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 × 10(3) 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.

Les objectifs de la présente étude étaient d'utiliser une méthode non-équilibrée de fractionnement par flot sous champs gravitationnel (GrFFF), une méthode sans marquage immunologique de séparation des cellules souches mésenchymateuses (MSCs), afin de séparer les cellules souches mésenchymateuses dérivées du tissu musculaire équin (MMSCs) et les cellules souches mésenchymateuses provenant de la moelle osseuse (BMSCs) en sous-populations et de réaliser des essais afin de comparer leurs capacités ostéogéniques. Des cellules provenant d'un jeune cheval adulte furent isolées du muscle semi-tendineux gauche et d'aspirations de la moelle osseuse de la quatrième et cinquième strernèbre. Des aliquotes de 800 × 103 MSCs provenant de chaque source de tissu furent séparés en 5 fractions par GrFFF non-équilibré (système breveté GrFFF). Des fractions regroupées ont été mises en culture afin de proliférer pour utilisation dans des essais ostéogéniques, incluant la cytométrie en flux, l'histochimie, des essais de nodules osseux, et l'amplification en chaine quantitative par la polymérase (qPCR) pour l'expression des gènes de l'ostéocalcine (OCN), RUNX2, et osterix. Les MMSCs et BMSCs équins étaient séparés de manière constante en 5 fractions qui demeuraient viables pour utilisation dans des essais ostéogéniques additionnels. Les analyses statistiques ont confirmé une régulation à la hausse très significative pour OCN, RUNX2 et osterix pour la fraction 4 des BMSC (P < 0,00001). La cytométrie en flux a révélé une taille et une granularité différente pour la fraction 4 des BMSCs et la fraction 2 des MMSCs comparativement aux témoins non-séparés et aux autres fractions. L'histochimie et les essais de nodules osseux ont révélé des nodules se colorant positivement sans différence pour les tissus ou les fractions dans la moyenne de la surface du nodule, du périmètre, ou de l'intensité de la coloration. Étant donné qu'il y a différentes sous-populations de MSCs avec différentes capacités ostéogéniques parmi les sources dérivées du muscle et de la moelle osseuse, ces différences doivent être prises en compte lors de l'utilisation thérapeutique en médecine vétérinaire des cellules souches pour induire la guérison osseuse.(Traduit par Docteur Serge Messier).

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 × 10(3) 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.

L'objectif de la présente étude était de valider une méthode non-équilibrée de fractionnement par flot sous champs gravitationnel (GrFFF), une méthode sans marquage immunologique de séparation des cellules souches mésenchymateuses (MSCs) en sous-populations, pour utilisations avec des MSCs provenant de tissu musculaire, de tissu de périoste, de moelle osseuse, et de tissu adipeux de chevaux. Les cellules furent prélevées post-mortem à partir de six jeunes chevaux adultes. Les cellules furent isolées du muscle semi-tendineux gauche, du périoste de l'aspect disto-médial du tibia droit, d'aspirations de moelle osseuse de la quatrième et cinquième sternèbres, et du tissu adipeux sous-cutané de la région supra-glutéale gauche. Des aliquots de 800 × 103 MSCs de chaque tissu ont été séparés et injectés dans un appareil capillaire apparenté à un ruban par flot continu (système breveté GrFFF). Les cellules furent séparées en six fractions et les absorbances [densité optique (OD)] notées. Six fractions de chacun des six aliquots furent par la suite combinées afin de fournir des fractions poolées qui avaient des nombres adéquats de cellules pour ensemencer des concentrations égales dans les essais. Les MSCs provenant du tissu musculaire, du périoste, de la moelle osseuse, et du tissu adipeux étaient de manière constante séparées en six fractions qui sont demeurées viables pour utilisation dans des essais ultérieurs. La fraction 1 avait plus de cellules de morphologie cuboïde comparativement aux autres fractions. Les analyses statistiques des OD des fractions ont révélé une valeur de P < 0,05 lorsque les fractions 2 et 3 étaient comparées aux fractions 1, 4, 5, et 6. Il fut conclu que la méthode GrFFF non-équilibrée est une méthode valide pour séparer les MSCs équines dérivées des cellules musculaires, du périoste, de la moelle osseuse, et du tissu adipeux en sous-populations qui demeurent viables, assurant ainsi son potentiel pour utilisation en médecine vétérinaire et les applications avec les cellules souches équines.(Traduit par Docteur Serge Messier).

Evaluation of an in vivo heterotopic model of osteogenic differentiation of equine bone marrow and muscle mesenchymal stem cells in fibrin glue scaffold.

Autologous mesenchymal stem cells (MSCs) have been used as a potential cell-based therapy in various animal and human diseases. Their differentiation capacity makes them useful as a novel strategy in the treatment of tissue injury in which the healing process is compromised or delayed. In horses, bone healing is slow, taking a minimum of 6-12 months. The osteogenic capacity of equine bone marrow and muscle MSCs mixed with fibrin glue or phosphate-buffered saline (PBS) as a scaffold is assessed. Bone production by the following groups was compared: Group 1, bone marrow (BM) MSCs in fibrin glue; Group 2, muscle (M) MSCs in fibrin glue; Group 3, BM MSCs in PBS; Group 4, M MSCs in PBS and as a control; Group 5, fibrin glue without cells. BM and M MSCs underwent osteogenic stimulation for 48 h prior to being injected intramuscularly into nude mice. After 4 weeks, the mice were killed and muscle samples were collected and evaluated for bone formation and mineralization by using radiology, histochemistry and immunohistochemistry. Positive bone formation and mineralization were confirmed in Group 1 in nude mice based on calcium deposition and the presence of osteocalcin and collagen type I; in addition, a radiopaque area was observed on radiographs. However, no evidence of mineralization or bone formation was observed in Groups 2-5. In this animal model, equine BM MSCs mixed with fibrin glue showed better osteogenic differentiation capacity compared with BM MSCs in PBS and M MSCs in either carrier.

Characterization and osteogenic potential of equine muscle tissue- and periosteal tissue-derived mesenchymal stem cells in comparison with bone marrow- and adipose tissue-derived mesenchymal stem cells.

OBJECTIVE: To characterize equine muscle tissue- and periosteal tissue-derived cells as mesenchymal stem cells (MSCs) and assess their proliferation capacity and osteogenic potential in comparison with bone marrow- and adipose tissue-derived MSCs. SAMPLE: Tissues from 10 equine cadavers. PROCEDURES: 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 adipose tissue from the left subcutaneous region. Mesenchymal stem cells were characterized on the basis of morphology, adherence to plastic, trilineage differentiation, and detection of stem cell surface markers via immunofluorescence and flow cytometry. Mesenchymal stem cells were tested for osteogenic potential with osteocalcin gene expression via real-time PCR assay. Mesenchymal stem cell cultures were counted at 24, 48, 72, and 96 hours to determine tissue-specific MSC proliferative capacity. RESULTS: Equine muscle tissue- and periosteal tissue-derived cells were characterized as MSCs on the basis of spindle-shaped morphology, adherence to plastic, trilineage differentiation, presence of CD44 and CD90 cell surface markers, and nearly complete absence of CD45 and CD34 cell surface markers. Muscle tissue-, periosteal tissue-, and adipose tissue-derived MSCs proliferated significantly faster than did bone marrow-derived MSCs at 72 and 96 hours. CONCLUSIONS AND CLINICAL RELEVANCE: Equine muscle and periosteum are sources of MSCs. Equine muscle- and periosteal-derived MSCs have osteogenic potential comparable to that of equine adipose- and bone marrow-derived MSCs, which could make them useful for tissue engineering applications in equine medicine.

Scaffold effects on osteogenic differentiation of equine mesenchymal stem cells: an in vitro comparative study.

The in vitro viability, osteogenic differentiation, and mineralization of four different equine mesenchymal stem cells (MSCs) from bone marrow, periosteum, muscle, and adipose tissue are compared, when they are cultured with different collagen-based scaffolds or with fibrin glue. The results indicate that bone marrow cells are the best source of MSCs for osteogenic differentiation, and that an electrochemically aggregated collagen gives the highest cell viability and best osteogenic differentiation among the four kinds of scaffolds studied.

Comparison of isolation and expansion techniques for equine osteogenic progenitor cells from periosteal tissue.

Stem cell therapy and cell-based therapies using other progenitor cells are becoming the treatment of choice for many equine orthopedic lesions. Important criteria for obtaining autogenous equine progenitor cells in vitro for use in clinical cell-based therapy include the ability to isolate and expand cells repeatedly to high numbers (millions) required for therapy, in a clinically relevant time frame. Cells must also maintain their ability to differentiate into the tissue type of choice. The objective of this study was to compare isolation and expansion techniques for preparation of periosteal-derived osteogenic progenitor cells for use in commercial autogenous cell-based therapy. Cells were allowed to migrate spontaneously from periosteal tissue or were enzymatically released. Isolated cells were expanded using enzymatic detachment of cells and subsequent monolayer or dynamic culture techniques. Viable osteogenic progenitor cells from each group were counted at 2 weeks, and osteogenic potential determined. Cells isolated or expanded using the explant or bioreactor technique yielded cells at a much lower number per gram of tissue compared with that of enzyme digestion and monolayer expansion, but all cells were able to differentiate into the ostoblast phenotype. Osteogenic progenitor cells isolated by enzymatic release and expanded using monolayer culture reached the highest number of viable cells per gram of donor periosteal tissue while maintaining the ability to differentiate into bone forming cells in vitro. This technique would be an easy, consistent method of preparation of equine osteogenic cells for clinical cell based therapy for orthopedic conditions.

Isolation, characterization, and in vitro proliferation of canine mesenchymal stem cells derived from bone marrow, adipose tissue, muscle, and periosteum.

OBJECTIVE: To isolate and characterize mesenchymal stem cells (MSCs) from canine muscle and periosteum and compare proliferative capacities of bone marrow-, adipose tissue-, muscle-, and periosteum-derived MSCs (BMSCs, AMSCs, MMSCs, and PMSCs, respectively). SAMPLE: -7 canine cadavers. PROCEDURES: -MSCs were characterized on the basis of morphology, immunofluorescence of MSC-associated cell surface markers, and expression of pluripotency-associated transcription factors. Morphological and histochemical methods were used to evaluate differentiation of MSCs cultured in adipogenic, osteogenic, and chondrogenic media. Messenger ribonucleic acid expression of alkaline phosphatase, RUNX2, OSTERIX, and OSTEOPONTIN were evaluated as markers for osteogenic differentiation. Passage-1 MSCs were counted at 24, 48, 72, and 96 hours to determine tissue-specific MSC proliferative capacity. Mesenchymal stem cell yield per gram of tissue was calculated for confluent passage-1 MSCs. RESULTS: -Successful isolation of BMSCs, AMSCs, MMSCs, and PMSCs was determined on the basis of morphology; expression of CD44 and CD90; no expression of CD34 and CD45; mRNA expression of SOX2, OCT4, and NANOG; and adipogenic and osteogenic differentiation. Proliferative capacity was not significantly different among BMSCs, AMSCs, MMSCs, and PMSCs over a 4-day culture period. Periosteum provided a significantly higher MSC yield per gram of tissue once confluent in passage 1 (mean ± SD of 19,400,000 ± 12,800,000 of PMSCs/g of periosteum obtained in a mean ± SD of 13 ± 1.64 days). CONCLUSIONS AND CLINICAL RELEVANCE: -Results indicated that canine muscle and periosteum may be sources of MSCs. Periosteum was a superior tissue source for MSC yield and may be useful in allogenic applications.

Osteoprogenitor cell therapy in an equine fracture model.

OBJECTIVE: To compare the efficacy of osteoprogenitors in fibrin glue to fibrin glue alone in bone healing of surgically induced ostectomies of the fourth metacarpal bones in an equine model. STUDY DESIGN: Experimental. ANIMALS: Adult horses (n = 10). METHODS: Segmental ostectomies of the 4th metacarpal bone (MC4) were performed bilaterally in 10 horses. There was 1 treatment and 1 control limb in each horse. Bone defects were randomly injected with either fibrin glue and osteoprogenitor cells or fibrin glue alone. Radiography was performed every week until the study endpoint at 12 weeks. After euthanasia, bone healing was evaluated using radiography and histology. Analysis of radiographic data was conducted using a linear-mixed model. Analysis of histologic data was conducted using a general linear model. Statistical significance was set at P < .05. RESULTS: Radiographic grayscale data as a measure of bone healing revealed no significant difference between treatment and control limbs. Radiographic scoring results also showed that the treatment effect was not significant. Histologic analysis was consistent with radiographic analysis showing no significant difference between the area of bone present in treatment and control limbs. CONCLUSION: Injection of periosteal-derived osteoprogenitors in a fibrin glue carrier into surgically created ostectomies of MC4 does not accelerate bone healing when compared with fibrin glue alone.

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.

Evaluation of a technique for collection of cancellous bone graft from the proximal humerus in horses.

OBJECTIVES: To describe a technique for collecting cancellous bone graft from the proximal humerus in horses. STUDY DESIGN: Prospective evaluation of an experimental bone graft collection technique. ANIMAL POPULATION: Eight horses, 3-15 years, weighing 495-605 kg. METHODS: Horses were anesthetized and positioned in lateral recumbency. The lateral aspect of the proximal humerus was exposed by a 7-10-cm incision extending distally from the greater humeral tubercle, followed by sharp dissection through the omotransversarius muscle and between the infraspinatus and deltoideus muscles. A 12-mm cortical defect was incrementally created in the lateral proximal humerus. Human bone graft harvesting equipment (Acumed, Beaverton, OR) was drilled through this defect to collect a core of cancellous bone. In five horses additional cancellous bone was then collected with conventional instruments. Bone samples were weighed and histologically examined. Horses were monitored and graded for quality of anesthetic recovery, incisional complications, and postoperative lameness. RESULTS: Total mean (+/-SD) surgical time for harvesting bone with the Acumed system and traditional techniques (n=5) was 38+/-6 minutes (range, 32-47 minutes). Mean cancellous bone weight collected with the Acumed system was 3.6+/-0.8 g (range, 2.0-4.6 g), and cancellous bone collected conventionally was 25.6+/-7.5 g (range, 16.8-34.2 g). Minimal incisional complications or postoperative lameness were observed. Mortality was 12.5%; one horse fractured the operated humerus during anesthetic recovery. CONCLUSION: The Acumed system provided limited cancellous bone when used with the technique described. However, the quantity of cancellous bone collected with traditional harvesting instruments was comparable to other sites used in horses. The procedure was associated with minimal postoperative incisional complications or lameness, but because one horse suffered a catastrophic humeral fracture further research is required to assess the effects of this procedure on humeral breaking strength. CLINICAL RELEVANCE: Based on the risk of catastrophic fracture, this technique cannot be recommended for use in clinical cases, especially if an unassisted recovery from general anesthesia is planned.

In vitro comparison of equine cancellous bone graft donor sites and tibial periosteum as sources of viable osteoprogenitors.

OBJECTIVE: To compare the osteogenic potential of cancellous bone of conventional graft sites with that of one nonconventional site (fourth coccygeal vertebra) and to investigate the tibial periosteum as a donor site with respect to osteogenic potential. STUDY DESIGN: In vitro osteogenic cell culture system. SAMPLE POPULATION: Eight adult horses. METHODS: Cancellous bone or tibial periosteum was aseptically collected and cut into bone chips or periosteal strips of 1 to 2 mm(3) for primary explant cultures. After 2 weeks, primary tissue cultures that yielded a population of osteogenic cells were counted and subcultured at 1 x 10(5) cells/35-mm dish in osteogenic media. After 7 to 10 days, subcultures were stained with Von Kossa (VK) to assess mineralized bone nodule formation. VK-positive bone nodules were counted as osteoprogenitors and compared among 3 donor sites, which provided consistent primary osteogenic cells (tuber coxae, fourth coccygeal vertebra, periosteum) using ANOVA (P <.05). RESULTS: Sternal and tibial bone yielded viable osteogenic cells from 25% and 50% of horses, respectively, whereas yields from tuber coxae, coccygeal vertebra, and periosteum were 75%, 100%, and 100%, respectively. Tuber coxae and periosteum had significantly greater numbers of osteoprogenitors compared with fourth coccygeal vertebra. CONCLUSIONS: Among the conventional donor sites, tuber coxae most consistently yielded viable osteogenic cells with an acceptable percentage of osteoprogenitors. Sternal and tibial sites were unreliable in providing osteogenic cells. Two new donor sites, the fourth coccygeal vertebra and tibial periosteum, were tissues with good osteogenic potential. CLINICAL RELEVANCE: When a source of transplantable viable osteoprogenitor cells is desired, use of the tuber coxae as a conventional donor site is warranted. Use of tibial periosteum or fourth coccygeal vertebra as reliable sources of transplantable osteoprogenitors should be considered.