Name: लेखक स्पॉटलाइट: कण्डरा इम्पिंजमेंट के मेकेनोबायोलॉजी को उजागर करना - एक बहुअक्षीय म्यूरिन हिंद लिंब एक्सप्लांट मॉडल
Uploaded: 2023-12-08T13:30:00
Description: Watch this Scientific Journal Video about Unraveling the Mechanobiology of Tendon Impingement – A Multiaxial Murine Hind Limb Explant Model at JoVE.com

लेखक रोचेस्टर सेंटर फॉर मस्कुलोस्केलेटल रिसर्च के हिस्टोलॉजी, बायोकैमिस्ट्री और आणविक इमेजिंग (एचबीएमआई) कोर के जेफ फॉक्स और विद्या वेंकटरमानी द्वारा प्रदान किए गए समर्थन और सहायता के लिए आभारी हैं, जो P30AR06965 द्वारा भाग में वित्त पोषित है। इसके अतिरिक्त, लेखक मल्टीफोटन माइक्रोस्कोपी के साथ सहायता के लिए रोचेस्टर मेडिकल सेंटर विश्वविद्यालय में सेंटर फॉर लाइट माइक्रोस्कोपी और नैनोस्कोपी (CALMN) का शुक्रिया अदा करना चाहते हैं। इस अध्ययन को R01 AR070765 और R01 AR070765-04S1, साथ ही 1R35GM147054 और 1R01AR082349 द्वारा वित्त पोषित किया गया था।

सभी जानवरों के काम को पशु संसाधन पर रोचेस्टर समिति विश्वविद्यालय द्वारा अनुमोदित किया गया था।
1. टिशू कल्चर मीडिया की तैयारी
<ol><li>संस्कृति Dulbecco के संशोधित ईगल माध्यम (1x DMEM) में 1% वी / वी पेनि?...

कण्डरा प्रभाव को समझना - मेकेनोबायोलॉजी के अध्ययन के लिए मुराइन एक्सप्लांट मॉडल

इस अध्ययन में वर्णित टिशू कल्चर प्रोटोकॉल के साथ जोड़ा गया प्रायोगिक murine हिंद अंग एक्सप्लांट प्लेटफॉर्म Achilles कण्डरा सम्मिलन पर impingement संचालित fibrocartilage गठन के mechanobiology का अध्ययन करने के लिए एक उपयुक्त मॉडल प्रदा...

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Absorbent underpads</td>
			<td>VWR</td>
			<td>82020-845</td>
			<td>For benchtop dissection</td>
		</tr>
		<tr>
			<td>Acrylic bath</td>
			<td>Source One</td>
			<td>X001G46CB1</td>
			<td>Contains the explant platform submerged in culture media</td>
		</tr>
		<tr>
			<td>Autoclave bin</td>
			<td>Thermo Scientific</td>
			<td>13-361-20</td>
			<td>Used as secondary containment, holds two platforms</td>
		</tr>
		<tr>
			<td>Base</td>
			<td>-</td>
			<td>-</td>
			<td>3D printed from CAD files provided as Supplementary Files</td>
		</tr>
		<tr>
			<td>Braided line</td>
			<td>KastKing</td>
			<td>30lb test</td>
			<td>Used to wrap around paw and apply ankle dorsiflexion</td>
		</tr>
		<tr>
			<td>Clip</td>
			<td>-</td>
			<td>-</td>
			<td>3D printed from CAD files provided as Supplementary Files</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Fisherbrand</td>
			<td>12-541-034</td>
			<td>Rectangular, No. 2, 50 mm x 24 mm</td>
		</tr>
		<tr>
			<td>Cytoseal XYL</td>
			<td>VWR</td>
			<td>8312-4</td>
			<td>Xylene-based mounting media for coverslipping Toluidine blue stained tissue sections</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>MP Biomedical LLC</td>
			<td>194561</td>
			<td>CAS#50-02-2</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO), anhydrous</td>
			<td>Invitrogen by ThermoFisher</td>
			<td>D12345</td>
			<td>CAS#67-68-5, use to solubilize dexamethasone into concentrated stock solutions</td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>Scotch Brand</td>
			<td>34-8724-5195-9</td>
			<td>To attach sandpaper to Grip platens</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (1X DMEM)</td>
			<td>Gibco by ThermoFisher</td>
			<td>11965092</td>
			<td>high glucose, (-) pyruvate, (+) glutamine</td>
		</tr>
		<tr>
			<td>EDTA tetrasodium salt dihydrate</td>
			<td>Thermo Scientific Chemicals</td>
			<td>J15700.A1</td>
			<td>CAS#10378-23-1, used to make 14% EDTA solution for sample decalcifcation</td>
		</tr>
		<tr>
			<td>Ethanol, 200 proof</td>
			<td>Thermo Scientific</td>
			<td>T038181000</td>
			<td>CAS#64-17-5, 1 L supply</td>
		</tr>
		<tr>
			<td>Foam biopsy pads</td>
			<td>Leica</td>
			<td>3801000</td>
			<td>Used with processing cassettes, help hold ankle joints in desired position during fixation and decalcification</td>
		</tr>
		<tr>
			<td>Forceps, #SS Standard Inox</td>
			<td>Dumont</td>
			<td>11203-23</td>
			<td>Straight, smooth, fine tips</td>
		</tr>
		<tr>
			<td>Forceps, Micro-Adson 4.75&#34;</td>
			<td>Fisherbrand</td>
			<td>13-820-073</td>
			<td>Straight, fine tips with serrated teeth</td>
		</tr>
		<tr>
			<td>Garnet Sandpaper, 50-D Grit</td>
			<td>Norton</td>
			<td>M600060 01518</td>
			<td>Or other coarse grit sandpaper</td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Fisher Chemical</td>
			<td>A38S-500</td>
			<td>CAS#64-19-7, for adjusting pH of sodium acetate buffer used for Toluidine blue histology, as well as 14% EDTA decalcification solution</td>
		</tr>
		<tr>
			<td>Grips</td>
			<td>ADMET</td>
			<td>GV-100NT-A4</td>
			<td>Stainless steel vice grips, screws and springs described in the protocol are included</td>
		</tr>
		<tr>
			<td>Histobond Adhesive Microscope Slides</td>
			<td>VWR</td>
			<td>16005-108</td>
			<td>Sagittal sections of hind limbs explants reliably adhere to these slides through all staining protocols</td>
		</tr>
		<tr>
			<td>In situ Cell Death Detection Kit, TMR Red</td>
			<td>Roche</td>
			<td>12156792910</td>
			<td>TUNEL assay</td>
		</tr>
		<tr>
			<td>Labeling tape</td>
			<td>Fisherbrand</td>
			<td>15-959</td>
			<td>Or any other labeling tape of preference</td>
		</tr>
		<tr>
			<td>L-ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4544-100G</td>
			<td>CAS#50-81-7, for culture media formulation</td>
		</tr>
		<tr>
			<td>Neutral buffered formalin, 10%</td>
			<td>Leica</td>
			<td>3800600</td>
			<td>For sample fixation, 5 gallon supply</td>
		</tr>
		<tr>
			<td>Nunc petri dishes</td>
			<td>Sigma-Aldrich</td>
			<td>P7741-1CS</td>
			<td>100 mm diameter x 25 mm height, maintain explants submerged in 70 mL of culture media as described in protocol</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (100X)</td>
			<td>Gibco by ThermoFisher</td>
			<td>15140122</td>
			<td>Add 5 mL to 500 mL 1X DMEM for 1% v/v (1X) working concentration</td>
		</tr>
		<tr>
			<td>Polylactic acid (PLA) 1.75 mm filament</td>
			<td>Hatchbox</td>
			<td>-</td>
			<td>Choose filament diameter compatible with your 3D printer extruder, in color of choice.</td>
		</tr>
		<tr>
			<td>Processing cassettes</td>
			<td>Leica</td>
			<td>3802631</td>
			<td>For fixation, decalcification and paraffin embedding</td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade Reagent with DAPI</td>
			<td>Invitrogen by ThermoFisher</td>
			<td>P36931</td>
			<td>Mounting media for coverslipping tissue sections after TUNEL</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Fisher BioReagents</td>
			<td>BP1700-50</td>
			<td>CAS#39450-01-6, used for antigen retrieval in TUNEL protocol</td>
		</tr>
		<tr>
			<td>Scissors, Fine</td>
			<td>FST</td>
			<td>14094-11</td>
			<td>Straight, sharp</td>
		</tr>
		<tr>
			<td>Slide Staining Set, 12-place</td>
			<td>Mercedes Scientific&#160;</td>
			<td>MER 1011</td>
			<td>Rack with 12 stain dishes and slide dippers for Toluidine blue histology</td>
		</tr>
		<tr>
			<td>Sodium acetate, anhydrous</td>
			<td>Thermo Scientific Chemicals</td>
			<td>A1318430</td>
			<td>CAS#127-09-3, used to make buffer for Toluidine blue histology</td>
		</tr>
		<tr>
			<td>Tissue-Tek Accu-Edge Low Profile Microtome Blades</td>
			<td>VWR</td>
			<td>25608-964</td>
			<td>For paraffin sectioning</td>
		</tr>
		<tr>
			<td>Toluidine Blue O</td>
			<td>Thermo Scientific Chemicals</td>
			<td>348601000</td>
			<td>CAS#92-31-9</td>
		</tr>
		<tr>
			<td>Volume Reduction Insert</td>
			<td>-</td>
			<td>-</td>
			<td>3D printed from CAD files provided as Supplementary Files</td>
		</tr>
		<tr>
			<td>Xylenes</td>
			<td>Leica</td>
			<td>3803665</td>
			<td>4 gallon supply for histological staining</td>
		</tr>
	</tbody>
</table>

murine hind limb explant model for studying the mechanobiology of achilles tendon impingement

हड्डी पर कण्डरा प्रभाव स्पष्ट रूप से ऊंचा अनुप्रस्थ संपीड़ित तनाव के साथ एक बहुअक्षीय यांत्रिक तनाव वातावरण उत्पन्न करता है, जो ग्लाइकोसामिनोग्लाइकन (जीएजी) -समृद्ध मैट्रिक्स के संचय और कोलेजन नेटवर्क के रीमॉडेलिंग द्वारा विशेषता वाले स्थानीयकृत फाइब्रोकार्टिलेज फेनोटाइप को प्राप्त करता है। जबकि फाइब्रोकार्टिलेज स्वस्थ टेंडन के प्रभावित क्षेत्रों में एक सामान्य विशेषता है, अतिरिक्त जीएजी जमाव और कोलेजन नेटवर्क की अव्यवस्था टेंडिनोपैथी की पहचान विशेषताएं हैं। तदनुसार, टेंडिनोपैथी की दीक्षा और प्रगति में एक महत्वपूर्ण बाहरी कारक के रूप में चिकित्सकीय रूप से मान्यता प्राप्त है। फिर भी, कण्डरा टकराव अंतर्निहित मैकेनोबायोलॉजी का अध्ययन नहीं किया गया है। कण्डरा टकराव के लिए सेलुलर प्रतिक्रिया को स्पष्ट करने के पूर्व प्रयासों ने कोशिकाओं के लिए एकअक्षीय संपीड़न लागू किया है और इन विट्रो में कण्डरा एक्सप्लांट्स को उत्तेजित किया है। हालांकि, पृथक कोशिकाओं में मेकेनोरेस्पॉन्स के लिए महत्वपूर्ण त्रि-आयामी बाह्य वातावरण की कमी होती है, और इन विट्रो और एक्सक्साइड एक्सप्लांट अध्ययन दोनों विवो में कण्डरा टकराव द्वारा उत्पन्न बहुअक्षीय तनाव वातावरण को पुन: प्राप्त करने में विफल रहते हैं, जो कि प्रभावित क्षेत्र की शारीरिक विशेषताओं पर निर्भर करता है। इसके अलावा, कण्डरा टकराव के विवो मॉडल में यांत्रिक तनाव पर्यावरण पर नियंत्रण की कमी होती है। इन सीमाओं को दूर करने के लिए, हम एक उपन्यास murine हिंद अंग explant मॉडल प्रस्तुत Achilles कण्डरा impingement के mechanobiology का अध्ययन करने के लिए उपयुक्त है. यह मॉडल स्थानीय शरीर रचना को संरक्षित करने के लिए एच्लीस कण्डरा को सीटू में बनाए रखता है और अपने मूल वातावरण के भीतर कोशिकाओं को बनाए रखते हुए निष्क्रिय रूप से लागू टखने के डॉर्सिफ्लेक्सियन के दौरान कैल्केनस पर एच्लीस कण्डरा सम्मिलन के प्रभाव से उत्पन्न बहुअक्षीय तनाव वातावरण को पुन: पेश करता है। हम इस मॉडल के अभिन्न अंग एक टिशू कल्चर प्रोटोकॉल का वर्णन करते हैं और 7 दिनों में निरंतर एक्सप्लांट व्यवहार्यता स्थापित करने वाले डेटा प्रस्तुत करते हैं। प्रतिनिधि परिणाम बढ़ाया हिस्टोलॉजिकल जीएजी धुंधला प्रदर्शित करते हैं और कोलेजन फाइबर संरेखण माध्यमिक को प्रभावित करने के लिए कम हो जाते हैं, ऊंचा फाइब्रोकार्टिलेज गठन का सुझाव देते हैं। इस मॉडल को आसानी से विभिन्न यांत्रिक लोडिंग रेजिमेंस की जांच करने के लिए अनुकूलित किया जा सकता है और प्रभाव के जवाब में एच्लीस कण्डरा में फेनोटाइपिक परिवर्तन की मध्यस्थता करने वाले तंत्र की पहचान करने के लिए ब्याज के आणविक मार्गों में हेरफेर की अनुमति देता है।

A multitude of tendons, including the Achilles tendon and rotator cuff tendons, experience bony impingement due to normal anatomical positioning1,2,3,4. Tendon impingement generates compressive strain directed transversely to the longitudinal fiber axis5,6,7. Regions of tendon impingement demonstrate a unique fibrocartilage phenotype in which shrunken, round cells (fibrochondrocytes) are embedded within a disorganized collagen network with markedly increased glycosaminoglycan (GAG) content2,3,4,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24. Prior studies suggest the disparate mechanical environment produced by tendon impingement sustains this GAG-rich matrix by driving the deposition of large aggregating proteoglycans, most notably aggrecan, although the underlying mechanisms are unclear1,3,12,13,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39. While fibrocartilage is a normal feature in impinged regions of healthy tendons, aberrant proteoglycan metabolism associated with excessive fibrocartilage formation is a hallmark feature of tendinopathy, a common and debilitating disease that disproportionately emerges in chronically impinged tendons1,40,41,42,43,44,45,46,47,48,49. Accordingly, tendon impingement is clinically recognized as an important extrinsic factor driving several of the most common tendinopathies, including rotator cuff disease and insertional Achilles tendinopathy (IAT)50,51,52. Currently, treatment of tendinopathy is inefficient. For example, approximately 47% of patients with IAT require surgical intervention after failed conservative management, with variable postoperative outcomes53,54,55,56. Despite the apparent relationship between impingement and tendinopathy, the mechanobiological mechanisms by which cells in impinged tendon sense and respond to their mechanical environment are poorly described, which obscures understanding of tendinopathy pathogenesis and results in inadequate treatment.
Explant models are useful tools in the study of tendon mechanobiology57,58. As a first step towards understanding the mechanobiology of tendon impingement, several prior studies have explored cellular response following the application of simple uniaxial compression to cells or excised tendon explants27,29,30,31,32,33,34,39. However, cells in vitro lack extracellular and pericellular matrices that facilitate strain transfer, sequester important growth factors and cytokines released by mechanical deformation, and provide substrate for focal adhesion complexes that play a role in mechanotransduction57,59. Additionally, both in vitro and excised explant studies fail to recapitulate the multiaxial mechanical strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region5,6. In the context of the impinged Achilles tendon insertion, this includes surrounding tissues such as the retrocalcaneal bursa and Kager&#39;s fat pad60,61,62,63. Conversely, in vivo models of tendon impingement25,28,36,37,38,64,65,66 allow minimal control over the magnitude and frequency of load applied directly to the tendon, which is a well-recognized limitation of in vivo models for studying tendon mechanobiology57,58,67,68,69,70. Given challenges in measuring tendon strain in vivo, the internal strain environment generated within these models is often poorly characterized.
In this manuscript, we present a custom experimental platform that recreates impingement of the Achilles tendon insertion upon the calcaneus within whole murine hind limb explants that, when paired with this tissue culture protocol, maintains viability over 7 days in explant culture and allows for study of the biologic sequelae of tendon impingement. The platform is built upon a 3D printed polylactic acid (PLA) base that provides the foundation for the attachment of the grips and 3D printed PLA volume reduction insert. The grips are used to clamp the upper leg and knee proximal to the Achilles myotendinous junction with the caudal aspect of the hind limb facing upward, allowing the Achilles tendon to be imaged from above using an ultrasound probe or inverted microscope (Figure 1A). The volume reduction insert&#160;slides along a track on the base and reduces the required volume of tissue culture media. A braided line wrapped around the hind paw is routed out of the platform utilizing the base design and a 3D printed PLA clip. By pulling on the string, the hind paw is dorsiflexed, and the Achilles tendon insertion is impinged against the calcaneus, resulting in elevated transverse compressive strain5,6 (Figure 1A). The platform is contained within an acrylic bath that maintains the hind limb explants submerged in tissue culture media. Securing the taut string to the outside of the bath with adhesive tape maintains ankle dorsiflexion to produce static impingement of the Achilles tendon insertion. CAD files for 3D printed components are provided in multiple formats (Supplementary File 1), allowing import into a range of commercial and free, open-source CAD software for modification to suit experimental needs. If access to 3D printers is not available for fabrication, CAD files can be provided to online 3D printing services that will print and ship the parts at low cost.
Importantly, the triceps surae-Achilles musculotendinous complex spans both the knee and ankle joints71,72,73. Consequently, tensile strain in the Achilles tendon is influenced by knee flexion. Knee extension places the Achilles tendon under tension, whereas knee flexion reduces tension. By first extending the knee and then passively dorsiflexing the ankle, compressive strains at the impinged insertion can be superimposed upon tensile strains. Conversely, by passively dorsiflexing the ankle with the knee flexed, tensile strain is reduced, and compressive strain remains. The current protocol explores three such conditions. 1) For static impingement, the foot is dorsiflexed to &#60; 110&#176; with respect to the tibia to impinge the insertion, with the knee flexed to reduce tension. 2) For the baseline tension group, the ankle is extended above 145&#176; of dorsiflexion with the knee extended, generating predominately tensile strain at the insertion. 3) For the unloaded group, explants are cultured in a Petri dish with the knee and ankle in neutral positions in the absence of externally applied load. The angles referred to above are photographically measured relative to a coordinate system where the foot and tibia are parallel at an angle of 180&#176; and perpendicular at an angle of 90&#176;.
Key steps of the protocol include 1) dissection of hind limb explants and careful removal of the skin and plantaris tendon; 2) explant culture following a 48 h dexamethasone pretreatment; 3) tissue sectioning and histological staining; and 4) color image analysis to assess fibrocartilage formation. Following dissection, each hind limb explant is pretreated for 48 h in culture media&#160;supplemented with dexamethasone74. Contralateral limbs from each mouse are assigned to separate experimental groups for pairwise comparison, which helps control biological variability. After pretreatment, explants are positioned into platforms as described above and cultured for 7 more days (Figure 1B). Additional comparisons are made to a pretreated (day 0) group in which explants are removed immediately following the 48 h pretreatment.
After explant culture, hind limbs are trimmed down, formalin fixed, decalcified and embedded in paraffin. Serial sectioning in sagittal orientation provides visualization of the Achilles tendon from the myotendinous junction to the calcaneal insertion while allowing section depth to be tracked through the entire tendon. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP X-nick labeling (TUNEL) is used to visualize DNA damage secondary to apoptosis and assess viability. Toluidine blue histology and custom color image analysis are performed to quantify changes in GAG staining. Toluidine blue stained tissue sections are then used for SHG imaging to characterize alterations in collage fiber organization (Figure 1B).
The provided representative results suggest altered histological staining of the GAG-rich matrix and disorganization of the extracellular collagen network generated by 7 days of static impingement within the model. This model can be utilized to explore molecular mechanisms underlying impingement-driven fibrocartilaginous change.

लेखक स्पॉटलाइट: कण्डरा इम्पिंजमेंट के मेकेनोबायोलॉजी को उजागर करना - एक बहुअक्षीय म्यूरिन हिंद लिंब एक्सप्लांट मॉडल 

Achilles कण्डरा Impingement के मेकेनोबायोलॉजी का अध्ययन करने के लिए Murine हिंद लिंब एक्सप्लांट मॉडल

We study the mechanobiology underlying tendon impingement and the process by which this unique mechanical demand drives localized fiber cartilage formation in health and disease. Here we seek to characterize matrix for modeling relative to spatially heterogeneous patterns of multiaxial mechanical strain, generated by impingement and to identify molecular mechanisms mediating this response. In vitro models for studying impingement mechanobiology have applied simple compression to isolated tendon cells or artificial uniaxial compression to partial and whole tendon explants.Established animal models of tendon impingement, manipulating external source of tendon impingement in vivo most often surgically and explore biology following resuming physical activity. In vitro models present significant limitations as isolated cells lack their three dimensional extracellular environment, crucial to mechano-response. While excised explant models circumvent this limitation, both fail to recreate multiaxial strain patterns generated by impingement in vivo.Conversely, animal models offer limited ability to measure or control internal tissue strains. Our Murine Hind Limb Explant Model for studying impingement mechanobiology maintains cells within their extracellular environment and preserves local anatomy of the impinged achilles tendon insertion in situ, allowing controlled prescription of impingement through passively applied joint motion to recreate multiaxial patterns of tissue strain that are measurable and well-characterized.

ट्यूनल सना हुआ ऊतक वर्गों की प्रतिनिधि छवियां प्रयोगात्मक समूहों(चित्रा 2ए)में एक्सप्लांट कल्चर के 7 दिनों के बाद अकिलीज़ कण्डरा के शरीर के भीतर न्यूनतम एपोप्टोटिक नाभिक प्रदर्शित करती है?...

Watch this Scientific Journal Video about Unraveling the Mechanobiology of Tendon Impingement – A Multiaxial Murine Hind Limb Explant Model at JoVE.com

हम एक कस्टम प्रयोगात्मक मंच और टिशू कल्चर प्रोटोकॉल प्रस्तुत करते हैं जो निरंतर सेल व्यवहार्यता के साथ murine हिंद अंग explants में Achilles कण्डरा सम्मिलन के प्रभाव से प्रेरित फाइब्रोकार्टिलाजिनस परिवर्तन को फिर से बनाता है, जो कण्डरा टकराव के mechanobiology की खोज के लिए उपयुक्त मॉडल प्रदान करता है।

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a ...

हम कण्डरा टकराव के अंतर्निहित मेकेनोबायोलॉजी का अध्ययन करते हैं और जिस प्रक्रिया से यह अद्वितीय यांत्रिक मांग स्वास्थ्य और बीमारी में स्थानीयकृत फाइबर उपास्थि गठन को चलाती है। यहां हम बहुअक्षीय यांत्रिक तनाव के स्थानिक रूप से विषम पैटर्न के सापेक्ष मॉडलिंग के लिए मैट्रिक्स को चिह्नित करना चाहते हैं, जो कि प्रभाव से उत्पन्न होता है और इस प्रतिक्रिया की मध्यस्थता करने वाले आणविक तंत्र की पहचान करता है। इन विट्रो मॉडल में प्रभाव का अध्ययन करने के लिए मैकेनोबायोलॉजी ने पृथक कण्डरा कोशिकाओं या कृत्रिम एकअक्षीय संपीड़न को आंशिक और पूरे कण्डरा एक्सप्लांट्स के लिए सरल संपीड़न लागू किया है।कण्डरा टकराव के स्थापित पशु मॉडल, विवो में कण्डरा टकराव के बाहरी स्रोत में हेरफेर करना सबसे अधिक बार शल्य चिकित्सा और शारीरिक गतिविधि को फिर से शुरू करने के बाद जीव विज्ञान का पता लगाना। इन विट्रो मॉडल में महत्वपूर्ण सीमाएं मौजूद हैं क्योंकि पृथक कोशिकाओं में उनके तीन आयामी बाह्य वातावरण की कमी होती है, जो मेकोनो-प्रतिक्रिया के लिए महत्वपूर्ण है। जबकि एक्साइज़्ड एक्सप्लांट मॉडल इस सीमा को दरकिनार करते हैं, दोनों विवो में प्रभाव से उत्पन्न बहुअक्षीय तनाव पैटर्न को फिर से बनाने में विफल रहते हैं।इसके विपरीत, पशु मॉडल आंतरिक ऊतक उपभेदों को मापने या नियंत्रित करने की सीमित क्षमता प्रदान करते हैं। इम्पिंगमेंट मेकेनोबायोलॉजी का अध्ययन करने के लिए हमारा म्यूरिन हिंद लिम्ब एक्सप्लांट मॉडल उनके बाह्य वातावरण के भीतर कोशिकाओं को बनाए रखता है और सीटू में इम्पिंग्ड एच्लीस टेंडन सम्मिलन की स्थानीय शारीरिक रचना को संरक्षित करता है, जिससे ऊतक तनाव के बहुअक्षीय पैटर्न को फिर से बनाने के लिए निष्क्रिय रूप से लागू संयुक्त गति के माध्यम से प्रभाव के नियंत्रित नुस्खे की अनुमति मिलती है जो औसत दर्जे का और अच्छी तरह से विशेषता है।

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JoVE Journal

Bioengineering

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a localized fibrocartilage phenotype characterized by accumulation of glycosaminoglycan (GAG)-rich matrix and remodeling of the collagen network. While fibrocartilage is a normal feature in impinged regions of healthy tendons, excess GAG deposition and disorganization of the collagen network are hallmark features of tendinopathy. Accordingly, impingement is clinically recognized as an important extrinsic factor in the initiation and progression of tendinopathy. Nevertheless, the mechanobiology underlying tendon impingement remains understudied. Prior efforts to elucidate the cellular response to tendon impingement have applied uniaxial compression to cells and excised tendon explants in vitro. However, isolated cells lack a three-dimensional extracellular environment crucial to mechanoresponse, and both in vitro and excised explant studies fail to recapitulate the multiaxial strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region. Moreover, in vivo models of tendon impingement lack control over the mechanical strain environment. To overcome these limitations, we present a novel murine hind limb explant model suitable for studying the mechanobiology of Achilles tendon impingement. This model maintains the Achilles tendon in situ to preserve local anatomy and reproduces the multiaxial strain environment generated by impingement of the Achilles tendon insertion upon the calcaneus during passively applied ankle dorsiflexion while retaining cells within their native environment. We describe a tissue culture protocol integral to this model and present data establishing sustained explant viability over 7 days. The representative results demonstrate enhanced histological GAG staining and decreased collagen fiber alignment secondary to impingement, suggesting elevated fibrocartilage formation. This model can easily be adapted to investigate different mechanical loading regimens and allows for the manipulation of molecular pathways of interest to identify mechanisms mediating phenotypic change in the Achilles tendon in response to impingement.

We present a custom experimental platform and tissue culture protocol that recreates fibrocartilaginous change driven by impingement of the Achilles tendon insertion in murine hind limb explants with sustained cell viability, providing a model suitable for exploring the mechanobiology of tendon impingement.

Explant Dissection to Generate Murine Hind Limb Explant Model to Study Tendon Impingement

To begin, transfer 70 milliliters of pre-warmed Dulbecco's Modified Eagle Medium into a Petri dish in a sterile biosafety cabinet and add dexamethasone at a concentration of 100 nano molar. Assemble the surgical tools required for the dissection, which includes smooth, straight, fine tip forceps. Straight fine tip forceps with serrated teeth and straight sharp fine scissors.To dissect hind limb explants. Locate the hip joint of a euthanized mouse positioned supine on the bench top lined with absorbent under pads. Use fine scissors to create a small incision on the skin covering the proximal and anterior aspects of the upper leg.Then use fingers to pull the incision apart to expand and pinch the exposed upper leg. Carefully pull the skin distally to de-glove the hind limb to the level of the ankle. Insert one scissor blade gently under the skin along the dorsal aspect of the foot.Then make an incision that extends towards the toes and continue pulling the skin distally until it is completely removed. Position the mouse to have a clear view of the Achilles tendon insertion onto the posterior aspect of the calcaneus near the ankle. To remove the plant terrace tendon, insert one tip of the smooth fine tip forceps between the two tendons.Pass the tip medially under the plantaris tendon. Then pull it proximally to tear through the plantaris muscle. Then using fine tip serrated forceps, grasp the detached proximal end of the plantaris tendon and pull it distally to completely remove it.At the hip joint, employ fine scissors to precisely sever through the pelvis, isolating the hind limb. Use the scissors to gently remove the remaining pelvis, revealing the femoral head. In the biosafety cabinet transfer the hind limb explant into the dish containing cell culture media and dexamethasone.Relocate the dish to the incubator and subject it to pre-treatment for 48 hours.

कण्डरा प्रभाव का अध्ययन करने के लिए म्यूरिन हिंद लिंब एक्सप्लांट मॉडल उत्पन्न करने के लिए एक्सप्लांट विच्छेदन

Explant Culture and Platforms Loading to Study the Biologic Sequelae of Tendon Impingement

To begin, preheat an adequate amount of sterile culture media while the 48-hour dexamethasone pretreatment of murine hindlimb explant is about to complete. Once the pretreatment ends, move the pretreated explants to the biosafety cabinet. For the unloaded group, aspirate the pretreatment media and transfer the explants to fresh Petri dishes.Add 70 milliliters of culture media to each dish before returning them to the incubator. To prepare the explant platforms for the baseline tension and static impingement group, cut pieces of sandpaper to match the size of the grip platens. For the static impingement group, cut pieces of braided line and pret tie a loose overhand knot halfway along the length of the line.Tear pieces of aluminum foil to cover each acrylic bath and spray them with 70%ethanol. Transfer the prepared components to the biosafety cabinet. Arrange the components such as acrylic bath, base, volume reduction inserts, clip, and grips for platform assembly.Place platform components inside secondary containers of sufficient volume to contain any potential culture media leaks. Submerge the containers in a solution of at least 10%bleach for a minimum of one hour. Rinse the bleach solutions with tap water.After transferring the rinsed components to the biosafety cabinet, use M3 screws and compression springs to affix the platens to the grips. Securely attach the grips to the base employing an M5 screw and insert the M6 screws until they contact the platens. Enhance the platens'functionality by affixing sandpaper using double-sided tape.Close the grips to facilitate the adhesion of the sandpaper to the platens. To load a platform, fully open the grips and use forceps to carefully position the upper leg and knee between the platens. Close the grips gently to hold the limb in place.Use forceps to grasp the exposed femoral head or foot and adjust the knee flexion angle, while gradually closing the forceps to secure it in place. For the baseline tension group, extend the knee joint while closing the grips. In the case of the static impingement group, flex the knee joint while closing the grips.For the static impingement group, place the overhand knot of the string around the distal paw and tighten it securely. Route the string through a slot in the base underneath the explant and through the clip hole. Position the base into the acrylic bath and fasten the clip to the top edge of the bath.To dorsiflex the foot, pull the string until it reaches at least 110 degrees relative to the tibia. Use a permanent marker to mark the string as it exits the clip. Capture a photograph of the explant in this dorsiflexed position for future quantification of the dorsiflexion angle.Remove the base from the setup and attach the volume reduction insert by sliding it along a track on the base. Place the base back into the acrylic bath and reposition the clip on the top edge. Use the marked string as a guide.Pull it to restore the foot to its original dorsiflexion angle and secure the string to the exterior of the bath using tape to maintain a static dorsiflexed position. For the baseline tension group, once the explant is positioned between the grips, take a photograph to quantify the dorsiflexion angle. Then slide the volume reduction insert onto the base and place it into the acrylic bath.Add 125 milliliters of prewarmed culture media to each platform to submerge the explants. After covering the top of the bath with aluminum foil, place the entire setup into a secondary container. Relocate the secondary container to the incubator and culture for seven days.