The procedures are performed using the Lipogems System.

All procedures performed in the studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee, and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
1. Medical History
<ol>
	<li>Start clinical examination with a detailed patient history. 
	NOTE: An OLT must always be suspected in case of instability of the ankle with repeated sprains associated with swelling, stiffness...

<ol>
	<li>D'Ambrosi, R., Maccario, C., Serra, N., Liuni, F., Usuelli, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteochondral+Lesions+of+the+Talus+and+Autologous+Matrix-Induced+Chondrogenesis:+Is+Age+a+Negative+Predictor+Outcome?.">Osteochondral Lesions of the Talus and Autologous Matrix-Induced Chondrogenesis: Is Age a Negative Predictor Outcome?.</a> Arthroscopy. 33 (2), 428-435 (2017).</li><li>Becher, 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=T2-mapping+at+3+T+after+microfracture+in+the+treatment+of+osteochondral+defects+of+the+talus+at+an+average+follow-up+of+8+years.">T2-mapping at 3 T after microfracture in the treatment of osteochondral defects of the talus at an average follow-up of 8 years.</a> Knee Surg. SportsTraumatol. Arthrosc. 23 (8), 2406-2412 (2015).</li><li>Polat, G., 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=Long-term+results+of+microfracture+in+the+treatment+of+talus+osteochondral+lesions.">Long-term results of microfracture in the treatment of talus osteochondral lesions.</a> Knee Surg. Sports Traumatol. Arthrosc. 24 (4), 1299-1303 (2016).</li><li>van Bergen, C. 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=Arthroscopic+treatment+of+osteochondral+defects+of+the+talus:+outcomes+at+eight+to+twenty+years+of+follow-up.">Arthroscopic treatment of osteochondral defects of the talus: outcomes at eight to twenty years of follow-up.</a> J. Bone Joint Surg. Am. 95 (6), 519-525 (2013).</li><li>van Eekeren, I. 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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=Endothelial+progenitor+cells+(EPCs)+and+mesenchymal+stem+cells+(MSCs)+in+bone+healing.">Endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) in bone healing.</a> Curr. Stem Cell. Res. Ther. 7 (4), 293-301 (2012).</li><li>Leigheb, 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=Italian+translation,+cultural+adaptation+and+validation+of+the+American+Orthopaedic+Foot+and+Ankle+Society's+(AOFAS)+ankle-hindfoot+scale.">Italian translation, cultural adaptation and validation of the American Orthopaedic Foot and Ankle Society's (AOFAS) ankle-hindfoot scale.</a> Acta Biomed. 87 (1), 38-45 (2016).</li><li>Ware, J., Kosinski, M., Keller, S. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosing,+planning+and+evaluating+osteochondral+ankle+defects+with+imaging+modalities.">Diagnosing, planning and evaluating osteochondral ankle defects with imaging modalities.</a> World. J. Orthop. 6 (11), 944-953 (2015).</li><li>van Dijk, C. N., Reilingh, M. L., Zengerink, M., van Bergen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteochondral+defects+in+the+ankle:+why+painful?.">Osteochondral defects in the ankle: why painful?.</a> Knee Surg. Sports Traumatol. Arthrosc. 18 (5), 570-580 (2010).</li><li>Madry, H., van Dijk, C. N., Mueller-Gerbl, 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+basic+science+of+the+subchondral+bone.">The basic science of the subchondral bone.</a> Knee Surg. Sports Traumatol. Arthrosc. 18 (4), 419-433 (2010).</li><li>Mintz, D. N., Tashjian, G. S., Connell, D. A., Deland, J. T., O'Malley, M., Potter, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteochondral+lesions+of+the+talus:+a+new+magnetic+resonance+grading+system+with+arthroscopic+correlation.">Osteochondral lesions of the talus: a new magnetic resonance grading system with arthroscopic correlation.</a> Arthroscopy. 19 (4), 353-359 (2003).</li><li>Leumann, 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=A+novel+imaging+method+for+osteochondral+lesions+of+the+talus--comparison+of+SPECT-CT+with+MRI.">A novel imaging method for osteochondral lesions of the talus--comparison of SPECT-CT with MRI.</a> Am. J. Sports Med. 39 (5), 1095-1101 (2011).</li><li>Kim, Y. S., Park, E. H., Kim, Y. C., Koh, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+outcomes+of+mesenchymal+stem+cell+injection+with+arthroscopic+treatment+in+older+patients+with+osteochondral+lesions+of+the+talus.">Clinical outcomes of mesenchymal stem cell injection with arthroscopic treatment in older patients with osteochondral lesions of the talus.</a> Am. J. Sports Med. 41 (5), 1090-1099 (2013).</li><li>Kim, Y. S., Lee, H. J., Choi, Y. J., Kim, Y. I., Koh, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+an+injection+of+a+stromal+vascular+fraction+containing+adipose-derived+mesenchymal+stem+cells+influence+the+outcomes+of+marrow+stimulation+in+osteochondral+lesions+of+the+talus?+A+clinical+and+magnetic+resonance+imaging+study.">Does an injection of a stromal vascular fraction containing adipose-derived mesenchymal stem cells influence the outcomes of marrow stimulation in osteochondral lesions of the talus? A clinical and magnetic resonance imaging study.</a> Am. J. Sports Med. 42 (10), 2424-2434 (2014).</li><li>Kim, Y. S., Koh, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+Mesenchymal+Stem+Cells+as+a+Supplementary+Strategy+of+Marrow+Stimulation+Improves+Cartilage+Regeneration+After+Lateral+Sliding+Calcaneal+Osteotomy+for+Varus+Ankle+Osteoarthritis:+Clinical+and+Second-Look+Arthroscopic+Results.">Injection of Mesenchymal Stem Cells as a Supplementary Strategy of Marrow Stimulation Improves Cartilage Regeneration After Lateral Sliding Calcaneal Osteotomy for Varus Ankle Osteoarthritis: Clinical and Second-Look Arthroscopic Results.</a> Arthroscopy. 32 (5), 878-889 (2016).</li><li>Kim, Y. S., Lee, M., Koh, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additional+mesenchymal+stem+cell+injection+improves+the+outcomes+of+marrow+stimulation+combined+with+supramalleolar+osteotomy+in+varus+ankle+osteoarthritis:+short-term+clinical+results+with+second-look+arthroscopic+evaluation.">Additional mesenchymal stem cell injection improves the outcomes of marrow stimulation combined with supramalleolar osteotomy in varus ankle osteoarthritis: short-term clinical results with second-look arthroscopic evaluation.</a> J. Exp. Orthop. 3 (1), 12 (2016).</li><li>Hanke, C. W., Bernstein, G., Bullock, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+of+tumescent+liposuction+in+15,336+patients.+National+survey+results.">Safety of tumescent liposuction in 15,336 patients. National survey results.</a> Dermatol Surg. 21 (5), 459-462 (1995).</li><li>Illouz, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complications+of+liposuction.">Complications of liposuction.</a> Clin Plast Surg. 33 (1), 129-163 (2006).</li><li>Dixit, V. V., Wagh, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unfavourable+outcomes+of+liposuction+and+their+management.">Unfavourable outcomes of liposuction and their management.</a> Indian J Plast Surg. 46 (2), 377-392 (2013).</li><li>Lehnhardt, M., Homann, H. H., Daigeler, A., Hauser, J., Palka, P., Steinau, H. 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In recent years, clinical and preclinical trials have focused their attention on the effect of ADSCs to treat different musculoskeletal pathologies. The aim of this article is to describe the protocol for the treatment of osteochondral lesions of the talus using microfractured and purified adipose tissue in association with arthroscopic microperforations. The protocol involves several critical steps with high risks of complications. During fat harvesting, complications can be divided into local or systemic complications....

Arthroscopy is the gold standard for the treatment of osteochondral lesions of the talus (OLTs) with the aim of pain relief, restoring functionality, and improving quality of life, especially in young and active patients.
Currently, arthroscopic techniques can be classified in three ways. The reparative technique stimulates cells derived from bone marrow through a debridement and microperforations at the level of lesion. The reconstructive technique replaces the lesion using an autologous or heterologous ostechondral graft. The regenerative technique exploits the ability of multipotent cells to differentiate and replicate to reconstruct the damaged tissue1,2,3,4,5,6.
In recent years , regenerative techniques have been the subject of numerous in vitro and in vivo studies for the treatment of OLTs, and particularly mesenchymal stem cells derived from adipose tissue (ADSCs)7,8,9. These mesenchymal stem cells exhibit morphological and functional characteristics similar to other multipotent cells, isolated from other tissues; they also have the ability to differentiate into several and different cellular lines both in vitro and in vivo10,11,12,13. The focus on research regarding these cells is mainly due to their localization, in fact they represent from 10% to 30% of normal body weight with a concentration of 5,000 cells per gram of tissue13,14. On the other hand, a factor that limits the use of these cells is related to their handling during laboratory procedures. The lipoaspirate containing aggregates of adipocytes, collagen fibers, and normal vascular components is enzymatically processed with collagen A type I, and subjected to hemolysis before culture. The aim here is to describe the protocol for the treatment of osteochondral lesions of the talus using microfractured and purified adipose tissue.

<table>
	<tbody>
		<tr>
			<td>PROCESS KIT - PROCESSING KIT FOR FAT TISSUE</td>
			<td>LIPOGEMS</td>
			<td>LG PK 60</td>
			<td>Lipogems Kit to obtain microfractured and purified ADSCs</td>
		</tr>
		<tr>
			<td>HINTERMANN SPREADER</td>
			<td>INTEGRA</td>
			<td>119654</td>
			<td>The spreader allow to access most of the talar dome, in particular in case of posterior lesion</td>
		</tr>
		<tr>
			<td>CUP CURETTE</td>
			<td>ARTHREX</td>
			<td>AR-8655-02</td>
			<td>To remove the damaged cartilage and necrotic and sclerotic bone</td>
		</tr>
		<tr>
			<td>CHONDRAL PICK 30&#176; TIP / 60&#176; TIP</td>
			<td>ARTHREX</td>
			<td>AR-8655-05 
			AR-8655-06</td>
			<td>To perfrom microperforation at the level of the lesion, stimulating bleeding from the subchondral bone</td>
		</tr>
		<tr>
			<td>SHAVER</td>
			<td>ARTHREX</td>
			<td>AR-7300SR</td>
			<td>To clean the joint and aspirate water</td>
		</tr>
	</tbody>
</table>

autologous microfractured and purified adipose tissue for arthroscopic management of osteochondral lesions of the talus

In recent years, regenerative techniques have been increasingly studied and used to treat osteochondral lesions of the talus. In particular, several studies have focused their attention on mesenchymal stem cells derived from adipose tissue. Adipose-derived stem cells (ADSCs) exhibit morphological characteristics and properties similar to other mesenchymal cells, and are able to differentiate into several cellular lines. Moreover, these cells are also widely available in the subcutaneous tissue, representing 10 - 30% of the normal body weight, with a concentration of 5,000 cells per gram of tissue.
In the presented technique, the first step involves harvesting ADSCs from the abdomen and a process of microfracture and purification; next, the surgical procedure is performed entirely arthroscopically, with less soft tissue dissection, better joint visualization, and a faster recovery compared with standard open procedures. Arthroscopy is characterized by a first phase in which the lesion is identified, isolated, and prepared with microperforations; the second step, performed dry, involves injection of adipose tissue at the level of the lesion.
Between January 2016 and September 2016, four patients underwent arthroscopic treatment of osteochondral lesion of the talus with microfractured and purified adipose tissue. All patients reported clinical improvement six months after surgery with no reported complications. Functional scores at the latest follow-up are encouraging and confirm that the technique provides reliable pain relief and improvements in patients with osteochondral lesion of the talus.

Between January 2016 and September 2016, four patients underwent arthroscopic treatment of osteochondral lesion of the talus with microfractured and purified adipose tissue. All patients reported clinical improvement six months after surgery. Preliminary clinical results are reported in Table 1. No complications were reported.
In recent years, the use of ADSCs for the treatment of foot and ankle pathologies has ...

Watch this Scientific Journal Video about Autologous Microfractured and Purified Adipose Tissue for Arthroscopic Management of Osteochondral Lesions of the Talus at JoVE.com

Autologous Microfractured and Purified Adipose Tissue for Arthroscopic Management of Osteochondral Lesions of the Talus

The aim of this study is to report a protocol for the arthroscopic treatment of osteochondral lesions of the talus using microfractured and purified adipose-derived stem cells.

In recent years, regenerative techniques have been increasingly studied and used to treat osteochondral lesions of the talus. In particular, several ...

The overall goal of this surgical procedure is to treat osteochondral lesions of the talus through the induced regeneration of talar cartilage using autologous microfractured and purified adipose tissue in conjunction with microperforations. This method can help answer key questions in the regenerative medicine field about how to improve the quality of neo-formed tissues. The main advantage of this technique is that it is completely arthroscopic, with little soft tissue dissection, better joint visualization, and a faster patient recovery.The implications of this technique extend beyond the ankle to the treatment of lesions on any another joint in the body. Visual demonstration of this method is critical, as the positioning of the Hintermann spreader is difficult to learn without observation of the open posterior lesion treatment position. To harvest the adipose tissue, first use a scalpel to make two 0.5-centimeter periumbilical incisions.Then, use a 60-milliliter syringe equipped with an 18-gauge needle to inject approximately 300 milliliters of Klein solution through the incisions into the subcutaneous fat tissue of the abdomen. When all of the solution has been delivered, use a 13-gauge blunt catheter attached to a 20-milliliter syringe to harvest 40 to 45 milliliters of adipose-derived stem cells, or ADSCs, from the periumbilical area, and apply a compression bandage onto the incision. Add the cell suspension into the large filter of a processing kit, and press 100 to 300 milliliters of the lipoaspirate through the filter into the kit device to obtain the first cluster reduction.A corresponding volume of saline solution will exit the kit into waste bag. Next, add stainless steel marbles to the kit to achieve a temporary emulsion between the oil, blood, and saline in the aspirate, and use a gravity counter-flow of saline solution to remove the contaminating oil residue and blood from the sample. When the eluate is clear and the lipoaspirate is yellow, stop the saline flux and reverse the device so that the gray cap is facing up.Then, connect a 10-milliliter syringe to the upper opening of the device, and use a second 10-milliliter syringe to push the floating adipose clusters from below through the second cutting hexagonal filter into the first syringe to achieve the second adipose cluster reduction. To prepare the lesion for adipose-derived stem cell injection, place the patient in the supine position under spinal anesthesia with a tourniquet at the thigh level to decrease the bleeding and to allow a better arthroscopic visualization. Using a dermographic pen, mark both the lateral and medial malleoli, the anterior joint line, the tibialis anterior and Achilles tendons, the great saphenous vein, and the superficial peroneal nerve.When all of the landmarks have been notated, use a scalpel to make a shallow skin incision just medial to the tibialis anterior tendon, and perforate the capsule by blunt dissection, taking care to avoid the saphenous nerve and the great saphenous vein. For arthroscopic treatment of the cartilage, place a wide-angle, 2.7-millimeter arthroscope with a 30-degree viewing angle into the anterior recess of the joint. After checking the joint line, create the anterolateral portal, medial to the lateral malleolus and lateral to the extensor digitorum tendon, as just demonstrated for the approach portal.Assess the size and position of the articular cartilage within the portal, using a palpator to evaluate the condition and the quality of the tissue. In the case of a posterior lesion, position one K-wire on the tibia and one K-wire on the talar bone, and percutaneously apply the opening level arm of a Hintermann spreader onto the K-wires to distract the joint and to allow exposure of the lesion. Use a standard arthroscopic curette to remove the damaged and unstable cartilage, the calcified layer, and any necrotic or sclerotic bone, resulting in a regular-shaped, contained lesion with shouldered borders.Next, use a chondral pick to create microperforations about three millimeters apart from the outside to the inside of the lesion to stimulate the bone marrow stem cells from the subchondral bone and to induce microfractures on the healthy subchondral bone beneath the defect. Then, use a shaver to remove any intraarticular water, followed by swabbing with a clean, cotton sponge. When the joint is completely dry, inject five to seven milliliters of adipose-derived stem cells into the ankle joint through either of the two portals, and release the tourniquet.Between January and September of 2016, four patients underwent arthroscopic treatment of osteochondral lesions of the talus with induced microfractures and purified adipose tissue, as just demonstrated. All patients reported clinical improvement six months after surgery, with no complications. Once mastered, this technique can be completed in 30 minutes if it is performed properly.Following this procedure, other surgical procedures such as lateral ligament reconstruction can be performed to improve ankle stability. After its development, this technique paved the way for researcher in the field of regenerative medicine to explore cartilage healing in orthopedics pathologies. After watching this video, you should have a good understanding of how to use arthroscopy to deliver autologous microfractured adipose-derived stem cell into the ankle joint.Although mesenchymal stem cell therapies are becoming more common in the clinic, it's important to remember to take extra safety precautions when using ADSCs in regenerative medicine methods/protocols.

autologous-microfractured-purified-adipose-tissue-for-arthroscopic

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JoVE Journal

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10.2K vues.  IRCCS Istituto Ortopedico Galeazzi - C.A.S.C.O. Piede e Caviglia. The overall goal of this surgical procedure is to treat osteochondral lesions of the talus through the induced regeneration of talar cartilage using autologous microfractured and purified adipose tissue in conjunction with microperforations. This method can help answer key questions in the regenerative medicine field about how to improve the quality of neo-formed tissues. The main advantage of this technique is that it is completely arthroscopic, with little soft tissue dissection, better joi...

Vidéo: Autologous Microfractured and Purified Adipose Tissue for Arthroscopic Management of Osteochondral Lesions of the Talus

The aim of this study is to report a protocol for the arthroscopic treatment of osteochondral lesions of the talus using microfractured and purified adipose-derived stem...

clinical protocol of producing adipose tissue-derived stromal vascular fraction for potential cartilage regeneration

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

human adipose tissue micro-fragmentation for cell phenotyping and secretome characterization

Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization

Here, we present human adipose tissue enzyme-free micro-fragmentation using a closed system device. This new method allows the obtainment of sub-millimeter clusters of adipose tissue suitable for in vivo transplantation, in vitro culture, and further cell isolation and...

dissection, histological processing, and gene expression analysis of murine supraclavicular brown adipose tissue

Author Spotlight: Optimizing Supraclavicular Brown Adipose Tissue Extraction for Genetic Analysis

Here, we provide a practical procedure for dissecting and performing histological and gene expression analyses of murine supraclavicular brown adipose...

Morphological and Molecular Characterization of the scBAT in Mice

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matrix-assisted autologous chondrocyte transplantation for remodeling and repair of chondral defects in a rabbit model

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation...

adipo-clear: a tissue clearing method for three-dimensional imaging of adipose tissue

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for...

isolation of nuclei from human intermuscular adipose tissue and downstream single-nuclei rna sequencing

Author Spotlight: Deciphering the Cellular Mysteries of Intermuscular Adipose Tissue in Humans

The biology of intermuscular adipose tissue (IMAT) is largely unexplored due to the limited accessibility of human tissue. Here, we present a detailed protocol for nuclei isolation and library preparation of frozen human IMAT for single nuclei RNA sequencing to identify the cellular composition of this unique adipose...

Single Cell Nuclei Isolation from IMAT for Studying Gene Expression Profiles 

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infrared thermography for the detection of changes in brown adipose tissue activity

Infrared Thermography for the Detection of Changes in Brown Adipose Tissue Activity

Here, we present a protocol for measuring brown adipose tissue activity after a meal in humans and laboratory...

isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue

Author Spotlight: The Significance of Isolation, Culture, and Adipogenic Induction of SVF-Derived Preadipocytes from Mouse Perivascular Adipose Tissue 

Here, we describe the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, allowing for the study of perivascular adipose tissue function and its relationship with vascular...

Culturing Preadipocytes Isolated from Murine Perivascular Adipose Tissue

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comprehensive characterization of tissue mineralization in an ex vivo model

Author Spotlight: Advanced Techniques for Characterizing Tissue Mineralization in Bone Regeneration Research

The proposed protocol entails a global approach to assess bone formation in the context of bone regeneration using multimodal analyses. It aims to provide qualitative and quantitative information on new bone formation, enhancing the rigor and validity of basic and pre-clinical...

Multimodal Analysis of Bone Formation Using Explant Models

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white and brown adipose depot collection from mouse pup: a surgical procedure to harvest the white adipose tissue and brown adipose tissue from mouse pup

In this video, we demonstrate the surgical procedure to isolate the subcutaneous white adipose tissue and interscapular brown adipose tissue from a newborn...

White and Brown Adipose Depot Collection from Mouse Pup: A Surgical Procedure to Harvest the White Adipose Tissue and Brown Adipose Tissue from Mouse Pup

isolation of adipose tissue immune cells

Isolation of Adipose Tissue Immune Cells

Adipose tissue (AT) is a site of intense immune cell activation and interaction.  Almost all cells of the immune system are present in AT and their ratios are altered by obesity.  Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic...

semi-automated isolation of the stromal vascular fraction from murine white adipose tissue using a tissue dissociator

Author Spotlight: Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases...

Semi-Automated Isolation of Murine White Adipose Tissue

mouse adipose tissue collection and processing for rna analysis

Mouse Adipose Tissue Collection and Processing for RNA Analysis

The purpose of this paper is to present a step-by-step procedure to collect different white adipose tissues from mice, process the fat samples and extract RNA.

isolation of murine adipose tissue-derived microvascular fragments as vascularization units for tissue engineering

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue...

To collect interscapular brown adipose tissue, pull the skin from the shoulder blades over the head, and lift the deep red brown adipose tissue located between the shoulder blades. Carefully make incisions all around the adipose to detach it from the body, and check the tissue for consistency and color. After rinsing, place the tissue in a second 1.5 milliliter tube containing 250 microliters of PBS and 200 microliters of 2X isolation buffer on...

chromatin immunoprecipitation of murine brown adipose tissue

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in...

isolation, in vitro expansion, and characterization of mesenchymal stem cells from mouse epididymal adipose tissue

Author Spotlight: Advancing Treatment Approaches with Adipose-Derived Stem Cells

The adipose tissue is an excellent source of mesenchymal stem cells. Here, we bring the step-by-step extraction, cultivation, and characterization of adipose tissue-derived stem cells (ADSCs) from Swiss mice epididymal adipose tissue.

Primary Culture of Adipose Tissue Derived Stem Cells from Mouse Epididymis

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dissection and isolation of murine perivascular adipose tissue (pvat)

Dissection and Isolation of Murine Perivascular Adipose Tissue (PVAT)

isolation of adipose tissue nuclei for single-cell genomic applications

Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications

This publication describes a protocol for the isolation of nuclei from mature adipocytes, purification by fluorescence-activated sorting, and single-cell level...

Breathing-controlled Electrical Stimulation (BreEStim) for Management of Neuropathic Pain and Spasticity

Electrical stimulation (EStim) refers to the application of electrical current to muscles or nerves in order to achieve functional and therapeutic goals. It has been extensively used in various clinical settings. Based upon recent discoveries related to the systemic effects of voluntary breathing and intrinsic physiological interactions among systems during voluntary breathing, a new EStim protocol, Breathing-controlled Electrical Stimulation (BreEStim), has been developed to augment the effects of electrical stimulation. In BreEStim, a single-pulse electrical stimulus is triggered and delivered to the target area when the airflow rate of an isolated voluntary inspiration reaches the threshold. BreEStim integrates intrinsic physiological interactions that are activated during voluntary breathing and has demonstrated excellent clinical efficacy. Two representative applications of BreEStim are reported with detailed protocols: management of post-stroke finger flexor spasticity and neuropathic pain in spinal cord injury.

Breathing-controlled Electrical Stimulation BreEStim for Management of Neuropathic Pain and Spasticity

The purpose is to present a new method, breathing-control electrical stimulation (BreEStim) for management of neuropathic pain and spasticity.

Electrical stimulation (EStim) refers to the application of electrical current to muscles or nerves in order to achieve functional and therapeutic goals. ...

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

DTI of the Visual Pathway - White Matter Tracts and Cerebral Lesions

DTI is a technique that identifies white matter tracts (WMT) non-invasively in healthy and non-healthy patients using diffusion measurements. Similar to visual pathways (VP), WMT are not visible with classical MRI or intra-operatively with microscope. DTI will help neurosurgeons to prevent destruction of the VP while removing lesions adjacent to this WMT. We have performed DTI on fifty patients before and after surgery between March 2012 to January 2014. To navigate we used a 3DT1-weighted sequence. Additionally, we performed a T2-weighted and DTI-sequences. The parameters used were, FOV: 200 x 200 mm, slice thickness: 2 mm, and acquisition matrix: 96 x 96 yielding nearly isotropic voxels of 2 x 2 x 2 mm. Axial MRI was carried out using a 32 gradient direction and one b0-image. We used Echo-Planar-Imaging (EPI) and ASSET parallel imaging with an acceleration factor of 2 and b-value of 800 s/mm&#178;. The scanning time was less than 9 min.
The DTI-data obtained were processed using a FDA approved surgical navigation system program which uses a straightforward fiber-tracking approach known as fiber assignment by continuous tracking (FACT). This is based on the propagation of lines between regions of interest (ROI) which is defined by a physician. A maximum angle of 50, FA start value of 0.10 and ADC stop value of 0.20 mm&#178;/s were the parameters used for tractography.
There are some limitations to this technique. The limited acquisition time frame enforces trade-offs in the image quality. Another important point not to be neglected is the brain shift during surgery. As for the latter intra-operative MRI might be helpful. Furthermore the risk of false positive or false negative tracts needs to be taken into account which might compromise the final results.

Diffusion tensor imaging (DTI) was performed to try to depict the major parts of the visual pathway. The goal was to use an FDA approved standard commercial workstation which could be used for everyday routine in order to try to reduce postoperative damage of the visual pathway in the patients.

DTI is a technique that identifies white matter tracts (WMT) non-invasively in healthy and non-healthy patients using diffusion measurements. Similar to ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for ...

Fat-Covered Islet Transplantation Using Epididymal White Adipose Tissue

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this procedure. However, intraperitoneal islet transplantation has some limitations, including poor transplant efficacy, difficult graft detection ability, and a lack of graftectomy capability for post-transplant analysis. In this paper, "fat-covered islet transplantation", an intraperitoneal islet transplantation method that utilizes epididymal white adipose tissue, is used to assess the therapeutic effects of bioengineered islets. The simplicity of the method lies in the seeding of islets onto epididymal white adipose tissue and using the tissue to cover the islets. While this method can be categorized as an intraperitoneal islet transplantation technique, it shares characteristics with intra-adipose tissue islet transplantation. The fat-covered islet transplantation method demonstrates more robust therapeutic effects than intra-adipose tissue islet transplantation, however, including the improvement of blood glucose and plasma insulin levels and the potential for graft removal. We recommend the adoption of this method for assessing the mechanisms of islet engraftment into white adipose tissue and the therapeutic effects of bioengineered islets.

This fat-covered islet transplantation method is suitable for the detection of engrafted islets in the intraperitoneal cavity. Notably, it does not require the use of biobinding agents or suturing.

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this ...

Measuring brown adipose tissue (BAT) activity by positron emission tomography computed tomography (PET-CT) via the accumulation of 18F-fluorodeoxyglucose (FDG) after a meal or in obese or diabetic patients fails as the method of choice. The main reason is that 18F-FDG competes with the postprandial high glucose plasma concentration for the same glucose transporter on the membrane of BAT cells. In addition, BAT uses fatty acids as a source of energy as well, which is not visible with PET-CT and could be changed along with glucose concentration in obese and diabetic patients. Therefore, to estimate the physiological importance of BAT in animals and humans, a new infrared thermography method used in recent publications is applied.
After overnight fasting, BAT activity was measured by infrared thermography before and after a meal in human volunteers and&#160;female wild-type mice. The camera software calculates the object&#39;s temperature using distance from the object, skin emissivity, reflected room temperature, air temperature, and relative humidity. In mice, the shaved area above the BAT was a region of interest for which average and maximal temperatures were measured. The phase of the estrous cycle in female mice was determined after an experiment by vaginal smears stained with cresyl violet (0.1%) stain solution. In healthy volunteers, two skin areas of the neck were selected: the supraclavicular area (above the collarbone, where BAT cells are present) and the interclavicular area (between the collarbones, where there is no BAT tissue detected). BAT activity is determined by the subtraction of those two values. Also, the average and maximal temperatures of skin areas could be determined in animals and human participants.
Changes in BAT activity after a meal measured by infrared thermography, a non-invasive and more sensitive method, were shown to be sex, age, and phase of the estrous cycle dependent in laboratory animals. As part of diet-induced thermogenesis, BAT activation in humans was also proven to be sex, age, and body mass index dependent. Further determining the pathophysiological changes in BAT activity after a meal will be of great importance for participants with high glucose plasma concentrations (obesity and diabetes mellitus type 2), as well as in different laboratory animals (knock-out mice). This method is also a variable tool for determining possible activating drugs that could rejuvenate BAT activity.

Here, we present a protocol for measuring brown adipose tissue activity after a meal in humans and laboratory animals.

Measuring brown adipose tissue (BAT) activity by positron emission tomography computed tomography (PET-CT) via the accumulation of 18F-fluorodeoxyglucose ...