Peters and Watts Needle Advancement Increases MSC Concentration. mononuclear fraction in BMAC compared to BM has been well were performed in 12 healthy university owned horses and no. described in multiple species ranging from an increase of 2 4 to horses were euthanized for this study. 5 fold in people 6 7 fold in dogs 7 3 5 fold in pigs 8 and Bone marrow aspiration was performed from the sternal mar. 5 to 19 fold in the horse 9 The increase in MSC concentration row spaces 4 and 5 using an 11 gage 10 16 cm BM biopsy needle1. within the mononuclear fraction of BMAC versus unprocessed with mild sedation of horses xylazine2 0 4 mg kg body weight. BM is well accepted and relevant to therapeutic applications IV and application of local anesthetic in the subcutaneous tissues. In people with atrophic tibial diaphyseal non union treatment Figure 1 Selection of the site for the fourth and fifth space was. success with either BM or BMAC was related to the number of made adjacent to the point of the elbow in a square stance and. progenitors in the graft 10 3 4 cm caudal to that Collection from the fourth or fifth stern. Most BMAC systems for the horse require a minimum of ebrae was randomized for the SS and MS aspirations For the SS. 60 ml of BM 2 and anti coagulant for the system to separate collection the biopsy needle was advanced 2 cm from the ventral. nucleated cells and presumably the stem cells from other BM cortical surface of the sternum 13 the stylet was removed and. components However it is well known that the contamination 56 ml of BM was aspirated into a 60 ml syringe pre filled with. of peripheral blood in large volume BM aspirates results in a 5 000 U of heparin sodium injection 3 For the MS collection a. lower concentration of MSCs per milliliter of BM aspirated Thus technique similar to that reported by Hernigou et al was used. recommended BM aspirate volumes for patient side regenerative 10 The BM needle was advanced as for the SS but only 7 ml of. medicine applications in people are 2 10 ml of BM per aspira marrow was collected After the first 7 ml of BM the needle was. tion site 11 12 In the horse the same effect of MSC dilution rotated 45 and an additional 7 ml of BM was collected After the. in large volume BM aspirates was identified when the numbers first 14 ml of BM was collected the stylet was replaced and the. of colony forming unit fibroblasts CFU Fs in successive 5 ml biopsy needle was advanced 5 mm dorsally followed by collection. aliquots of BM were compared 13 Because of the MSC dilu of 7 ml of BM rotation of the needle and collection of 7 ml of. tion in large volume aspirates Ishihara et al tested a specialized BM This advancement and BM collection followed by rotation. needle that allows for collection of BM from several locations were repeated two additional times for a total BM collection of. within one sternal puncture in anesthetized horses Use of the 56 ml and a total distance from the ventral cortical surface of the. multidirectional needle for BM collection allowed them to collect sternum of 3 5 cm or 1 5 cm from the initial aspiration site. BM aspirates from three different sites within the marrow space. without removing replacing or advancing the needle beyond. their initial placement Instead of moving the needle to a new 1. Ranfac Corporation Avon MA USA, space within the sternal marrow space they twisted a second 2. VetOne Boise ID USA, cannula within the needle which redirected BM flow from and 3. Sagent Pharmaceuticals Schaumburg IL USA, to each of the three sites Use of their multidirectional needle. resulted in an increased frequency of BMAC samples character. ized as high in progenitor cells but no differences in the MSC. concentration in BM aspirate samples 9, Maximizing the number of MSCs in the BM aspirate prior to. concentration with a device would be important to enhance MSC. concentration of the final BMAC product We wanted to develop. a technique in standing and sedated horses that would collect. sufficient BM volume for most BMAC systems with a higher. concentration of MSCs and a reasonable number of bone punc. ture sites The objective of our study was to determine if multiple. advancements of the BM biopsy needle from the same sternal. puncture would result in a higher number of MSCs in 56 ml of. BM compared to collection from a single site SS We hypoth. esized that multiple advancements of the BM needle would result. in BM aspirates with higher total nucleated cell count TNCC. To test this we collected BM using two different techniques from. 12 horses and compared TNCC of BM aspirates the CFU F assay. of cultured BM and the number of culture expanded MSCs at. the first second and third passages from BM collected from a SS. versus multiple sites MS,MATERIALS AND METHODS, FIGURE 1 Photograph of bone marrow BM aspiration needle. Bone Marrow Collection showing A stylet B needle C aspiration holes and D centimeter. All animal procedures were approved by the institution s animal marks used to measure 2 cm distance from the ventral cortical. surface of the sternum, care and use committee IACUC 2013 0097 BM aspirations. Frontiers in Veterinary Science www frontiersin org 2 March 2016 Volume 3 Article 23. Peters and Watts Needle Advancement Increases MSC Concentration. Colony Forming Unit Fibroblast Assay 175 cm2 washed counted and reseeded at 5 000 MSCs cm2 until. The number of CFU Fs per milliliter of BM from SS and MS was passage 3 when the final expanded MSC number was determined. determined by seeding 1 ml of undiluted and unmanipulated. BM to a 10 cm tissue culture dish4 246 cm2 containing 9 ml Validation of MSCs. culture medium Dulbecco s modified Eagle s medium DMEM 5 At the conclusion of isolation and expansion the MSCs from MS. 1 g l glucose supplemented with 10 000 U ml Penicillin 10 mg and SS were combined and the cell surface marker expression for. streptomycin sulfate 25 g ml amphotericin B6 2 5 HEPES MHCII 17 CD44 see text footnote 17 CD29 18 CD45 19 and CD90. buffer7 10 g ml human recombinant basic fibroblastic growth see text footnote 19 were determined for MSCs from each horse. factor see text footnote 7 and 10 fetal bovine serum FBS 8 using flow cytometry with antibodies that have been previously. and maintained at 37 C in 5 CO2 humidified air Media was validated in the horse 15 16 Cryopreserved cells were thawed. exchanged three times per week Plates were inspected and pho and the concentration was adjusted to make aliquots of one mil. tographed daily with an Olympus CKX419 microscope When lion cells suspended in DPBS Blocking was performed by 20 min. colonies approached coalescence media was removed and the incubation with 10 l undiluted goat serum MSCs were pelleted. colonies were stained with 3 crystal violet10 similar to Franken by centrifugation at 400 g for 5 min followed by incubation with. et al 14 After plates dried for 24 h colonies were manually antibody dilutions For MHCII and CD29 and CD44 a 1 100 dilu. counted without magnification by an observer masked to treat tion with conjugated primary antibody was used For CD90 and. ment group CD45 a 1 400 dilution was used with primary and conjugated. secondary antibody Unstained MSCs were used as controls. Total Nucleated Cell Count Multipotency was tested for SS and MS combined MSCs from. Red blood cell lysis on 10 ml of BM was performed to obtain an all 12 horses and reported as positive or negative Tri lineage dif. initial TNCC BM was mixed with a sterile red blood cell lysis ferentiation was induced by techniques that have been described. solution 7 7 mg ml NH4CL11 2 06 mg ml hydroxymethane previously 17 19 For chondrogenic differentiation aliquots of. aminomethane12 pH 7 2 for 2 min and then centrifuged at MSCs 500 000 were centrifuged at 300 g for 5 min to pellet the. 300 g for 10 min at 4 C After aspirating supernatant the cell cells To induce chondrogenic differentiation supernatant was. pellet was resuspended and mixed with lysis solution and centri aspirated and 1 ml of chondrogenic induction media DMEM20. fuged at 300 g for 10 min at 4 C The cell pellet was washed with 4 5 g l glucose supplemented with 10 000 U ml Penicillin. DPBS13 and then centrifuged for 5 min at 300 g at 4 C Cells 10 mg streptomycin sulfate 25 g ml amphotericin B 2 5. were resuspended in isolation medium at a volume equal to the HEPES buffer 0 2 transforming growth factor 21 301 89 g. original volume of BM aspirate or 10 ml Samples were diluted dexamethasone 22 50 g ml l ascorbic acid see text footnote 22. 1 10 and a 100 l sample was used for manual counting of nucle 40 g ml proline see text footnote 22 1 ITS premix see text. ated cells with fluorescein diacetate14 and propidium iodide see footnote 7 and 1 FBS was added on top of the pellet Media. text footnote 14 was exchanged three times per week for 21 days Pellets were fixed. in 4 formaldehyde see text footnote 22 for 10 min followed. Isolation and Expansion of MSCs by routine processing embedding sectioning and staining with. Bone marrow aspirate was mixed at a 1 1 ratio with culture toluidine blue see text footnote 22. medium described above and seeded directly onto tissue cul Adipogenic differentiation of MSCs was induced by seeding a. ture flasks15 at 28 ml BM per 175 cm2 and maintained at 37 C 10 cm tissue culture dish with 1 000 MSCs cm2 At 70 conflu. in 5 CO2 humidified air After 24 h the same volume of media ence existing media was removed and replaced with adipogenic. was added again to the BM media mixture and half the resultant induction media DMEM Ham s F12 1 1 23 supplemented with. volume was transferred to a new flask doubling the number of 10 000 U ml penicillin 10 mg streptomycin sulfate 25 g. flasks Medium was exchanged three times per week Cells were ml amphotericin B 5 rabbit serum see text footnote 21. detached from tissue culture flasks when colonies or monolayers 33 M l biotin see text footnote 22 17 M pantothenate see. approached 70 80 of confluence with 5 ml Trypsin EDTA16 per text footnote 22 1 M l insulin see text footnote 22 1 M l. dexamethasone 225 l isobutylmethylxanthine see text footnote. 22 89 l rosiglitazone see text footnote 22 and 3 FBS for. Bio Basic Amherst NY USA 3 days Media was replaced with adipogenic maintenance media. DMEM 1 g l glucose Mediatech Manassas VA USA adipogenic induction media without isobutylmethylxanthine. Mediatech Inc Manassas VA USA and rosiglitazone for an additional 3 days followed by staining. Corning Corning NY USA with oil red O see text footnote 22. HyClone Thermo Scientific Logan UT USA,Olympus Center Valley PA USA. Crystal Violet Sigma Aldrich St Louis MO USA, Avantor Performance Materials Macron Fine Chemicals Center Valley PA USA 17. Bio Rad Raleigh NC USA,Sigma St Louis MO USA 18,Beckman Coulter Brea CA USA. Lonza Walkersville MD USA 19,VMRD Inc Pullman WA USA. EMD Chemicals Inc Calbiotech San Diego CA USA 20,DMEM 4 5 g l glucose Mediatech Manassas VA USA. 175 cm2 Polystyrene Tissue Culture Flask with Vented Caps Corning Corning 21. Life Technologies Grand Island NY USA,Sigma Aldrich St Louis MO USA. Trypsin EDTA Corning Corning NY USA 23,DMEM F12 Hams 1 1 mix Mediatech Manassas VA USA. Frontiers in Veterinary Science www frontiersin org 3 March 2016 Volume 3 Article 23. Peters and Watts Needle Advancement Increases MSC Concentration. For osteogenic differentiation MSCs were seeded on 10 cm. culture dishes at 1 000 MSCs cm2 At 70 confluence media was. exchanged for osteogenic induction media DMEM Ham s F12. 1 1 supplemented with 10 000 U ml penicillin 10 mg strepto. mycin sulfate 25 g ml amphotericin B 20 nM l dexamethasone. 50 g ml ascorbic acid 10 M l glycerophosphate see text foot. note 22 and 10 FBS and cultures were maintained for 14 and. 21 days Media was exchanged three times per week Cells were. stained with 2 alizarin red see text footnote 22,Statistical Analysis. Data were imported to a commercially available statistical analy. sis program Statistix 9 24 and tested for normality Paired data for. each horse were compared using Wilcoxon signed rank test or. paired t test as appropriate for data normality Differences were FIGURE 2 Colony forming unit fibroblast CFU F assay from bone. considered significant when p 0 05 one tailed marrow aspirates using a single site SS or a multiple site MS. technique during bone marrow aspiration mean standard deviation. There were significantly more CFU Fs from bone marrow collected with the. RESULTS MS technique p 0 02 paired t test,BM Collection. Twelve female horses ranging in age from 7 to 16 years were used. for BM collection They were all of Quarter Horse type BM col. lection went well without incident Horses were under our care. for the following 6 months No adverse events were recorded. CFU F Assay, The number of CFU F from the MS technique was significantly. mean SD higher than from the SS technique SS 33 27 6 MS. 51 36 4 p 0 02 Figure 2,Total Nucleated Cell Count. The TNCC from the MS technique was significantly mean SD. higher than from the SS technique SS 14 4 106 7 6 106 MS. 20 9 106 7 0 106 p 0 01 Figure 3, FIGURE 3 Total nucleated cell count mean standard deviation per. Isolation and Expansion of MSCs milliliter of raw bone marrow aspirated using a single site SS or a. The number of MSCs at the first passage was significantly multiple site MS technique during bone marrow aspiration There. median interquartile range higher at the first passage with MS were significantly more nucleated cells in BM collected with the MS technique. compared to SS aspiration SS 1 5 106 0 52 106 5 16 106 p 0 01 paired t test. MS 3 15 106 1 65 106 10 98 106 p 0 02 Figure 4 at, the second passage SS 4 95 106 2 17 106 11 77 106 MS. 7 0 106 3 16 106 13 57 106 p 0 1 Figure 4 and at All MSCs were positive for tri lineage differentiation. the third passage SS 7 84 106 5 95 106 18 7 106 MS Figure 5. 15 45 106 9 15 106 21 97 106 p 0 2 Figure 4 there was. no significant difference in the number of cells between the two DISCUSSION. aspiration techniques, Our objective was to determine whether multiple advance. Validation of Cell MSCs ments MS within the same sternal puncture would result in. The cell surface marker profile was consistent with previous a greater number of MSCs in a 56 ml BM aspirate compared. reports in the horse 15 MSCs from 8 of 12 horses were negative to collection of 56 ml BM from a single site SS We found a. for MHCII expression 11 of the 12 were positive for CD90 and significantly higher number of nucleated cells in the BM aspi. CD29 and 12 of 12 were negative for CD45 MSCs from one horse rate and after culture a significantly higher number of CFU Fs. displayed mixed expression of both CD90 and CD29 and a significantly higher number of MSCs at the first passage. from MS compared to SS aspirates The increase in CFU F. number and MSC number at the first passage supports that the. Statistix Analytical Software Tallahassee FL USA, increased TNCC was due at least in part to a higher number of. Frontiers in Veterinary Science www frontiersin org 4 March 2016 Volume 3 Article 23. Peters and Watts Needle Advancement Increases MSC Concentration. FIGURE 4 Total number of mesenchymal stem cells MSCs mean standard deviation at the each passage from bone marrow aspirates using a single. site SS or a multiple site MS technique during bone marrow BM aspiration There were significantly more MSCs from BM collected with the MS technique. p 0 02 Wilcoxon signed rank test The number of MSCs at passages 2 and 3 were not significantly different between SS and MS techniques p 0 1 p 0 2. FIGURE 5 Examples of MSCs after A adipogenic B osteogenic and C chondrogenic induction and staining with resulting in lipid calcium and. proteoglycan accumulation Original magnification 200 A C and 40 B scale bar 100 m A C or 500 m B. MSCs in the BM aspirate At later passages the total number MSCs in the aspirate combined with the efficiency of the BMAC. of MSCs was not different between SS and MS We think this system ultimately determines the number of MSCs in the BMAC. is because of the rapid growth 4 of MSCs that once isolated product Therefore increasing the concentration of MSCs in the. quickly overcome differences in initial MSC concentration This BM aspirate is a step the clinician can take to maximize MSC. information is important to the equine veterinarian using BM number in the final BMAC preparation. in patient side BMAC applications where large BM volumes To maximize progenitor concentration using recommenda. are required most BMAC kits use a total BM volume of 60 ml tions from human medicine of 2 ml BM per aspiration site as. and a higher concentration of MSCs in the BM aspirate prior to suggested by Muschler et al 12 and 10 ml BM per aspiration. concentration in the kit could result in higher MSC numbers in site as suggested by Hernigou et al 21 30 and 6 separate aspira. the final BMAC product tions respectively would be required to collect 60 ml which is. Estimates of MSC concentration in BM are 0 001 0 01 20 the typical BM volume for currently available veterinary BMAC. This is probably an underestimate of the MSC concentration kits 3 7 9 To minimize peripheral blood contamination with. because it was determined using aspirate volumes of 10 30 ml a more reasonable number of bone punctures Thoesen et al. of BM aspirate per site 20 Regardless the total number of collected 7 ml of BM from five separate aspiration sites from. Frontiers in Veterinary Science www frontiersin org 5 March 2016 Volume 3 Article 23. Peters and Watts Needle Advancement Increases MSC Concentration. each humerus in dogs 7 To achieve the same goal with even increase in the white blood cell population but rather an increase. fewer cortical bone penetrations in the horse another group in progenitor populations including MSCs This is supported by. described a specialized needle that increased the frequency of the increase in numbers of colonies in the CFU F assay from. BMAC samples characterized as high in progenitor cells but MS samples that reflect a higher concentration of MSCs in the. resulted in no differences in MSC concentration of BM aspirates original aspirate and the higher number of MSCs from MS at the. 9 We think our MS technique is similar to the multidirectional first passage 22. needle in that BM is collected from different sites within a single In conclusion we tested two techniques for collection of. sternal puncture however in contrast to Ishihara et al we found 56 ml of BM from the equine sternum In our 12 horses use of. significant differences in MSC concentration in the original BM the MS technique resulted in a higher concentration of MSCs. aspirate Our MS technique with multiple manual advancements in the BM aspirate as compared to the SS technique The higher. of the biopsy needle might be more efficient at entering naive concentration of MSCs was determined by comparing the TNCC. lacunae than the multidirectional needle Increased efficiency and the CFU F ability of BM aspirates as well as the total number. could have resulted in less peripheral blood contamination and of MSCs at the first passage during culture expansion Based on. a higher MSC concentration in the BM aspirate even without a our findings the MS technique for BM aspiration might be use. BMAC processing step ful in same day applications such as BMAC or BM grafting if. We think the 3 advancements of 5 mm each of the biopsy an increased number of MSCs in the BMAC product is desired. needle are responsible for the increased MSC concentration Future studies could test whether increased MSC concentration. due to reduced peripheral blood contamination Histological using the MS technique causes a greater treatment effect of. specimens after BM collection have demonstrated a minimal BMAC compared to using BM from the SS technique The MS. number of lacunae are penetrated during BM aspiration and technique may also be useful if the clinician wanted to use very. each lacuna is filled with hemorrhage 13 Without biopsy nee early passage MSCs. dle advancement we think continued aspiration from the same. site simply collects hemorrhage from the penetrated lacunae. rather than additional progenitors as has been demonstrated AUTHOR CONTRIBUTIONS. previously 13 By advancing the biopsy needle between. AP participated in study design performed laboratory work. aspirations new bony trabeculae are penetrated allowing. performed statistical analysis and drafted the manuscript AW. BM collection of MSC rich BM rather than BM hemorrhage. conceived designed and coordinated the study and assisted in. Additionally the 45 twist of the needle at each site of aspira. drafting and revising the manuscript All authors contributed to. tion repositions the holes along the side of the needle and could. data interpretation and all authors read and approved the final. also communicate with new lacunae,manuscript, One concern about the MS technique might be the increased. risk of cardiac puncture with multiple advancements of the. biopsy needle The dorsoventral dimension of the marrow space ACKNOWLEDGMENTS. not including the cortical bone thickness within the fourth and. fifth sternebrae of the horse is approximately 6 0 and 4 5 cm We would like to thank Dr AmandaJo Joswig and Ms Alexis. respectively 13 Routine technique for the aspiration of BM Mitchell for technical assistance. from the equine sternum is to advance the biopsy needle to a. depth of 2 cm from the ventral cortex 13 This depth would. leave a minimum additional 4 and 2 cm respectively of mar FUNDING. row space that the needle could be advanced into prior to. This study was funded by the Link Endowment for equine research. contacting the dorsal cortex Our MS technique results in an. at Texas A M University and by the Department of Large Animal. additional advancement of 1 5 cm after the initial placement. Clinical Sciences faculty grant program at Texas A M University. 2 cm from the ventral surface of the sternum Therefore with. accurate placement of the biopsy needle on midline and careful. needle advancement in 5 mm increments we think the risk of. SUPPLEMENTARY MATERIAL, penetration of the dorsal cortex is very low Certainly we did. not have any complications in this group of 12 horses where we The Supplementary Material for this article can be found online. utilized this technique and we have not had complications on the at http journal frontiersin org article 10 3389 fvets 2016 00023. approximately 120 clinical horses where we used the technique. TABLE S1 Colony forming unit fibroblasts CFU Fs expressed in. for BM aspiration number of colonies per milliliter of raw bone marrow aspirates and total. A limitation of our study is that we only have indirect proof nucleated cell count TNCC per milliliter of raw bone marrow expressed. of increased MSC number with the MS technique However the as number of cells 106 from single site SS and multiple site MS. increased TNCC in MS samples is unlikely to be caused by an techniques. Frontiers in Veterinary Science www frontiersin org 6 March 2016 Volume 3 Article 23. Peters and Watts Needle Advancement Increases MSC Concentration. REFERENCES 13 Kasashima Y Ueno T Tomita A Goodship AE Smith RK Optimisation. of bone marrow aspiration from the equine sternum for the safe. 1 Herthel D Enhanced suspensory ligament healing in 100 horses by stem cells recovery of mesenchymal stem cells Equine Vet J 2011 43 3 288 94. and other bone marrow components Proc Am Ass Eq Pract 2001 47 319 21 doi 10 1111 j 2042 3306 2010 00215 x. 2 Fortier LA Potter HG Rickey EJ Schnabel LV Foo LF Chong LR et al 14 Franken NA Rodermond HM Stap J Haveman J van Bree C Clonogenic assay. Concentrated bone marrow aspirate improves full thickness cartilage repair of cells in vitro Nat Protoc 2006 1 5 2315 9 doi 10 1038 nprot 2006 339. compared with microfracture in the equine model J Bone Joint Surg Am 15 Schnabel LV Pezzanite LM Antczak DF Felippe MJ Fortier LA Equine bone. 2010 92 10 1927 37 doi 10 2106 jbjs i 01284 marrow derived mesenchymal stromal cells are heterogeneous in MHC class. 3 Godwin EE Young NJ Dudhia J Beamish IC Smith RK Implantation of bone II expression and capable of inciting an immune response in vitro Stem Cell. marrow derived mesenchymal stem cells demonstrates improved outcome in Res Ther 2014 5 1 13 doi 10 1186 scrt402. horses with overstrain injury of the superficial digital flexor tendon Equine 16 Radcliffe CH Flaminio MJ Fortier LA Temporal analysis of equine bone mar. Vet J 2012 44 1 25 32 doi 10 1111 j 2042 3306 2011 00363 x row aspirate during establishment of putative mesenchymal progenitor cell. 4 Ikebe C Suzuki K Mesenchymal stem cells for regenerative therapy opti populations Stem Cells Dev 2010 19 2 269 82 doi 10 1089 scd 2009 0091. mization of cell preparation protocols Biomed Res Int 2014 2014 951512 17 Grogan SP Barbero A Winkelmann V Rieser F Fitzsimmons JS O Driscoll S. doi 10 1155 2014 951512 et al Visual histological grading system for the evaluation of in vitro generated. 5 Connolly J Guse R Lippiello L Dehne R Development of an osteogenic neocartilage Tissue Eng 2006 12 8 2141 9 doi 10 1089 ten 2006 12 2141. bone marrow preparation J Bone Joint Surg Am 1989 71 5 684 91 18 English A Jones EA Corscadden D Henshaw K Chapman T Emery P et al. 6 Hermann PC Huber SL Herrler T von Hesler C Andrassy J Kevy SV et al A comparative assessment of cartilage and joint fat pad as a potential source of. Concentration of bone marrow total nucleated cells by a point of care device cells for autologous therapy development in knee osteoarthritis Rheumatology. provides a high yield and preserves their functional activity Cell Transplant Oxford 2007 46 11 1676 83 doi 10 1093 rheumatology kem217. 2008 16 10 1059 69 doi 10 3727 000000007783472363 19 Zhu Y Ouyang Y Chang Y Luo C Xu J Zhang C et al Evaluation of the. 7 Thoesen MS Berg Foels WS Stokol T Rassnick KM Jacobson MS Kevy SV proliferation and differentiation behaviors of mesenchymal stem cells with. et al Use of a centrifugation based point of care device for production of partially converted borate glass containing different amounts of strontium. canine autologous bone marrow and platelet concentrates Am J Vet Res 2006 in vitro Mol Med Rep 2013 7 4 1129 36 doi 10 3892 mmr 2013 1341. 67 10 1655 61 doi 10 2460 ajvr 67 10 1655 20 Pittenger MF Mackay AM Beck SC Jaiswal RK Douglas R Mosca JD et al. 8 Hakimi M Grassmann JP Betsch M Schneppendahl J Gehrmann S Hakimi Multilineage potential of adult human mesenchymal stem cells Science 1999. AR et al The composite of bone marrow concentrate and PRP as an alterna 284 5411 143 7 doi 10 1126 science 284 5411 143. tive to autologous bone grafting PLoS One 2014 9 6 e100143 doi 10 1371 21 Hernigou P Homma Y Flouzat Lachaniette CH Poignard A Allain J. journal pone 0100143 Chevallier N et al Benefits of small volume and small syringe for bone mar. 9 Ishihara A Helbig HJ Sanchez Hodge RB Wellman ML Landrigan MD row aspirations of mesenchymal stem cells Int Orthop 2013 37 11 2279 87. Bertone AL Performance of a gravitational marrow separator multidirec doi 10 1007 s00264 013 2017 z. tional bone marrow aspiration needle and repeated bone marrow collections 22 J ger M Herten M Fochtmann U Fischer J Hernigou P Zilkens C et al. on the production of concentrated bone marrow and separation of mesen Bridging the gap bone marrow aspiration concentrate reduces autologous. chymal stem cells in horses Am J Vet Res 2013 74 6 854 63 doi 10 2460 bone grafting in osseous defects J Orthop Res 2011 29 2 173 80. ajvr 74 6 854 doi 10 1002 jor 21230, 10 Hernigou P Mathieu G Poignard A Manicom O Beaujean F Rouard H. Percutaneous autologous bone marrow grafting for nonunions Surgical Conflict of Interest Statement The authors declare that the research was con. technique J Bone Joint Surg Am 2006 88 Suppl 1 Pt 2 322 7 doi 10 2106 ducted in the absence of any commercial or financial relationships that could be. jbjs f 00203 construed as a potential conflict of interest. 11 Lee DH Ryu KJ Kim JW Kang KC Choi YR Bone marrow aspirate concen. trate and platelet rich plasma enhanced bone healing in distraction osteogen Copyright 2016 Peters and Watts This is an open access article distributed under. esis of the tibia Clin Orthop Relat Res 2014 472 12 3789 97 doi 10 1007 the terms of the Creative Commons Attribution License CC BY The use distribu. s11999 014 3548 3 tion or reproduction in other forums is permitted provided the original author s. 12 Muschler GF Boehm C Easley K Aspiration to obtain osteoblast progenitor or licensor are credited and that the original publication in this journal is cited in. cells from human bone marrow the influence of aspiration volume J Bone accordance with accepted academic practice No use distribution or reproduction is. Joint Surg Am 1997 79 11 1699 709 permitted which does not comply with these terms. Frontiers in Veterinary Science www frontiersin org 7 March 2016 Volume 3 Article 23.
outcomes, and c) relatively brief administration time required. Therefore, the CIRCLE system was developed by the staff of the Childrens Learning Institute to meet these instructional needs. We have collaborated with various software developers (e.g., Wireless
Etiology and Pathophysiology Food cue reactivity and craving predict eating and weight gain: a meta-analytic review Rebecca G. Boswell1 and Hedy Kober1,2 1Department of Psychology, Yale University, New Haven, CT, USA, and 2Department of Psychiatry, Yale School of Medicine, Yale
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Africa is south of Europe, between the Atlantic and Indian Oceans.To the north is the Mediterranean Sea. Africa is the second largest continent on Earth, after Asia. It can be divided into four main regions: West Africa, North Africa, Central and South Africa, and East Africa. Several vegetation zones form belts across Africa (see the second map
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