central nervous system and associated formations. Annotated CT images from this study are intended as a reference for clinical CT imaging studies of the equine head.     

Musculoskeletal responses of 2-year-old Thoroughbred horses to early training. 8. Quantitative back-scattered electron scanning electron microscopy and confocal fluorescence microscopy of the epiphysis of the third metacarpal bone

AIM: To characterise and explain the increase in density evident by computerised tomography (CT) and radiography in companion studies as a response to training, in bone in the palmar and dorsal regions of the condyles of the third metacarpal bone (Mc3) of 2-year-old Thoroughbred horses. METHODS: Compositional back-scattered electron (BSE) imaging in scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM) were conducted on polymethyl methacrylate (PMMA)-embedded mediolateral slices of the right distal Mc3 from seven 2-year-old Thoroughbred horses trained on a racetrack and seven untrained horses kept at pasture. One left Mc3 from each group was studied in transverse section planes. This study focussed on regions of Mc3 found to differ in density between the trained and untrained horses in companion studies using CT and radiography. RESULTS: The increase of bone density in the condyles of Mc3 in trained horses compared with untrained horses occurred, without prior osteoclastic resorption, via the deposition of new bone on pre-existing internal surfaces. Within prior marrow spaces of cancellous bone, there was also rapid formation of immature strands and fronds of bone which were more cellular and mineralised, and more lamellar bone tissue was deposited on these new scaffolding elements in the trained horses. Both resulted in increased bone volume fraction (BVF). The microscopic mineralisation density of the bulk of the new tissue was lower than in pre-existing bone, and CT and radiography underestimated the increase in BVF. The new tissue was thus probably less stiff at the microscopic scale than pre-existing bone, though its addition would stiffen the global structure. CONCLUSIONS: In Mc3 of all the trained horses, there were obvious differences in microscopic structure compared with those from the untrained horses. Moderate, industry-standard levels of exercise used to prepare young horses for racing induced the formation of new bone in non-bone spaces in bone tissue, such that the bone organ should better withstand later increased levels of exercise.