<meta charset="utf8"></head><body>Aksolotllarda kemik iyileşmesini ve uzuv rejenerasyonunu incelemek için plaka fiksasyonunun kullanılması

<ol>
	<li>Amamoto, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+axolotls+can+regenerate+original+neuronal+diversity+in+response+to+brain+injury.">Adult axolotls can regenerate original neuronal diversity in response to brain injury.</a> Elife. 5, 13998 (2016).</li><li>Butler, E. G., Ward, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+spinal+cord+following+ablation+in+urodele+larvae.">Reconstitution of the spinal cord following ablation in urodele larvae.</a> J Exp Zool. 160 (1), 47-65 (1965).</li><li>Echeverri, K., Tanaka, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectoderm+to+mesoderm+lineage+switching+during+axolotl+tail+regeneration.">Ectoderm to mesoderm lineage switching during axolotl tail regeneration.</a> Science. 298 (5600), 1993-1996 (2002).</li><li>Vargas-Gonzalez, A., Prado-Zayago, E., Leon-Olea, M., Guarner-Lans, V., Cano-Martinez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+regeneration+in+Ambystoma+mexicanum+after+surgical+injury.">Myocardial regeneration in Ambystoma mexicanum after surgical injury.</a> Arch Cardiol Mex. 75 (3), S321-S329 (2005).</li><li>Vieira, W. A., Wells, K. M., McCusker, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancements+to+the+axolotl+model+for+regeneration+and+aging.">Advancements to the axolotl model for regeneration and aging.</a> Gerontology. 66 (3), 212-222 (2020).</li><li>Song, F., Li, B., Stocum, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphibians+as+research+models+for+regenerative+medicine.">Amphibians as research models for regenerative medicine.</a> Organogenesis. 6 (3), 141-150 (2010).</li><li>McCusker, C., Bryant, S. V., Gardiner, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+axolotl+limb+blastema:+Cellular+and+molecular+mechanisms+driving+blastema+formation+and+limb+regeneration+in+tetrapods.">The axolotl limb blastema: Cellular and molecular mechanisms driving blastema formation and limb regeneration in tetrapods.</a> Regeneration (Oxf). 2 (2), 54-71 (2015).</li><li>Einhorn, T. A., Gerstenfeld, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture+healing:+Mechanisms+and+interventions).">Fracture healing: Mechanisms and interventions).</a> Nat Rev Rheumatol. 11 (1), 45-54 (2015).</li><li>Polikarpova, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+specialist+in+regeneration-the+Axolotl-a+suitable+model+to+study+bone+healing.">The specialist in regeneration-the Axolotl-a suitable model to study bone healing.</a> NPJ Regen Med. 7 (1), 35 (2022).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+axolotl+fibula+as+a+model+for+the+induction+of+regeneration+across+large+segment+defects+in+long+bones+of+the+extremities.">The axolotl fibula as a model for the induction of regeneration across large segment defects in long bones of the extremities.</a> PLoS One. 10 (6), e0130819 (2015).</li><li>Cosden-Decker, R. S., Bickett, M. M., Lattermann, C., MacLeod, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+analysis+of+intra-articular+interzone+tissue+in+axolotl+salamanders.">Structural and functional analysis of intra-articular interzone tissue in axolotl salamanders.</a> Osteoarthritis Cartilage. 20 (11), 1347-1356 (2012).</li><li>Williams, J. N., Li, Y., Valiya Kambrath, A., Sankar, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+generation+of+closed+femoral+fractures+in+mice:+A+model+to+study+bone+healing.">The generation of closed femoral fractures in mice: A model to study bone healing.</a> J Vis Exp. (138), e58122 (2018).</li><li>Cheung, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+externally+fixed+femoral+fracture+model+for+mice.">An externally fixed femoral fracture model for mice.</a> J Orthop Res. 21 (4), 685-690 (2003).</li><li>Jiang, S., Knapstein, P., Donat, A., Tsitsilonis, S., Keller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+protocol+for+a+standardized,+femoral+osteotomy+model+to+study+fracture+healing+in+mice.">An optimized protocol for a standardized, femoral osteotomy model to study fracture healing in mice.</a> STAR Protoc. 2 (3), 100798 (2021).</li><li>Matthys, R., Perren, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+fixator+for+use+in+the+mouse.">Internal fixator for use in the mouse.</a> Injury. 40, S103-S109 (2009).</li><li>Manassero, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+segmental+femoral+critical-size+defect+model+in+mice+stabilized+by+plate+osteosynthesis.">Establishment of a segmental femoral critical-size defect model in mice stabilized by plate osteosynthesis.</a> J Vis Exp. (116), e52940 (2016).</li><li>Gunderson, Z. J., Campbell, Z. R., McKinley, T. O., Natoli, R. M., Kacena, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+review+of+mouse+diaphyseal+femur+fracture+models.">A comprehensive review of mouse diaphyseal femur fracture models.</a> Injury. 51 (7), 1439-1447 (2020).</li><li>Riquelme-Guzman, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postembryonic+development+and+aging+of+the+appendicular+skeleton+in+Ambystoma+mexicanum.">Postembryonic development and aging of the appendicular skeleton in Ambystoma mexicanum.</a> Dev Dyn. 251 (6), 1015-1034 (2022).</li><li>Gentz, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medicine+and+surgery+of+amphibians.">Medicine and surgery of amphibians.</a> ILAR J. 48 (3), 255-259 (2007).</li><li>Lang, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+I-based+scaffolds+negatively+impact+fracture+healing+in+a+mouse-osteotomy-model+although+used+routinely+in+research+and+clinical+application.">Collagen I-based scaffolds negatively impact fracture healing in a mouse-osteotomy-model although used routinely in research and clinical application.</a> Acta Biomater. 86, 171-184 (2019).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.66 mm Gigly wire saw&#160;</td>
			<td>RISystem</td>
			<td>RIS.590.120</td>
			<td></td>
		</tr>
		<tr>
			<td>7.0 Optilene suture&#160;</td>
			<td>Braun</td>
			<td>C3090538</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzocaine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>E1501</td>
			<td>dilute to 0.03%&#160; prior to using</td>
		</tr>
		<tr>
			<td>Butorphanol (Butomidor 10 mg/mL)</td>
			<td>Richter Pharma AG</td>
			<td>-</td>
			<td>dilute to 0.5 mg/L&#160; prior to using</td>
		</tr>
		<tr>
			<td>Drill bit 0.30 mm</td>
			<td>RISystem</td>
			<td>RIS.590.200</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps - Standard/Inox</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand drill</td>
			<td>RISystem</td>
			<td>RIS.390.130</td>
			<td>better to have at least 3 pieces</td>
		</tr>
		<tr>
			<td>Micro CT data analyzer</td>
			<td>Bruker, Billerica, MA, USA</td>
			<td>SkyScan NRecon software</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro CT specimen scanner</td>
			<td>Bruker, Billerica, MA, USA</td>
			<td>SkyScan 1172&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Moria MC31b Iris forceps - smooth, curved, 10 cm</td>
			<td>Fine Science Tools</td>
			<td>11373-12FST</td>
			<td>2 pieces</td>
		</tr>
		<tr>
			<td>MouseFix Drill-&#38;Saw guide 1.75 mm, rigid</td>
			<td>RISystem</td>
			<td>RIS.301.102</td>
			<td></td>
		</tr>
		<tr>
			<td>MouseFix plate 4 hole, rigid</td>
			<td>RISystem</td>
			<td>RIS.401.110</td>
			<td></td>
		</tr>
		<tr>
			<td>MouseFix screw, L =2.00 mm</td>
			<td>RISystem</td>
			<td>RIS.401.100</td>
			<td>need 4 per bone</td>
		</tr>
		<tr>
			<td>Narrow Pattern Forceps</td>
			<td>VWR</td>
			<td>FSCI11002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Ring forceps</td>
			<td>Fine Science Tools</td>
			<td>11103-09</td>
			<td></td>
		</tr>
		<tr>
			<td>scalpel #15</td>
			<td>B Braun, Thermo Fischer Scientific</td>
			<td>5518032</td>
			<td></td>
		</tr>
		<tr>
			<td>Square box wrench 0.50 mm</td>
			<td>RISystem</td>
			<td>RIS.590.111</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile bone wax, 2.5 g</td>
			<td>Ethicon, Johnson &#38; Johnson</td>
			<td>W810</td>
			<td></td>
		</tr>
		<tr>
			<td>Student Fine Scissors - Straight/11.5cm</td>
			<td>Fine Science Tools</td>
			<td>91460-11</td>
			<td></td>
		</tr>
	</tbody>
</table>

stabilizing a femur osteotomy with a plate fixation in ambystoma mexicanum

The axolotl (Ambystoma mexicanum) is an important model for organ regeneration, including the tail, spinal cord, brain, heart, gills, and limbs1,2,3,4,5. Detailed studies of axolotl limb regeneration uncovered mechanisms of cell dedifferentiation and the formation of a stem cell pool, blastema, at the amputation site. Due to the ability of the blastema cells to reconstruct all missing limb parts, including a patterned skeleton6,7, the axolotl appears to be an attractive model organism for bone healing studies. Recently, several studies focused more on bone biology in axolotls, describing skeletal morphology, cellular composition, and ossification dynamics.
It was found in mammals that the bone healing process in long bones occurs via endochondral ossification and consists of several stages: hematoma, granulation tissue, and soft callus formation, callus ossification into hard callus and woven bone, and bone remodeling8. A recent study has shown that similar stages can be observed in axolotl bone healing9.
Until now, axolotl fractures were studied in a non-stabilized system, whereby bone is simply cut with iridectomy scissors. The large fractures were created in the zeugopod, where osteotomy is performed on one of the bones, whereas the other serves as support10,11. In contrast, fractures are routinely studied in mammals, including rats and mice, using reliable fixation systems, such as intramedullary pin and bone-aligning plates, to control fracture size and ensure bone alignment.
Thus, the method aims to ensure stabilized and uniform fixation of the axolotl femur prior to osteotomy. In order to make axolotl studies more comparable to mammals, including mice and humans, intramedullary pin12, external plate fixator13,14, and internal bone aligning plate15,16,17&#160;fixation were considered. The latter was shown to ensure proper bone fixation and allow for creating a gap of a certain size by using one or two cuts with a Gigly saw of a specific diameter. As axolotls represent the aquatic larvae of Ambystoma mexicanum, the external fixator might have caused post-surgical complications due to the open wound and contact with water. As axolotls do not develop secondary ossification centers even until very late in their development (20 years old18), and thus the standard intramedullary nail used in mice might not be prevented from puncturing the epiphyses, a decision was made to apply an internal plate fixation method to large axolotls. In large axolotls, the femur size and degree of ossification resemble that of an adult mouse, thus allowing for mid-diaphyseal osteotomy with titanium plate fixation1.
The fracture gap size largely determines the healing dynamics and outcome. For example, in a mouse, a 0.25 mm stabilized fractures heal mostly through intramembranous&#160;ossification due to their small size and rigid stabilization; a 0.7 mm fracture heals by endochondral ossification, with the formation of a cartilaginous callus around the fracture; large defects, such as 3.5 mm critical-sized defects do not heal completely and thus are used to model bone fracture non-union16. In this study, the plate fixation protocol of the axolotl femur prior to the osteotomy using the example of a 0.7 mm fracture gap was established with the ultimate goal of comparing axolotl bone healing to that of the mouse9.
After osteotomy, the fractures underwent the process of endochondral ossification, albeit slower than in mice, possibly due to the aquatic lifestyle of axolotls and slower cell division rates. In the method presented here, the 0.7 mm gap osteotomy with rigid plate fixation is shown; however, other gap sizes and semi-flexible fixators, as well as plates of different materials, are potentially possible. Overall, the method presented here can be used for standardized bone fixation and will be helpful for studies comparing axolotl limb regeneration to bone healing or studying bone healing in axolotls under different conditions to ensure standardized fracture fixation.

<meta charset="utf8"></head><body>Yazar Spotlight: Hücresel ve Moleküler Mekanizmalar, Kemik İyileşmesi ve İnsan Terapileri için Çıkarımlar Yoluyla Aksolotllarda Rejenerasyonu Keşfetmek

Ambystoma mexicanum'da Plak Fiksasyonu ile Femur Osteotomisinin Stabilize Edilmesi

We study tetrapod regeneration and use the axolotl as a top-tier regenerative species, comparing it to frogs and mice to discover cellular and molecular mechanisms of regeneration. The development of transgenics in genome editing, assembly of the axolotl genome, and application of RNA-Seq, ATAC-Seq, spatial transcriptomics and proteomics allow us to deeper understand the regeneration microenvironment and compare it to less regenerative species, including humans. We established the cellular contribution to tissues in the regenerating limb and spinal cord, and major regulators of initial cell proliferation and positional identity in the blastema.In a recent paper, we showed axolotl bone healing going through endochondral ossification, and the cellular level was similar to that of mammals. In contrast to previous techniques, where the axolotl fracture was either unfixed or the neighboring bone served as a support, in the current protocol, the bone is fixated with a plate, thus allowing to create a reproducible and aligned fracture and enabling a sound comparison to the studies in mice. The protocol allows for stable fracture with fixed gap size, broadening the studies on plate fixated fractures to amphibians.Despite their extreme regeneration abilities and full limb restoration upon amputations, axolotl surprisingly cannot heal large bone fractures with critical size defects. We aim to define the pro-regenerative blastema factors to treat bone non-union in critical size defects.

Read this Scientific Article Protocol about Author Spotlight: Exploring Regeneration in Axolotls Through Insights in Cellular and Molecular Mechanisms, Bone Healing, and Implications for Human Therapi

The axolotl (Ambystoma mexicanum) is a promising model organism for regenerative medicine due to its remarkable ability to regenerate lost or damaged ...

stabilizing-femur-osteotomy-with-plate-fixation-ambystoma

Research

JoVE Journal

Biology

Stabilizing a Femur Osteotomy with a Plate Fixation in Ambystoma mexicanum

406 Görüntüleme.  Research Institute of Molecular Pathology. Tetrapod rejenerasyonunu inceliyoruz ve aksolotl'u en üst düzey rejeneratif tür olarak kullanıyoruz, rejenerasyonun hücresel ve moleküler mekanizmalarını keşfetmek için kurbağalar ve farelerle karşılaştırıyoruz. Genom düzenlemede transgeniklerin geliştirilmesi, aksolotl genomunun birleştirilmesi ve RNA-Seq, ATAC-Seq, uzamsal transkriptomik ve proteomiklerin uygulanması, rejenerasyon mikro ortamını daha derinlemesine anlamamıza ve onu in...

Video: Yazar Spotlight: Hücresel ve Moleküler Mekanizmalar, Kemik İyileşmesi ve İnsan Terapileri için Çıkarımlar Yoluyla Aksolotllarda Rejenerasyonu Keşfetmek