Bone marrow grafting, either as unconcentrated or concentrated bone marrow, has been applied to treat various orthopedic disorders as a point-of-care transplantation. Bone marrow tissue is a rich autologous source of bone marrow-derived mesenchymal stem cells that are highly proliferative cells with self-renewal and multilineage differentiation capacity, including various mesenchymal tissues such as bone, cartilage, fat, muscle, and tendon.1 Bone marrow is also known to contain various growth factors, such as transforming growth factor-β, platelet-derived growth factor, and vascular endothelial growth factor,2 that are within the α-granules of platelets or secreted by mesenchymal stem cells.3,4 Autologous grafting of bone marrow or concentrated bone marrow promotes healing in nonunion or delayed union of long bone fractures.5–8 Also, direct injection of concentrated bone marrow has been used to promote cartilage healing in an experimental study9 in horses.
Bone marrow tissue has been harvested to isolate bone marrow-derived mesenchymal stem cells that are cultured, expanded, and injected back into the host (autologous cell therapy) to promote osseous or soft tissue healing. Equine bone marrow-derived mesenchymal stem cells have been expanded in culture and experimentally evaluated to promote healing of articular cartilage10,11 and tendon.12,13 Cultured equine bone marrow-derived mesenchymal stem cells have been applied clinically for the treatment of tendinitis14,15 and suspensory desmitis.16
Production of concentrated bone marrow and separation of bone marrow-derived mesenchymal stem cells have been performed with several density gradient solutions17,18 or point-of-care centrifugation systems.5,7 Although these methods are proficient, use of gravitational devices designed to enhance efficiency in the separation and concentration of bone marrow elements can improve platelet and bone marrow-derived mesenchymal stem cell number, concentration, frequency of rich fractions, and viability as well as avoid exposure to chemicals, multiple cell washing steps, risk of contamination, or sample mix-up during multistep handling. Marrow separator systems could be used to obtain a mixture of bone marrow-derived mesenchymal stem cells and platelet- or plasma-derived growth factors19 that can be applied for a 1-step, nonlaboratory, point-of-care application of concentrated bone marrow.
Adult stem cells such as bone marrow-derived mesenchymal stem cells can lose their multipotent capacity over cell passages, unlike embryonic stem cells.20 Therefore, bone marrow may need to be harvested more than once for multiple stem cell treatments for chronic conditions, such as delayed unions or nonunions, osteoarthritis, or cartilage defects. However, it is unclear whether repeated bone marrow collection during a short interval would affect the number or viability of bone marrow-derived mesenchymal stem cells.
One of the most important limiting factors of bone marrow-derived mesenchymal stem cell concentration is the number of recovered mononuclear cells in the bone marrow aspirate. In a previous study,21 collecting four 1-mL aspirates resulted in approximately twice as many osteoblast progenitor cells as did collecting one 4-mL aspirate because of sample dilution by peripheral blood. Therefore, novel aspiration needle designs, such as a multidirectional bone marrow aspiration needlea that is modified to harvest smaller volumes of bone marrow from several locations and depths, may improve the bone marrow-derived mesenchymal stem cell number or the frequency with which bone marrow-derived mesenchymal stem cell-rich samples are harvested. Also, a closed-end design may minimize dilution by peripheral blood.
The objectives of the study reported here were to determine the efficiency of a novel gravitational marrow separator system to obtain concentrated bone marrow from bone marrow, to assess the effect of repeated bone marrow collections on the numbers and viability of bone marrow-derived mesenchymal stem cells in the concentrated bone marrow, and to compare the quality of concentrated bone marrow between the novel multidirectional bone marrow aspiration needle and traditional bone marrow aspiration needle. Our hypotheses were that the novel gravitational marrow separator system could be used to obtain concentrated bone marrow with high bone marrow-derived mesenchymal stem cell count, when a large volume of bone marrow is aspirated from the same location within a short interval; that bone marrow-derived mesenchymal stem cell number would be lower with the second aspiration; and that use of the novel multidirectional bone marrow aspiration needle would improve the quality of concentrated bone marrow by increasing the number or frequency of bone marrow-derived mesenchymal stem cells obtained.
Materials and Methods
Study design—Eight healthy adult 5-year-old female Thoroughbreds with a mean body weight of 476 kg (range, 420 to 551 kg) were used in this noneuthanasia study approved by The Ohio State University Institutional Animal Care Committee. All horses were clinically normal in physical and lameness examinations and had no abnormalities in the sternum on palpation. Each of the 8 horses was used for bone marrow aspiration twice at a 1-month interval by use of general anesthesia (total, 16 collections). At each collection, bone marrow aspiration was performed from 4 sternal bodies, specifically the third, fourth, fifth, and sixth sternal bodies (total, 64 bone marrow aspiration samples). In each bone marrow aspiration sample, 66 mL of anticoagulated bone marrow was collected from each sternal body with 1 of 2 bone marrow aspiration needles: a traditional bone marrow aspiration needleb (control needle; n = 32 bone marrow aspirations) or a multidirectional, closed-end, double-cannulated bone marrow aspiration needlea (design needle; 32 bone marrow aspirations). Each needle was assigned to be used in each of the 4 sternebrae of each horse by use of a blocked design, such that each needle design was used in an equal number of aspirations for each sternebra (3 to 6) and each needle was used in the first and second collection times at each sternebra. Of the 66 mL in each aspirate, 6 mL was allocated to be analyzed as a bone marrow sample (n = 64), and the remaining 60 mL was concentrated by a gravitational marrow separatorc (Figure 1) into the final volume of 6 to 8 mL that was analyzed as a concentrated bone marrow sample (64). All bone marrow and concentrated bone marrow samples were analyzed for total WBC count, total platelet count, WBC differential count by use of an automated hematology analyzer and cytologic evaluation, cell viability via flow cytometry, and mesenchymal stem cell count via CFU analysis including fibroblasts and, in the second aspirate only, endothelial progenitor cells.
Illustrative representation of relative quantities of cell populations in peripheral blood, bone marrow, and bone marrow concentrated with a gravitational marrow separator. Notice that concentrated bone marrow contains a higher number of platelets, WBCs, precursor cells, and mesenchymal stem cell as well as fewer RBCs, compared with nonconcentrated bone marrow or peripheral blood.
Citation: American Journal of Veterinary Research 74, 6; 10.2460/ajvr.74.6.854
Bone marrow aspiration and concentration—Horses were premedicated with xylazine,d and general anesthesia was induced by administration of diazepame and ketamine hydrochloride.f Horses were positioned in dorsal recumbency, and anesthesia was maintained with isofluraneg and oxygen. Mechanical ventilation was used to maintain breathing, and routine intraoperative monitoring was performed, including ECG and arterial blood pressure monitoring. The aspiration sites (center of the third to sixth sternal bodies) were identified by use of ultrasonography and skin marker needles. Following aseptic preparation, skin stab incisions were made on the midline at the 4 aspiration sites, and the assigned bone marrow aspiration needle (control or design needle) was inserted into the marrow cavity at a 2-cm depth from the cortex. A 60-mL syringe containing 5.5 mL of heparinh (1,000 U/mL) was attached to the bone marrow aspiration needle. For the control needle, the needle was rotated 90° for every 5 to 6 mL to collect 33 mL of bone marrow from 2 directions at the same depth. For the design needle, an inner trocar was inserted and rotated 120° for every 5 to 6 mL to collect 33 mL of bone marrow at 3 directions at 3 depths because of the needle design (Figure 2). For both needle designs, a second syringe was attached and another 33 mL of bone marrow was aspirated with a similar method. The aspiration time (time between start and completion of bone marrow withdrawal per syringe) was recorded for each syringe per site. The bone marrow samples in the 2 syringes (total, 66 mL/site) were combined and kept on a rocker machine during the collection procedure. Bone marrow aspiration was performed from caudal to cranial aspiration sites, from the sixth sternal body, and continued to the third sternal body. Immediately after all bone marrow aspirations were obtained from the 4 sites, 60 of 66 mL of bone marrow/site was transferred into a marrow separator and processed. The centrifugation of the gravitation marrow separator was performed at room temperature (approx 22°C) with a centrifugea designed as a companion to the marrow separator system and was spun at approximately 2,000 × g for 15 minutes. Following centrifugation, concentrated bone marrow samples were collected from the port that draws off the separated concentrated bone marrow, per manufacturer's instructions, and the final volumes of separated concentrated bone marrow (between 6 and 8 mL) were recorded.
Photographs of a traditional bone marrow aspiration needle (Control needle) and a multidirectional double-cannulated bone marrow needle (Design needle) used to obtain bone marrow from the sternum of horses. In the close-up view of the design needle, notice the 3 groups of openings oriented in different directions that permit aspiration at 3 depths from a large area as the inner cannula is rotated (images to right of the close-up view) and the closed-end trocar tip that minimizes contamination with blood.
Citation: American Journal of Veterinary Research 74, 6; 10.2460/ajvr.74.6.854
Bone marrow and concentrated bone marrow sample analysis—All bone marrow and concentrated bone marrow samples were analyzed with a hematology analyzeri for an automated total WBC count, total platelet count, and WBC differential counts; gated; and validated for equine lymphocytes, neutrophils, monocytes, eosinophils, and basophils. Direct smear slides were made with 1 drop of bone marrow and concentrated bone marrow with a push technique. Slides were air-dried and stained with Wright stain for manual WBC differential counting. Cells were categorized as lymphocytes, neutrophils, monocytes, eosinophils, basophils, WBC precursors, RBC precursors, or megakaryocytes. All the cytologic assessments were made by 2 evaluators (AI and RBSH) in a masked fashion, without knowledge of needle design. As a comparison, venous blood samples were collected from the jugular vein and analyzed with a hematology analyzer and via manual cytologic WBC differential counts. The rest of the bone marrow and concentrated bone marrow samples were suspended in a standard RBC lysis buffer (0.15M NH4Cl, 1.0M KHCO3, and 0.1M EDTA), centrifuged, and washed twice in PBS solutionj containing 10% fetal bovine serum.k For each bone marrow and concentrated bone marrow sample, total RBC precursor and WBC precursor counts were calculated with the WBC counts (on the basis of the hematology analyzer result) and percentages of RBC and WBC precursors (the sum of percentages of RBC precursors and WBC precursors). On the basis of the mononuclear cell counts (the sum of RBC and WBC precursor, lymphocyte, monocyte, eosinophil, and basophil counts) obtained by the hematology analyzer, 1 million mononuclear cells from each bone marrow and concentrated bone marrow sample were transferred into a separate tube, stained with the 7-aminoactinomycin D fluorescent chemicall for 2 hours, washed twice with PBS solution, and analyzed via flow cytometry.m Cell viability was determined by detecting a fraction of 7-aminoactinomycin D-positive cells expressed as percentage for 4 cell populations: the all-WBC population, progenitor and plasma cell population, lymphocyte population, and granulocyte and monocyte population. For the CFU fibroblast assays, 0.5, 1, and 2 million mononuclear cells were seeded in duplicate cell culture flasks (area, 25 cm2)n with mesenchymal stem cell culture medium,° incubated for 2 weeks (37°C; 0.5% CO2), and stained with Giemsa stain.j The CFU endothelial progenitor cells assays were performed on the second aspirate only, and the 1 million mononuclear cells were seeded in duplicate cell culture flasks with basal medium,p incubated for 5 days (37°C and 0.5% CO2), and stained with Giemsa stain. For both CFU fibroblast and CFU endothelial progenitor cells assays, the number of visible colonies (diameter, 1 to 8 mm) was counted and expressed as CFU fibroblast and CFU endothelial progenitor cell counts per million mononuclear cells (on the basis of proportions of the numbers of mononuclear cells seeded); the means were calculated among the flasks.
Data analysis—Commercial statistical softwareq was used for all data analyses. Repeated-measures ANOVA was used to compare the outcome measurements between bone marrow and concentrated bone marrow samples, between the 2 needle designs, among aspiration sites (third to sixth sternal bodies), and between the first and second bone marrow aspiration. Repeated variables were considered to be nested within horse. In addition, the CFU fibroblast and CFU endothelial progenitor cell counts were categorized as low (< 10 CFUs/1 million mononuclear cells), medium (10 to 100 CFUs/1 million mononuclear cells), or high (> 100 CFUs/1 million mononuclear cells), and the frequencies of low, medium, and high CFU fibroblast CFU endothelial progenitor cell counts were compared via χ2 tests between bone marrow and concentrated bone marrow samples, between needle designs, among aspiration sites, and between the first and second bone marrow aspiration. Values of P < 0.05 were considered significant for all analyses.
Results
The concentrated bone marrow samples had significantly greater (7- to 19-fold) CFU fibroblast counts (Figure 3) and CFU endothelial progenitor cell counts (P < 0.006 for both), compared with original bone marrow samples. Colony-forming unit fibroblast counts of bone marrow and concentrated bone marrow samples were significantly (P < 0.01 for both) greater in the second bone marrow aspiration, compared with the first bone marrow aspiration. The design needle had significantly (P = 0.04) greater frequency of obtaining greater CFU fibroblast counts, compared with the control needles.
Results of equine bone marrow aspirations performed with the same needles as in Figure 2. A—Mesenchymal stem cell count based on CFU fibroblast (CFU-F) assay. B—Endothelial stem cell count based on CFU-endothelial progenitor cell (CFU-EPC) assay. The second aspiration was performed 1 month after the first aspiration and from the same aspiration sites. C—Photographs of results of CFU fibroblast assays and frequencies of low, medium, and high CFU fibroblast counts in bone marrow and concentrated bone marrow sample (data were combined) obtained by use of control and design needles. *Significant (P ≤ 0.05) difference between bone marrow and concentrated bone marrow samples, between first and second aspirations, or between needle designs. MNCs = Mononuclear cells. N/A = Not applicable.
Citation: American Journal of Veterinary Research 74, 6; 10.2460/ajvr.74.6.854
The concentrated bone marrow samples had significantly greater numbers (5- to 7-fold) of RBC and WBC precursors (Figure 4), WBCs, and platelets, compared with bone marrow samples (P < 0.02 for each). The concentrated bone marrow samples had significantly lower (one-fourth to one-sixth) RBC counts, compared with bone marrow samples. There were no significant differences in RBC and WBC precursor, WBC, platelet, or RBC counts between the 2 needle designs, among aspiration sites, or between the first and second bone marrow aspiration.
Results of RBC or WBC precursor (A), WBC (B), platelet (C), and RBC (D) counts of bone marrow and concentrated bone marrow samples obtained with the same needles as in Figure
Citation: American Journal of Veterinary Research 74, 6; 10.2460/ajvr.74.6.854
For differential counts obtained via manual cytologic counts, the total bone marrow cells (ie, sum of WBC precursors, RBC precursors, and megakaryocytes) in both bone marrow and concentrated bone marrow samples had a mean of approximately 40% precursor cells, which was significantly greater than whole blood from the same horse and indicated successful aspiration of bone marrow (Figure 5). Precursor percentages were significantly (P = 0.007) lower in the second bone marrow aspiration, compared with the first bone marrow aspiration. Also, the total bone marrow cells were significantly (P = 0.04) lower in concentrated bone marrow, compared with bone marrow in the second bone marrow aspiration. There were no significant differences in cell differential counts obtained by manual methods, between needle designs, or among sternebrae aspiration sites.
Results of cell differential counts of bone marrow and concentrated bone marrow samples obtained with the same needles as in Figure 2 and performed by use of cytologic examination or an automated hematology analyzer. The second aspiration was performed 1 month after the first aspiration and from the same aspiration sites. *Significant (P ≤ 0.05) difference between bone marrow and concentrated bone marrow samples or between first and second aspirations. The hematology analyzer appeared to count precursor cells within the neutrophil gating range.
Citation: American Journal of Veterinary Research 74, 6; 10.2460/ajvr.74.6.854
For cell differential counts obtained by use of a hematology analyzer, there were no significant differences in any cell fractions between bone marrow and concentrated bone marrow, between needle designs, among aspiration sites, or between first and second bone marrow aspirations. Interestingly, the bone marrow and concentrated bone marrow had relatively greater cell counts in the size gating for neutrophils (> 89% in bone marrow and > 82% in concentrated bone marrow), compared with manual counts for neutrophils in both the first and second bone marrow aspirations (Figure 5), which were also significantly (P < 0.001) greater than neutrophil counts in venous blood.
The concentrated bone marrow samples had significantly (P = 0.03) lower cell viability than bone marrow samples for all 4 cell populations in both the first and second bone marrow aspirations (Figure 6). The concentrated bone marrow samples in the second bone marrow aspiration had significantly (P < 0.04) lower cell viability for all 4 cell populations, compared with the concentrated bone marrow samples in the first bone marrow aspiration or bone marrow samples in the second bone marrow aspiration. There were no significant differences in cell viability between needle designs or among aspiration sites.
Percentage cell viability of various cell populations in bone marrow and concentrated bone marrow samples obtained with the same needles as in Figure 2. The second aspiration was performed 1 month after the first aspiration and from the same aspiration sites. a–cDifferent superscript letters indicate significant (P ≤ 0.05) differences between bone marrow and concentrated bone marrow samples or between first and second aspirations.
Citation: American Journal of Veterinary Research 74, 6; 10.2460/ajvr.74.6.854
The aspiration time was significantly (P = 0.02) longer with use of the design needle, compared with use of the control needle (Figure 7), because of the greater number of manipulations. Also, with use of the design needle, aspiration time was significantly (P = 0.04) longer in the second syringe, compared with the first syringe in the first bone marrow aspiration.
Results of use of the same needles as in Figure 2 for sternal bone marrow aspiration in horses. The second aspiration was performed 1 month after the first aspiration and from the same aspiration sites. A—Aspiration times. *Significant (P ≤ 0.05) difference between needle designs. #Significant (P ≤ 0.05) difference between the first and second syringes. B—Representative ECG of iatrogenic arrhythmia associated with bone marrow needle insertion into the sixth sternal body of a horse. Lower panel represents arterial blood pressure. C—Frequency of intraoperative complications (number of incidents/number of procedures performed) associated with use of control and design bone marrow needles. *Significant (P ≤ 0.05) difference between needle designs. #Significantly (P ≤ 0.05) greater prevalence associated with the sixth sternal body, compared with the third, fourth, and fifth sternal bodies.
Citation: American Journal of Veterinary Research 74, 6; 10.2460/ajvr.74.6.854
Several complications associated with sternal bone marrow aspiration occurred, including insufficient withdrawal volume that required needle repositioning, loss of negative pressure caused by air migration, and iatrogenic arrhythmia. The iatrogenic cardiac arrhythmia, including ventricular tachycardia and fibrillation, was consistently observed via ECG when bone marrow aspiration needles were inserted into the sixth sternal body and was significantly (P = 0.03) more frequent, compared with the frequency of arrhythmia when needles were inserted into the third, fourth, or fifth sternebral bodies. In addition, the design needle had more frequent (P = 0.04) loss of negative pressure but less frequent need for repositioning.
Discussion
This study revealed that a novel point-of-care gravitational marrow separating system can effectively obtain concentrated bone marrow and concentrated bone marrow-derived mesenchymal stem cells from bone marrow, in addition to providing concentration of platelets and reduction in RBCs (Figures 1, 3, and 4). The concentrated bone marrow samples had significantly greater numbers of bone marrow-derived mesenchymal stem cells, RBC and WBC precursors, WBC, and platelets, compared with bone marrow samples or peripheral blood. The centrifuge equipment for this marrow separator system is light and portable and can be used for point-of-care, nonlaboratory, stall-side, and ambulatory-based application of concentrated bone marrow as a mixture of bone marrow-derived mesenchymal stem cells, platelets, and plasma. Reportedly, including in horses, platelet counts are strongly correlated with the concentrations of growth factors, including transforming growth factor-β and platelet-derived growth factor in platelet-rich plasma,22 and concentrated bone marrow would be anticipated to have similar increases in growth factors. Further work is needed to confirm this for concentrated bone marrow.
Use of the marrow separator can also facilitate a simple, 1-step, consistent, technically undemanding method for bone marrow-derived mesenchymal stem cell separation. The marrow separator has been optimized for maximal separation of the platelet, mature WBC, and progenitor cell layer in a concentrated and separated form suspended in plasma.23,r After centrifugation, the platelet layer is located directly below the WBC layer and within the top RBC layer. The larger progenitor cells are located at the top of the WBC layer because of their larger size and density. Test tubes designed to separate WBCs (buffy coats) from RBCs will not collect the platelets in the top RBC layer. Decanting or pipetting the plasma from the surface of the test tube results in plasma currents that disturb the top progenitor cell layer and reduce efficiency of progenitor separation. Specifically, in this marrow separator, the buoy shape, density, and material composition were extensively studied,23 in combination with the optimal physical effects of centrifugation, to provide consistent and maximal viability, function, separation, and concentration of the platelets and cells of interest from bone marrow in a consistent, repeatable manner. The marrow product is separated such that it is drawn off without potential for removal of the other layers during handling. Few devices designed to isolate platelets from blood (platelet-rich plasma devices) have been reported to effectively concentrate the bone marrow elements.9
Laboratory methods for separation of bone marrow cell elements use a density gradient reagent, such as sucrose-rich polymer, and supercentrifugation. Advantages of use of a gravitational separator over laboratory techniques include those for test tube separation as well as lack of stem cell exposure to chemicals (eg, density gradient reagents) that may diminish cell viability or be incompletely washed out of the sample; elimination of multiple cell wash steps, reducing the risk of bacterial contamination; and point-of-care convenience. Additionally, the final bone marrow product includes not only cell fractions (eg, bone marrow-derived mesenchymal stem cells), but also the plasma contents (a source of various growth factors). In the gravitation separator system, the bone marrow-derived mesenchymal stem cells are maintained in the plasma component of the bone marrow, an optimal environment for the viability of bone marrow-origin cells, for the entire process of cell separation, concentration, and subsequent application of concentrated bone marrow. With human bone marrow, the gravitational marrow separatorc increases the efficiency of collection of bone marrow total cells and increases efficiency of the separation of mesenchymal stem cells (CFU fibroblast) by 2.89-fold, compared with the gradient technique.s It is likely, although not reported, to the author's knowledge, that this holds true for equine bone marrow as well. Importantly, the focus of the present study was to report the performance and quality of mesenchymal stem cell separation of one of the systems under study for potential use in horses and humans and investigate the function of a novel needle design. Additional work comparing systems and other laboratory methods of bone marrow separation for horses would be of value.
In a subsequent study,t the bone marrow-derived mesenchymal stem cells from the marrow separator differentiated into 3 standard mesenchymal lineages, including osteogenic, adipogenic, and chondrogenic pathways. The population of bone marrow-derived mesenchymal stem cells in the present study was 0.0023% of all mononuclear cells (1 bone marrow-derived mesenchymal stem cell in 4.3 × 104 mononuclear cells), whereas previous work with other systems found bone marrow-derived mesenchymal stem cell populations in bone marrow mononuclear cells of 0.001% to 0.0001% in humans,24 0.024% in horses,25 0.004% in dogs,26 and 0.00026% in cats.27 The reasons for the variability in bone marrow-derived mesenchymal stem cell populations seen in the present study and a previous equine study25 are unknown but might include the differences in methods, ages and breeds of horses, or volumes of bone marrow aspirates.
To the authors' knowledge, the present study was the first to evaluate the effect of repeated bone marrow collections on the number and viability of bone marrow-derived mesenchymal stem cells isolated. Large volumes of bone marrow (66 mL × 4 sites = 264 mL) were collected twice at a 1-month interval from the same aspiration sites, and the concentrations of bone marrow-derived mesenchymal stem cells (on the basis of CFU fibroblast counts) in the second bone marrow aspiration were approximately 4 times those obtained in the first bone marrow aspiration (Figure 3). The cell differential analysis revealed that bone marrow samples in the second bone marrow aspiration contained a higher proportion of total bone marrow cells than the samples in first bone marrow aspiration, which implies a process of restoration at the time of second bone marrow aspiration. In combination, these results may suggest that actively hematopoietic bone marrow could contain a higher number of bone marrow-derived mesenchymal stem cells than resting bone marrow, resulting in greater numbers of bone marrow-derived mesenchymal stem cells collected. The centrifugation process of the second bone marrow aspiration (Figures 5 and 6) resulted in a diminished number and viability of total bone marrow cells, compared with the first bone marrow aspiration samples, but the total number of bone marrow-derived mesenchymal stem cells was greater. This result may imply that adolescent bone marrow cells in recuperating bone marrow may be more fragile and delicate. On the basis of these results, we conclude that bone marrow collection for concentrated bone marrow can be performed repeatedly from the same aspiration site to obtain comparable, or even greater, numbers of bone marrow-derived mesenchymal stem cells.
In the present study, both automated analyzer and manual cytologic evaluations were performed for blood, bone marrow, and concentrated bone marrow samples. The automated analyzer was gated for equine blood and synovial fluid analysis and was not adjusted or validated to identify progenitor cells in bone marrow. Progenitor cells are larger than peripheral venous WBCs and could be located outside the gating of the analyzer. The progenitors appeared to be recognized as neutrophils in the automated readout, considering that the automated neutrophil count was increased by approximately the same amount that the manual progenitor count was increased. Therefore, the true neutrophil counts were determined by cytologic hand-counting. The clinical relevance of such an observation is that clinicians in the field would not be able to rely on automated analysis of a bone marrow sample to identify progenitor separation. However, results of the present study suggested that an estimate of progenitor numbers could be obtained by comparing the neutrophil count in the blood versus the bone marrow automated analyzer neutrophil count, with the degree of increase representing an estimate of degree of bone marrow enrichment of progenitor cells.
The present results indicated that the novel multidirectional bone marrow aspiration needle harvested moderate- to high-quality bone marrow samples more frequently than did the control needle, as determined on the basis of the CFU fibroblast counts (Figure 3), even though there was no difference in counts of mean CFU fibroblast per milliliter between either first or second aspiration samples recovered by either needle, when the data were compared as groups. If the variability in CFU fibroblast counts is high, then the expected difference in mean counts may not be measureable. The novel needle was designed to promote a greater likelihood of obtaining a high-quality marrow sample on a given aspirate because of multiple aspiration sites in 3 directions at different depths, enabling bone marrow collection from a larger area, and the closed-end needle tip that minimized contamination with peripheral blood. For these reasons, use of the multidirectional needle may be helpful to avoid a suboptimal quality of bone marrow aspirate on a given occasion and increase the chance of getting a higher-quality sample. Handling of the prototype needles used in this study was somewhat cumbersome because of the weight of the metal prototype, friction between the inner and outer cannulae, and the arrangement of the inner trocar within the needle. The result was longer aspiration times, more manipulation, and loss of negative pressure during aspiration (Figure 7). Modification and commercial production of this prototype are expected to resolve these issues.
The present study revealed that the number and viability of bone marrow-derived mesenchymal stem cells were comparable among the 4 aspiration sites of the sternum (third to sixth sternal bodies). In horses, bone marrow aspiration has been commonly performed from the sternum, although the ilium and tibia are other options.28 A recent comprehensive study29 of bone marrow aspiration technique from the equine sternum determined that the fourth to sixth sternal bodies can be used to harvest bone marrow. Because there were no significant differences in bone marrow quality among the sternal bodies in the present results, the fifth sternebral body may be the optimal choice for aspiration because the ventral cortex is thin, permitting easy needle insertion, and the risk of cardiac arrhythmia is lower than for the sixth sternebral body. In this study, bone marrow aspiration needle insertion into the sixth sternal bodies of horses in dorsal recumbency resulted in iatrogenic ventricular tachycardia and fibrillation on ECG (Figure 7). This arrhythmia was temporary and quickly resolved as soon as the needle was retracted even a few millimeters. Once a normal rhythm returned, no abnormalities were evident on physical examinations, auscultations, or ECG during or after the procedure. No blood or fluid accumulation was evident via thoracic ultrasonography at 24 and 48 hours after surgery; thus, the iatrogenic arrhythmia apparently had no long-term adverse effects in these anesthetized horses. Further work determining the risk to standing horses receiving bone marrow aspiration in the sixth sternebra should be performed. This finding has prompted greater use of ECG by the authors during bone marrow aspirates in horses. Horses subjected to bone marrow aspirates performed during anesthesia are now often monitored for arrhythmia, and antiarrhythmics are administered by some clinicians if arrhythmias are identified. Anecdotal reports of horses collapsing during bone marrow aspiration have been mentioned among practitioners and may be related to cardiac arryhthmia. Although unsubstantiated currently, standing horses may be at greater risk because of the closer contact between the heart and the sternum in the standing position. Further work on the avoidance of risks of bone marrow aspiration is warranted. Use of the more cranial sternebra may be indicated.
Use of a gravitational marrow separator and multidirectional bone marrow aspiration needle effectively harvested bone marrow, concentrated the bone marrow, and yielded a greater frequency of obtaining more bone marrow-derived mesenchymal stem cells. This gravitational marrow separator was used in a 1-step, point-of-care, nonlaboratory method to obtain concentrated bone marrow as a mixture of platelets, progenitor cells, and bone marrow-derived mesenchymal stem cells suspended in plasma. When bone marrow aspiration was performed after a 1-month interval from the same aspiration sites, comparable (or even greater) numbers of bone marrow-derived mesenchymal stem cells could be collected. The novel multidirectional bone marrow aspiration needle can harvest bone marrow samples of greater quality more frequently than the traditional bone marrow aspiration needle.b
Biomet Biologics, Warsaw, Ind.
Jamshidi needle, Ranfac Corp, Avon, Mass.
MarrowStim Concentration Kit, Biomet Biologics, Warsaw, Ind.
Lloyd Laboratories, Shenandoah, Iowa.
Hospira Inc, Lake Forest, Ill.
Fort Dodge Animal Health, Fort Dodge, Iowa.
Abbott Animal Health, North Chicago, Ill.
APP Pharmaceuticals, Schaumburg, Ill.
Cell-Dyn 3500, Abbott Diagnostics, Mississauga, ON, Canada.
Invitrogen Corp, Carlsbad, Calif.
Hyclone, Thermo Scientific, Rockford, Ill.
BD Biosciences, San Jose, Calif.
Accuri Cytometer, Ann Arbor, Mich.
VWR International, Radnor, Pa.
MesenCult, StemCell Technologies, Vancouver, BC, Canada.
EndoCult, StemCell Technologies, Vancouver, BC, Canada.
SAS, SAS Institute Inc, Cary, NC.
Swift MJ, McKale JM, Greene J, et al. Concentration of white blood cell types with a commercially available platelet concentration system (abstr), in Proceedings. Tissue Eng Regen Med Int Soc North Am 2007;201.
Swift MJ, Welch ZR, McKale JM, et al. Efficient harvest of a mononuclear cell-rich fraction from aspirated bone marrow (abstr), in Proceedings. 53rd Annu Meet Orthop Res Soc 2007;1287.
Pigott JH, Ishihara A, Wellman ML, et al. Inflammatory and immune effects of autologous, allogeneic, xenogeneic and genetically modified autologous mesenchymal stem cells after intra-articular injection in horses (abstr), in Proceedings. Am Coll Vet Surg Symp 2012;1710.
References
1. Gómez-Barrena E, Rosset P & Müller I, et al. Bone regeneration: stem cell therapies and clinical studies in orthopaedics and trau-matology. J Cell Mol Med 2011; 15: 1266–1286.
2. Anitua E, Andia I & Ardanza B, et al. Autologous platelets as a source of proteins for healing and tissue regeneration. Thromb Haemost 2004; 91: 4–15.
3. Mehta S, Watson JT. Platelet rich concentrate: basic science and current clinical applications. J Orthop Trauma 2008; 22: 432–438.
4. Potier E, Ferreira E & Dennler S, et al. Desferrioxamine-driven upregulation of angiogenic factor expression by human bone marrow stromal cells. J Tissue Eng Regen Med 2008; 2: 272–278.
5. Garnavos C, Mouzopoulos G & Morakis E. Fixed intramedullary nailing and percutaneous autologous concentrated bone-marrow grafting can promote bone healing in humeral-shaft fractures with delayed union. Injury 2010; 41: 563–567.
6. Hernigou P, Poignard A & Beaujean F, et al. Percutaneous autologous bone-marrow grafting for nonunions. Influence of the number and concentration of progenitor cells. J Bone Joint Surg Am 2005; 87: 1430–1437.
7. Murawski CD, Kennedy JG. Percutaneous internal fixation of proximal fifth metatarsal jones fractures (zones II and III) with Charlotte Carolina screw and bone marrow aspirate concentrate: an outcome study in athletes. Am J Sports Med 2011; 39: 1295–1301.
8. Wilkins RM, Chimenti BT, Rifkin RM. Percutaneous treatment of long bone nonunions: the use of autologous bone marrow and allograft bone matrix. Orthopedics 2003; 26:s549–s554.
9. Fortier LA, Potter HG & Rickey EJ, et al. Concentrated bone marrow aspirate improves full-thickness cartilage repair compared with microfracture in the equine model. J Bone Joint Surg Am 2010; 92: 1927–1937.
10. Wilke MM, Nydam DV, Nixon AJ. Enhanced early chondrogenesis in articular defects following arthroscopic mesenchymal stem cell implantation in an equine model. J Orthop Res 2007; 25: 913–925.
11. Frisbie DD, Kisiday JD & Kawcak CE, et al. Evaluation of adipose-derived stromal vascular fraction or bone marrow-derived mesenchymal stem cells for treatment of osteoarthritis. J Orthop Res 2009; 27: 1675–1680.
12. Schnabel LV, Lynch ME & van der Meulen MC, et al. Mesenchymal stem cells and insulin-like growth factor-I gene-enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons. J Orthop Res 2009; 27: 1392–1398.
13. Violini S, Ramelli P & Pisani LF, et al. Horse bone marrow mesenchymal stem cells express embryo stem cell markers and show the ability for tenogenic differentiation by in vitro exposure to BMP-12. BMC Cell Biol 2009; 10:29.
14. Smith RK, Korda M & Blunn GW, et al. Isolation and implantation of autologous equine mesenchymal stem cells from bone marrow into the superficial digital flexor tendon as a potential novel treatment. Equine Vet J 2003; 35: 99–102.
15. Smith RK. Use of bone marrow-derived mesenchymal stem cells to enhance tendon and ligament healing, in Proceedings. Am Coll Vet Surg Vet Symp 2008;172–176.
16. Clegg PD. Biological therapies for treatment of tendon and ligament injuries, in Proceedings. 47th Br Equine Vet Assoc Cong 2008;263–264.
17. Dashtdar H, Rothan HA & Tay T, et al. A preliminary study comparing the use of allogenic chondrogenic pre-differentiated and undifferentiated mesenchymal stem cells for the repair of full thickness articular cartilage defects in rabbits. J Orthop Res 2011; 29: 1336–1342.
18. Granero-Moltó F, Myers TJ & Weis JA, et al. Mesenchymal stem cells expressing insulin-like growth factor-I (MSCIGF) promote fracture healing and restore new bone formation in Irs1 knockout mice: analyses of MSCIGF autocrine and paracrine regenerative effects. Stem Cells 2011; 29: 1537–1548.
19. Woodell-May J, Matuska A & Oyster M, et al. Autologous protein solution inhibits MMP-13 production by IL-1β and TNFα-stimulated human articular chondrocytes. J Orthop Res 2011; 29: 1320–1326.
20. Apel A, Groth A & Schlesinger S, et al. Suitability of human mesenchymal stem cells for gene therapy depends on the expansion medium. Exp Cell Res 2009; 315: 498–507.
21. Muschler GF, Boehm C & Easley K. Aspiration to obtain osteoblast progenitor cells from human bone marrow: the influence of aspiration volume. J Bone Joint Surg Am 1997; 79: 1699–1709.
22. McCarrel T & Fortier L. Temporal growth factor release from platelet-rich plasma, trehalose lyophilized platelets, and bone marrow aspirate and their effect on tendon and ligament gene expression. J Orthop Res 2009; 27: 1033–1042.
23. Woodell-May JE; inventor; Biomet Biologics LLP, assignee. Apparatus and method for separating and concentrating fluids containing multiple components. US patent application 2006/0278588 A1. Dec 14, 2006.
24. Pittenger MF, Mackay AM & Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284: 143–147.
25. Vidal MA, Kilroy GE & Johnson JR, et al. Cell growth characteristics and differentiation frequency of adherent equine bone marrow-derived mesenchymal stromal cells: adipogenic and osteogenic capacity. Vet Surg 2006; 35: 601–610.
26. Kadiyala S, Young RG & Thiede MA, et al. Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro. Cell Transplant 1997; 6: 125–134.
27. Martin DR, Cox NR & Hathcock TL, et al. Isolation and characterization of multipotential mesenchymal stem cells from feline bone marrow. Exp Hematol 2002; 30: 879–886.
28. Désévaux C, Laverty S & Doizé B. Sternal bone biopsy in standing horses. Vet Surg 2000; 29: 303–308.
29. Kasashima Y, Ueno T & Tomita A, et al. Optimisation of bone marrow aspiration from the equine sternum for the safe recovery of mesenchymal stem cells. Equine Vet J 2011; 43: 288–294.
