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, pain, and joint blockage. Moreover, in many cases OLT may be associated with a history of systemic disease, such as inflammatory or vascular disease, neuropathy or neurologic disease, and diabetes. The use of drugs or medical issues that may influence healing must be evaluated and taken into account.</li>
</ol>
2. Clinical Examination
<ol>
	<li>Evaluate the patient in an orthostatic position to highlight ankle or hind-foot deformity. Assess muscle and tendon function and ankle range of movement (ROM). A diffuse tenderness is often present, especially during maximum flexion and extension, and it is not uncommon to encounter a touch-sensitive area at the level of the articular joint.</li>
	<li>Perform an anterior and posterior drawer test to identify a concomitant lateral ankle instability.</li>
	<li>During the preoperative consultation, record the following clinical and functional scores: American Orthopedic Foot &#38; Ankle Society (AOFAS) ankle and hind-foot scores15, Visual analogue scale (VAS) pain score16, and 12-Item Short Form Health Survey (SF-12)17.</li>
</ol>
3. Radiological Assessment
<ol>
	<li>Perform a bilateral weight bearing radiograph of the foot and ankle. This consists of conventional weight-bearing anteroposterior (AP) mortise, and lateral weight-bearing views of the ankle. Perform AP mortise in neutral position, and with 15 degrees of internal rotation for a better visualization of the talus. 
	NOTE: Only 50% of OLT can be diagnosed with radiograph; in case of big lesions, an area of detached bone surrounded by radiolucency can be noted18.</li>
	<li>Perform a conventional computed tomography-scan (CT-scan) of the ankle. A CT-scan allows a precise location and size of the lesion, identifying also bone fragments in case of detachment. The weak point of the CT is the ability to show the status of the cartilage. A previous study showed a sensitivity and specificity of 0.81 and 0.99, respectively, for detecting OLTs on CT19,20.</li>
	<li>Perform magnetic resonance imaging (MRI) of the ankle. MRI is fundamental in cartilage and subchondral bone assessment. Moreover, the MRI does not involve ionizing radiation, and allows a better visualization of soft-tissue. The literature reports a sensitivity and specificity of 96% in detecting OLTs21,22.</li>
</ol>
4. Surgical Technique
<ol>
	<li>Harvesting and processing of the adipose tissue
	<ol>
		<li>Prepare Klein solution: 1 L of 9 g/L saline, 50 mL of 1% lidocaine, 1 mL of 1:1000 epinephrine, 10 mL of 8.4% sodium bicarbonate, and 0.1 mL of 10 mg/mL of triamcinolone acetonide.</li>
		<li>Create two para-umbilical incisions of about 0.5 cm with a scalpel blade. Inject approximately 300 mL of Klein solution into the subcutaneous fat tissue of the abdomen through the incisions using 60 mL syringes with a 18G needle (Figure 1).</li>
		<li>Harvest 40 - 45 mL of ADSCs using a 13G blunt cannula attached to a 20-mL syringe and introduced into the processing kit (Figure 2). Typically, perform the harvesting in the peri-umbilical area.</li>
		<li>Insert 100 - 130 mL of lipoaspirate in the closed system. Push the lipoaspirate into the device through a large filter to obtain a first cluster reduction; at the same time, a corresponding quantity of saline solution exits toward the wasting bag. A key role is played by stainless steel marbles to obtain a temporary emulsion between oil, blood, and saline. Remove oil residues and contaminated blood by a gravity counter-flow of saline solution.</li>
		<li>After this washing step (the flowing solution appears clear and the lipoaspirate yellow), stop the saline flux and reverse the device (gray cap up), leading to the second adipose cluster reduction. Obtain the reduction by pushing the floating adipose clusters through the second cutting hexagonal filter, pushing fluid from below with a 10-mL syringe. Collect the final product into a 10-mL syringe connected to the upper opening of the device. 
		NOTE: The processing kit for fat tissue improves standard lipofilling technique: in fact, the system consists of a closed, full-immersion, low-pressure cylindrical system, in order to obtain a fluid and uniform product containing a great number of pericytes. This procedure allows the processing of adipose cells exclusively via mild mechanical forces, and preserving the integrity of the stromal vascular niche. The process is the least traumatic possible, and makes the final product available in a short time (15 - 20 min), without enzymatic treatments or expansion. Undamaged vasculostromal niches help the healing process.</li>
		<li>Once the ADSCs have been harvested, apply a compression bandage on the abdomen.</li>
	</ol>
	</li>
	<li>Surgical procedure and Adipose Tissue Injection
	<ol>
		<li>Place the patient in a supine position under spinal anesthesia with a tourniquet, at a pressure of 250 mmHg, at the level of the thigh to decrease the bleeding and allow a better arthroscopic visualization1.</li>
		<li>Mark the anatomical landmarks on the skin with a dermographic pen. Landmarks are essential to avoid iatrogenic injuries. 
		Mark the following (Figure 3): 
		both malleoli (lateral (LM) and medial (MM)) 
		the anterior joint line (JL), identified with dorsi- and plantar-flexion of the ankle joint 
		the tibialis anterior tendon (TAT), and Achilles tendon 
		the great saphenous vein, which runs just ahead of the medial malleolus 
		the superficial peroneal nerve (SPN)</li>
		<li>First, perform the anteromedial portal just medial to the tibialis anterior tendon, coinciding with a soft spot. This portal represents the approach portal. In most cases, a depression with the ankle in dorsiflexion is visible and palpable.
		<ol>
			<li>Cut only the skin with a blade, and then perforate the capsule by blunt dissection. Take care to avoid the saphenous nerve and the great saphenous vein. The saphenous vein is located 9 mm laterally to the portal, while the nerve is about 7.4 mm lateral to the portal. This constitutes one of two primary viewing portals.</li>
		</ol>
		</li>
		<li>Check the joint line, place the anterolateral portal, medial to the lateral malleolus, and lateral to the extensor digitorum tendon. 
		NOTE: When performing the anterolateral portal, prevent any injuries to the intermediate dorsal cutaneous nerve (the lateral branch of the superficial peroneal nerve); for this reason, after cutting, the skin must be followed by blunt dissection.</li>
		<li>Inspect the articular cartilage to assess size and position. Evaluate the condition and the quality of the cartilage with the palpator. Osteochondral lesions of the talus are usually located either posteromedially or anterolaterally.
		<ol>
			<li>Perform arthroscopic treatment using wide-angle 2.7-mm arthroscopes with a 30&#176; viewing angle, though some surgeons will use a larger 4 mm arthroscope, and keep the instrument in the anterior recess of the joint. Non-invasive joint distraction techniques and hyper plantar flexion can be used to access most of the talar dome.</li>
			<li>In case of a posterior lesion, percutaneously place a Hintermann spreader to distract the joint and allow exposure of the lesion. The Hintermann spreader has an opening lever arm applied on two K-wires previously positioned in the tibia and talar bone medially or laterally, according to the lesion side. In the case of a lateral lesion, take care to insert the proximal K-wire in the tibial bone, avoiding the fibula, to achieve better distraction of the joint.</li>
		</ol>
		</li>
		<li>Prepare the lesion with a curette, removing the damaged and unstable cartilage, the calcified layer and necrotic and sclerotic bone in order to create a regular-shaped contained lesion with shouldered borders. For this step, use a standard arthroscopic curette (Figure 4).</li>
		<li>Stimulate bone marrow stem cells from subchondral bone performing microperforations roundly from the outside to the inside of the lesion.
		<ol>
			<li>Carry out perforations with intervals of about 3 mm between them. Induce microfractures using a Chondral Pick on the healthy subchondral bone underneath the defect (Figure 5). 
			NOTE: A hemorrhage results in the formation of a fibrin clot. Blood clotting products, via inflammatory cascade activation, release vasoactive mediators, growth factors, and cytokines. These factors have the power to stimulate vascular invasion and migration of mesenchymal stem cells into the chondral portion of the lesion. These pluripotent cells are stimulated to differentiate into fibroblasts, chondrocytes, and osteoblasts, and play an important role in the stimulation of repair of the lesion. Paracrine growth factors in the articular environment promote the formation of extracellular matrix and the production of fibrocartilage. At the same time, cells of the bone part of the lesion produce an immature bone tissue that is progressively replaced by a mature bone.</li>
		</ol>
		</li>
		<li>Remove the intra-articular water using the shaver in aspiration and the remaining liquid using a cotton sponge until the joint is completely dry.</li>
	</ol>
	</li>
	<li>Adipose-derived stem cell injection
	<ol>
		<li>Inject 5 - 7 mL of adipose-derived stem cells, previously prepared in Step 4.1.3, into the ankle joint using one of the two portals (anteromedial or anterolateral).</li>
		<li>Release the tourniquet.</li>
	</ol>
	</li>
</ol>
5. Postoperative Care
<ol>
	<li>Avoid ankle movements for 15 days, with the operated limb in full discharge.</li>
	<li>15 days after surgery, allow active and passive movements of the ankle, until full recovery and range of motion is reached.</li>
	<li>Allow charge 30 days after surgery.</li>
</ol>
6. Clinical and Radiographic Follow-Up
<ol>
	<li>Evaluate patients clinically and radiologically at six and twelve months and, thereafter, annually. The follow-up protocol consists of AOFAS ankle and hind-foot scores, VAS pain score, and SF-12 at each end-point15,16,17. Radiographic examination includes an MRI and CT-scan of the operated ankle.</li>
</ol>

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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+ankle+arthroscopy+for+the+treatment+of+anterolateral+soft-tissue+impingement.">Safety of ankle arthroscopy for the treatment of anterolateral soft-tissue impingement.</a> Arthroscopy. 30 (2), 256-259 (2014).</li><li>Suzangar, M., Rosenfeld, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ankle+arthroscopy:+is+preoperative+marking+of+the+superficial+peroneal+nerve+important?.">Ankle arthroscopy: is preoperative marking of the superficial peroneal nerve important?.</a> J. Foot. Ankle Surg. 51 (2), 179-181 (2012).</li><li>Kraeutler, M. 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=Current+Concepts+Review+Update:+Osteochondral+Lesions+of+the+Talus.">Current Concepts Review Update: Osteochondral Lesions of the Talus.</a> Foot Ankle Int. 38 (3), 331-342 (2017).</li><li>Looze, C. 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=Evaluation+and+Management+of+Osteochondral+Lesions+of+the+Talus.">Evaluation and Management of Osteochondral Lesions of the Talus.</a> Cartilage. 8 (1), 19-30 (2017).</li><li>Dragoo, J. L., 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=Healing+full-thickness+cartilage+defects+using+adipose-derived+stem+cells.">Healing full-thickness cartilage defects using adipose-derived stem cells.</a> Tissue Eng. 13 (7), 1615-1621 (2007).</li><li>Lee, S. Y., Kim, W., Lim, C., Chung, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+Lateral+Epicondylosis+by+Using+Allogeneic+Adipose-Derived+Mesenchymal+Stem+Cells:+A+Pilot+Study.">Treatment of Lateral Epicondylosis by Using Allogeneic Adipose-Derived Mesenchymal Stem Cells: A Pilot Study.</a> Stem Cells. 33 (10), 2995-3005 (2015).</li><li>Feisst, V., Meidinger, S., Locke, 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=From+bench+to+bedside:+use+of+human+adipose-derived+stem+cells.">From bench to bedside: use of human adipose-derived stem cells.</a> Stem Cells Cloning. 8, 149-162 (2015).</li></ol>

Federico Giuseppe Usuelli, MD, reports personal fees from Integra and Geistlich, and grants and personal fees from Zimmer, outside the submitted work.

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.
The most common postoperative complication is contour irregularities, with an incidence of 2.7%. This can be avoided using small cannulas, not performing superficial liposuction, and turning the suction off when exiting incisions. Rarely, skin conditions such as hyperpigmentation, necrosis, and erythema in patients with underlying connective tissue disease can be seen27,28. Seromas are often the result of aggressive liposuction due to the collection of serous fluid in a treated area leading to the formation of a single cavity; this is most common in patients with high BMI29. Infection is a very rare complication (&#60;1%) and this may be because of a combination of sterile technique, small incisions, and the antibacterial effects of lidocaine30.
The literature reports subsequent life-threatening complications after liposuction, such as pulmonary embolism, fat embolism, sepsis, necrotizing fasciitis, and perforation of abdominal organs. The most common cause of death is pulmonary thromboembolism. These complications are due to lack of sterility, poor patient compliance, and permissive post-operative discharge30.
Also during arthroscopy, there are critical steps that can lead to complications: in this protocol, all arthroscopic procedures are performed using an anteromedial and anterolateral portal31. The most frequent complication with this approach is a deficit of the superficial peroneal nerve, reported in 1.04% of patients, despite preoperative marking of the nerve and its branches32,33. Furthermore, the risk increases considering variations of this nerve: anatomical studies have demonstrated that 50% of the population present two branches, and these can reach up to 5 branches with an extremely variable width (1 to 13 mm).
The technique described in the protocol combines the effect of the microfracture in association with mesenchymal stem cells harvested from fat tissue. Microperforations stimulate the development of a reparative tissue: drilling the subchondral bone produces bleeding and leakage of mesenchymal cells, to produce fibrocartilage. The success of microfractures is strictly related to the size of the lesion. If lesions are less than 1.5 cm2, the neo-formed fibrocartilage tissue, although qualitatively different from hyaline cartilage and with lower mechanical properties, can provide adequate reparation, with resolution of the symptoms in a high percentage of cases34,35. ADSCs may form neo-tissue with a hyaline cartilage phenotype, if cultured in association with different growth factors (TGFb, GH, and FGF-2) and placed in a scaffold of fibrin glue36.
The described technique preserves the identity of the pericyte, leaving intact the stromal vascular niche, promoting osteochondral healing in this way. Moreover, ADSCs produce a variety of paracrine bioactive molecules and can activate the physiological healing process. The final product is available in less than 20 min, thanks to the gentle mechanical method. Finally, in accordance with the US Food and Drug Administration, ADSCs are minimally manipulated.
The use of MSCs is constantly increasing, and future research should focus on the use of allogeneic ADSCs as described by Lee37. The advantages of an allotransplant would be numerous. First of all, handling and standardization of the product would be easier. Liposuction and processing steps could be eliminated, and a healthy donor could be pre-selected, according to his cytokine and cell marker expression profile, improving the effect of MSCs38.

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&amp;deg; TIP / 60&amp;deg; TIP</td><td>ARTHREX</td><td>AR-8655-05&lt;br/&gt; 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 increased. In 2013, Kim et al.23 treated 65 elderly patients, older than 50 years for symptomatic OLT dividing for the type of treatment: 
&#8211;&#160;&#160;&#160;&#160;&#160;isolated marrow stimulation 
&#8211;&#160;&#160;&#160;&#160;&#160;marrow stimulation in association with ADSCs
At final follow-up, patients with a combined treatment showed significant clinical improvement compared to the isolated marrow stimulation treatment. A subsequent study, conducted by the same group24, for the treatment of OLT, confirmed how the combined treatment of SVF and marrow stimulation was superior to isolated microfracture.
In 2016, Kim et al.25 assessed the outcomes in 49 patients treated with marrow stimulation and lateral sliding calcaneal osteotomy. Twenty-six patients also underwent MSC injection. One year after surgery, patients treated with MSC reported a higher ICRS score and clinical outcomes. Recently, Kim et al.26 noted that patients treated with MSC injection after supramalleolar osteotomy and marrow stimulation reported higher clinical and radiological outcomes, compared to patients treated without MSC.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56395/56395fig1.jpg" /> 
Figure 1: Saline solution containing adrenaline and lidocaine is injected at the level of the abdomen using a 20 g cannula. For liposuction, use a 13 g cannula connected to a syringe. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56395/56395fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56395/56395fig2.jpg" /> 
Figure 2: The processing kit for fat tissue consists of a single-use kit for the lipoaspiration and purification of adipose tissue. All procedures are performed with the ADSCs immersed in a saline solution, avoiding any trauma and maintaining intact the vasculostromal niches containing mesenchymal stem cells and pericytes. The unit consists of a transparent plastic cylinder with filters and beads for the microfracturing of adipose tissue. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56395/56395fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56395/56395fig3.jpg" /> 
Figure 3: Outside views of ankle during surgical planning. With the patient in the supine position, it is useful to identify all the landmarks on the joint needed for the surgical procedure. 
AM = anteromedial portal 
AL = anterolateral portal 
MM = medial malleolus 
LM = lateral malleolus 
TAT = tibalis anterior tendon 
JL = joint line 
SPN = superficial peroneal nerve <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56395/56395fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56395/56395fig4.jpg" /> 
Figure 4: Arthroscopic view. The lesion is prepared using a curette removing the damaged cartilage and the necrotic and sclerotic bone <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56395/56395fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56395/56395fig5.jpg" /> 
Figure 5: Arthroscopic view. Microfractures, performed at the level of the osteochondral lesion, stimulate bleeding and leakage of mesenchymal stem cells from the subchondral bone. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56395/56395fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="3">Patients</td>
			<td colspan="4">Pre-operative</td>
			<td colspan="2">6 months after surgery</td>
		</tr>
		<tr>
			<td rowspan="2">AOFAS</td>
			<td rowspan="2">VAS</td>
			<td colspan="2">SF-12</td>
			<td rowspan="2">AOFAS</td>
			<td rowspan="2">VAS</td>
		</tr>
		<tr>
			<td>PCS</td>
			<td>MCS</td>
		</tr>
		<tr>
			<td>1</td>
			<td>44</td>
			<td>8</td>
			<td>31.1</td>
			<td>32.4</td>
			<td>88</td>
			<td>3</td>
		</tr>
		<tr>
			<td>2</td>
			<td>32</td>
			<td>7</td>
			<td>27.5</td>
			<td>42.1</td>
			<td>78</td>
			<td>2</td>
		</tr>
		<tr>
			<td>3</td>
			<td>52</td>
			<td>9</td>
			<td>40.1</td>
			<td>28.7</td>
			<td>87</td>
			<td>2</td>
		</tr>
		<tr>
			<td>4</td>
			<td>59</td>
			<td>8</td>
			<td>36.6</td>
			<td>41</td>
			<td>82</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Mean</td>
			<td>46.75</td>
			<td>8</td>
			<td>33.83</td>
			<td>36.05</td>
			<td>83.75</td>
			<td>2.25</td>
		</tr>
	</tbody>
</table>
Table 1: Clinical results at 6 months of follow-up. AOFAS: The American Orthopaedic Foot and Ankle Society Score; VAS: Visual Analogue Scale for Pain; SF-12: 12-Item Short Form Survey; MCS: Mental Component Score; PCS: Physical Component Score.

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Medicine

10.3K Views.  IRCCS Istituto Ortopedico Galeazzi - C.A.S.C.O. Piede e Caviglia. 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.

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

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

Treatment of Ankle Osteoarthritis with Total Ankle Replacement Through a Lateral Transfibular Approach

Total ankle replacement (TAR) is a valid option for the treatment of ankle osteoarthritis. The traditional surgical approach for TAR is the anterior approach. Recently, the lateral transfibular approach to the ankle has gained popularity since a new TAR implant was designed to be performed via this approach that results in an ideal visualization of the center of rotation of the ankle and curved resections that allow for sparing bone cuts. The aim of the present paper is to present our preoperative, operative, and postoperative protocols for the treatment of the osteoarthritis of the ankle with TAR via lateral approach. We present our preoperative clinical and radiographic protocol. In addition, we describe our surgical technique with some technical tips. Finally, we report our follow-up schedule that includes the collection of clinical, functional, and radiographic data. The results of this procedure are encouraging: TAR through a lateral transfibular approach provides reliable pain relief and improvements in functional outcomes in patients with ankle osteoarthritis.

We present our preoperative, operative, and postoperative protocols for the treatment of osteoarthritis of the ankle with total ankle replacement via lateral transfibular approach.

Total ankle replacement (TAR) is a valid option for the treatment of ankle osteoarthritis. The traditional surgical approach for TAR is the anterior ...

Behavior

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

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated cartilage lesions lead to ongoing pain, negatively affect the quality of life and predispose for osteoarthritis. During the last decades, several surgical techniques have been developed to treat such lesions. However, until now it was not possible to achieve a full repair in terms of covering the defect with hyaline articular cartilage or of providing satisfactory long-term recovery 2-4. Therefore, articular cartilage injuries remain a prime target for regenerative techniques such as Tissue Engineering. In contrast to other surgical techniques, which often lead to the formation of fibrous or fibrocartilaginous tissue, Tissue Engineering aims at fully restoring the complex structure and properties of the original articular cartilage by using the chondrogenic potential of transplanted cells. Recent developments opened up promising possibilities for regenerative cartilage therapies.
The first cell based approach for the treatment of full-thickness cartilage or osteochondral lesions was performed in 1994 by Lars Peterson and Mats Brittberg who pioneered clinical autologous chondrocyte implantation (ACI) 5. Today, the technique is clinically well-established for the treatment of large hyaline cartilage defects of the knee, maintaining good clinical results even 10 to 20 years after implantation 6. In recent years, the implantation of autologous chondrocytes underwent a rapid progression. The use of an artificial three-dimensional collagen-matrix on which cells are subsequently replanted became more and more popular 7-9.
MACT comprises of two surgical procedures: First, in order to collect chondrocytes, a cartilage biopsy needs to be performed from a non weight-bearing cartilage area of the knee joint. Then, chondrocytes are being extracted, purified and expanded to a sufficient cell number in vitro. Chondrocytes are then seeded onto a three-dimensional matrix and can subsequently be re-implanted. When preparing a tissue-engineered implant, proliferation rate and differentiation capacity are crucial for a successful tissue regeneration 10. The use of a three-dimensional matrix as a cell carrier is thought to support these cellular characteristics 11.
The following protocol will summarize and demonstrate a technique for the isolation of chondrocytes from cartilage biopsies, their proliferation in vitro and their seeding onto a 3D-matrix (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Switzerland). Finally, the implantation of the cell-matrix-constructs into artificially created chondral defects of a rabbit's knee joint will be described. This technique can be used as an experimental setting for further experiments of cartilage repair.

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 method.

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated ...

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...

Bioengineering

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

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

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.

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the ...

Multimodal Analysis of Bone Formation Using Explant Models

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous modalities that are currently available for analysis. Here, we propose a workflow for a comprehensive evaluation of new bone formation using a relevant large animal osseous ex vivo explant. A bone defect (diameter = 3.75 mm; depth = 5.0 mm) is created in an explanted sheep femoral head and injected with a macroporous bone substitute loaded with a pro-osteogenic growth factor (bone morphogenetic protein 2 - BMP2). Subsequently, the explant is maintained in culture for a 28-day period, allowing cellular colonization and subsequent bone formation. To evaluate the quality and structure of newly mineralized tissue, the following successive methods are set up: (i) Characterization and high-resolution 3D images of the entire explant using micro-CT, followed by deep learning image analyses to enhance the discrimination of mineralized tissues; (ii) Nano-indentation to determine the mechanical properties of the newly formed tissue; (iii) Histological examinations, such as Hematoxylin/Eosin/Saffron (HES), Goldner's trichrome, and Movat's pentachrome to provide a qualitative assessment of mineralized tissue, particularly with regard to the visualization of the osteoid barrier and the presence of bone cells; (iv) Back-scattering scanning electron microscopy (SEM) mapping with internal reference to quantify the degree of mineralization and provide detailed insights into surface morphology, mineral composition, and bone-biomaterial interface; (v) Raman spectroscopy to characterize the molecular composition of the mineralized matrix and to provide insights into the persistence of BMP2 within the cement through the detection of peptide bonds. This multimodal analysis will provide an effective assessment of newly formed bone and comprehensive qualitative and quantitative insights into mineralized tissues. Through the standardization of these protocols, we aim to facilitate interstudy comparisons and improve the validity and reliability of research findings.

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 investigations.

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous ...

Destabilization of the Medial Meniscus and Cartilage Scratch Murine Model of Accelerated Osteoarthritis

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are used to mimic many aspects of the disease. The present protocol describes the destabilization and cartilage scratch model (DCS) of post-traumatic osteoarthritis. Based on the widely used destabilization of the medial meniscus (DMM) model, DCS introduces three scratches on the cartilage surface. The current article highlights the steps to destabilize the knee by transecting the medial meniscotibial ligament followed by three intentional superficial scratches on the articular cartilage. The possible analysis methods by dynamic weight-bearing, microcomputed tomography, and histology are also demonstrated. While the DCS model is not recommended for studies that focus on the effect of osteoarthritis on the cartilage, it enables the study of osteoarthritis development in a shorter time window, with special focus on (1) osteophyte formation, (2) osteoarthritic and injury pain, and (3) the effect of cartilage damage in the whole joint.

The present protocol describes the controlled microblade scratches on the surface of the articular cartilage after destabilizing the mouse knee by cutting the medial miniscotibial ligament. This animal model presents an accelerated form of osteoarthritis (OA) suitable for studying osteophyte formation, osteosclerosis, and early-stage pain.

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are ...

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

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. Accordingly, techniques for harvesting and processing human adipose tissue have evolved in order to quickly and efficiently obtain large amounts of tissue. Among these, the closed system technology represents an innovative and easy-to-use system to harvest, process, and re-inject refined fat tissue in a short time and in the same intervention (intra-operatively). Adipose tissue is collected by liposuction, washed, emulsified, rinsed and minced mechanically into cell clusters of 0.3 to 0.8 mm. Autologous transplantation of mechanically fragmented adipose tissue has shown remarkable efficacy in different therapeutic indications such as aesthetic medicine and surgery, orthopedic and general surgery. Characterization of micro-fragmented adipose tissue revealed the presence of intact small vessels within the adipocyte clusters; hence, the perivascular niche is left unperturbed. These clusters are enriched in perivascular cells (i.e., mesenchymal stem cell (MSC) ancestors) and in vitro analysis showed an increased release of growth factors and cytokines involved in tissue repair and regeneration, compared to enzymatically derived MSCs. This suggests that the superior therapeutic potential of microfragmented adipose tissue is explained by a higher frequency of presumptive MSCs and enhanced secretory activity. Whether these added pericytes directly contribute to higher growth factor and cytokine production is not known. This clinically approved procedure allows the transplantation of presumptive MSCs without the need for expansion and/or enzymatic treatment, thus bypassing the requirements of GMP guidelines, and reducing the costs for cell-based therapies.

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 characterization.

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. ...

Biology

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...