Name: stromal vascular fraction-enriched fat grafting for the treatment of symptomatic end-neuromata
Uploaded: 2017-11-23T13:00:00.000+00:00
Duration: 7 min 58 s
Description: Watch this Scientific Journal Video about Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata at JoVE.com

The authors have no acknowledgements.

The protocol follows the guidelines of the University Hospital Zurich Human Research Ethics Committee.
1. Diagnoses of Neuromata and Patient Selection
<ol>
	<li>Perform preoperative nerve blocks proximal to the neuroma in order to confirm the diagnoses. Assess the blocks in the middle third of the forearm and exclude potential triggers of the overlapping innervation areas of the SBRN, such as the lateral antebrachial cutaneous nerve (LACN), posterior interosseous nerve (PIN), and a dorsal branch of ulnar nerve10.</li>
	<li>Perform surgery only in patients with considerable pain reduction (at least 5 points on the Visual Analog Scale (VAS) for pain)11.</li>
</ol>
2. Surgical Preparation
<ol>
	<li>Forbid the patient any food or liquid intake 6 h prior to the surgery.</li>
	<li>Place the patient in the middle of the surgical suite on a universal operative table in a beach chair position.</li>
	<li>Place the affected arm/hand on an arm rest.</li>
	<li>Ask the anesthetists to perform endotracheal anesthesia (fentanyl 2 &#181;g/kg, rocuronium 0.6 mg/kg, propofol 150 - 250 mg).</li>
	<li>Disinfect the skin with povidone iodine in the area of liposuction (e.g. inner thigh and/or upper/lower abdomen) and the site of neuroma excision.</li>
	<li>Cover the patient with sterile surgical drapes, leaving exposed the site of liposuction and the surgical site.</li>
</ol>
3. Tumescent Liposuction
<ol>
	<li>Make several small stab (puncture) incisions of approximately 3 - 5 mm diameter with a #11-blade scalpel in the area of liposuction. Incise as far as the subcutaneous layer is reached.</li>
	<li>Inject the tumescent solution containing a dilute lidocaine and epinephrine (500 mL Ringer&#39;s lactate solution, 20 mL of 1% lidocaine, and 1 mg epinephrine) via the 14-gauge infiltrator (infiltrating cannula) into the subcutaneous fat layer (3 mm in depth) in the region of fat harvesting.</li>
	<li>Wait for at least 20 min allowing the tumescent solution to take effect.</li>
	<li>Hook the 3 mm-collecting cannula (outer diameter 3 mm, length 26 cm) to a 60-mL Toomey syringe and start harvesting adipose via the stab incisions. 
	NOTE: The nondominant hand continually controls placement and course of the cannula.</li>
	<li>Pull back the plunger of the Toomey syringe to the 60-mL mark and place a mosquito clamp along the withdrawn plunger in order to maintain negative pressure in the syringe and avoid a continuous vacuum creation by the dominant hand.</li>
	<li>Harvest a minimum of 220 mL of adipose tissue by positioning the Toomey syringes in a vertical position in the provided Toomey syringe stand; this allows the sedimentation of the fat and separation from the fluid.</li>
	<li>Take special care to obtain a good quality fat graft without substantial amount of blood. 
	NOTE: Change the region of liposuction when augmented blood content is noticed.</li>
	<li>Suture the stab incisions with simple stitches and apply pressure as required (e.g. compressive belt).</li>
	<li>Transfer the lipoaspirate into a single-use sterile disposable set of the isolation system.</li>
	<li>Keep one Toomey syringe with lipoaspirate in the provided stand in order to obtain the fat fraction for later lipofilling.</li>
</ol>
4. Adipose Tissue Processing
<ol>
	<li>Close the sterile consumable set. 
	NOTE: The isolation system (see Table of Materials) confirms the completeness by performing a wet test.</li>
	<li>Follow the instructions on the display. 
	NOTE: The process is mostly software controlled and only requires the manual confirmation for the listed steps below by pressing the buttons &#34;next&#34; or &#34;back&#34; on the control panel. The lipoaspirate is transferred into the tissue collection canister. In the tissue collection canister, the lipoaspirate is weighed and automatically cleaned with Ringer&#39;s lactate solution in order to remove the residual blood cells and wetting solution.</li>
	<li>Add more fat if required. 
	NOTE: Depending on the weight of harvested adipose tissue the isolation system asks the user to add more fat or it calculates the amount of additional enzyme reagent to add.</li>
	<li>Add the required amount of enzyme reagent when prompted by the isolation system. 
	NOTE: During the process of enzymatic digestion, separation of lipid and the SVF is achieved. Thereby, the tissue is in oscillating motion. After the enzymatic digestion is completed, the SVF is automatically transferred into the centrifuge chamber for cell concentration. In the centrifuge chamber, the SVF is concentrated to a cell pellet (centrifuge speed is approximately 3,000 x g) . Finally, the cell pellet undergoes a series of washes with Ringer&#39;s lactate solution to reduce residual enzyme levels in the output. When the process is completed, 5 mL of clear fluid (SVF) is obtained.</li>
</ol>
5. Surgical Procedure
<ol>
	<li>To approach the neuroma, make a skin incision over the previously diagnosed neuroma site in the distal third of the forearm.</li>
	<li>Under the usage of surgical binocular loupes magnifier glasses (2.5 - 3.5X), continue to dissect the subcutaneous tissue and identify the involved nerve proximal to the neuroma.
	<ol>
		<li>Subsequently, follow the nerve distally and identify the neuroma (based on visual inspection).</li>
	</ol>
	</li>
	<li>Once the full extension of the neuroma is clear, begin to excise the neuroma by a straight trans-neural scissor cut. If no neuroma is identified, only neurolysis of the nerve is performed.</li>
	<li>Continue with the neurolysis of the remaining nerve stump until 3 cm proximal to the wrist. 
	NOTE: The end of the nerve stump should be located in the middle of the principal incision.</li>
	<li>Arrange two to four small puncture (stab) incisions (1 mm in diameter) with a scalpel through epidermal and dermal cutis around the principal approach.</li>
	<li>Place two to four blunt cannulas (16-gauge, length 9 cm) via the stab incisions perineural to the stump. 
	NOTE: The tip of the cannulas should be directed towards the nerve stump.</li>
	<li>Secure the cannulas in place by adhesive dressing.</li>
	<li>Place the stitches around the cannulas and leave the suture material in situ without securing the node.</li>
	<li>Suture the main incision tightly in order to prevent the outflow of grafting. 
	NOTE: Ensure that cannulas remain in situ.</li>
</ol>
6. Application of the SVF-enriched Fat Graft
NOTE: After approximately 1.5 h of processing, the 5 mL of the processed SVF is ready for further use.
<ol>
	<li>Aspirate 5 mL of SVF in a 10-mL communicating syringe.</li>
	<li>In the same manner, aspirate 2 mL of the sedimented lipid fraction in the opposing 10-mL syringe.</li>
	<li>Mix the 5 mL of SVF with the 2 mL of the sedimented lipid fraction by alternating pulling back the plunger of one syringe and pulling down the plunger of the communicating, opposing syringe until sufficient mixing is obtained.</li>
	<li>Divide the SVF-enriched fat graft equally in two to four 10 mL syringes.</li>
	<li>Distribute the prepared SVF-enriched fat graft one after the other equally around the nerve stump by connecting the syringes to the blunt cannulas. 
	NOTE: Before applying the graft, ensure that the cannulas are secured in the correct position and be careful not to dislocate the cannulas while applying the fat graft.</li>
	<li>Remove the cannulas carefully.</li>
	<li>Then, tape the small puncture incisions with steri-strips.</li>
	<li>Immobilize the hand by applying a splint in the intrinsic plus position (metacarpophalangeal (MCP) joints flexed at 60 -&#160;70&#176;, the interphalangeal (IP) joints fully extended, thumb in the fist projection, wrist in dorsal flexion of 25&#176;).&#160;If only neurolysis of the nerve is performed, no splint is needed and arm and wrist are bandaged for 10 days.</li>
</ol>
7. Postoperative Patient Handling
<ol>
	<li>Transfer the patient to the post-anesthesia recovery unit post-surgery, and allow for sufficient monitoring in the ward.</li>
	<li>Continue to immobilize the wrist for 10 days after the surgery.</li>
	<li>Perform the first change of bandages after 48 h post-surgery and continue at regular intervals (i.e. every second day until discharge).</li>
	<li>Remove the sutures 12 - 14 days postoperatively.</li>
	<li>Perform clinical assessment of pain during follow-up at 2, 6, 12, and 36 months.</li>
</ol>

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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=Multilineage+cells+from+human+adipose+tissue:+implications+for+cell-based+therapies.">Multilineage cells from human adipose tissue: implications for cell-based therapies.</a> Tissue Eng. 7 (2), 211-228 (2001).</li><li>Fraser, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Celution+System:+Automated+Processing+of+Adipose-Derived+Regenerative+Cells+in+a+Functionally+Closed+System.">The Celution System: Automated Processing of Adipose-Derived Regenerative Cells in a Functionally Closed System.</a> Adv Wound Care (New Rochelle). 3 (1), 38-45 (2014).</li><li>Guo, 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=Stromal+vascular+fraction:+A+regenerative+reality?+Part+2:+Mechanisms+of+regenerative+action.">Stromal vascular fraction: A regenerative reality? Part 2: Mechanisms of regenerative action.</a> J Plast Reconstr Aesthet Surg. 69 (2), 180-188 (2015).</li><li>Lutz, B. S., Ma, S. F., Chuang, D. C., Chan, K. H., Wei, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interposition+of+a+pedicle+fat+flap+significantly+improves+specificity+of+reinnervation+and+motor+recovery+after+repair+of+transected+nerves+in+adjacency+in+rats.">Interposition of a pedicle fat flap significantly improves specificity of reinnervation and motor recovery after repair of transected nerves in adjacency in rats.</a> Plast Reconstr Surg. 107 (1), 116-123 (2001).</li><li>Calcagni, 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=The+novel+treatment+of+SVF-enriched+fat+grafting+for+painful+end-neuromas+of+superficial+radial+nerve.">The novel treatment of SVF-enriched fat grafting for painful end-neuromas of superficial radial nerve.</a> Microsurgery. , (2016).</li><li>Hawker, G. A., Mian, S., Kendzerska, T., French, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measures+of+adult+pain:+Visual+Analog+Scale+for+Pain+(VAS+Pain),+Numeric+Rating+Scale+for+Pain+(NRS+Pain),+McGill+Pain+Questionnaire+(MPQ),+Short-Form+McGill+Pain+Questionnaire+(SF-MPQ),+Chronic+Pain+Grade+Scale+(CPGS),+Short+Form-36+Bodily+Pain+Scale+(SF-36+BPS),+and+Measure+of+Intermittent+and+Constant+Osteoarthritis+Pain+(ICOAP).">Measures of adult pain: Visual Analog Scale for Pain (VAS Pain), Numeric Rating Scale for Pain (NRS Pain), McGill Pain Questionnaire (MPQ), Short-Form McGill Pain Questionnaire (SF-MPQ), Chronic Pain Grade Scale (CPGS), Short Form-36 Bodily Pain Scale (SF-36 BPS), and Measure of Intermittent and Constant Osteoarthritis Pain (ICOAP).</a> Arthritis Care Res (Hoboken). 63 Suppl. 63, S240-S252 (2011).</li><li>Stokvis, A., van der Avoort, D. J., van Neck, J. W., Hovius, S. 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U., 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=Stromal+vascular+fraction+transplantation+as+an+alternative+therapy+for+ischemic+heart+failure:+anti-inflammatory+role.">Stromal vascular fraction transplantation as an alternative therapy for ischemic heart failure: anti-inflammatory role.</a> J Cardiothorac Surg. 6, 43 (2011).</li><li>Paik, K. 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=Studies+in+Fat+Grafting:+Part+V.+Cell-Assisted+Lipotransfer+to+Enhance+Fat+Graft+Retention+Is+Dose+Dependent.">Studies in Fat Grafting: Part V. Cell-Assisted Lipotransfer to Enhance Fat Graft Retention Is Dose Dependent.</a> Plast Reconstr Surg. 136 (1), 67-75 (2015).</li></ol>

The authors have nothing to disclose.

The purpose of the present study is to methodically illustrate the technical aspects of SVF-enriched fat grafting as a novel treatment for symptomatic end-neuromata.
Neuromata of the SBRN are associated with a surgical success rate of only 33%12. Vaienti et al. presented a series of favorable results in early follow-up using perineural fat grafting (PFG) without adding the SVF in 8 patients with painful neuromata of the upper limb<sup c...

The management of symptomatic end-neuromata of the SBRN remains challenging with a multitude of existing surgical treatments. Principally, the neuroma is excised and the remaining nerve stump is relocated into neighboring structures such as muscles1,2, bone3, or veins4. This is assumed to prevent the out-sprouting of the regenerating neural fascicles by mimicking the ambience of an end-organ. However, none of these techniques proved superior, as high rates of pain relapse and insufficient pain reduction were often observed.
Recently, fat grafting around the remaining nerve stump yielded promising results and was advocated as a new treatment option for painful end-neuromata of the upper limb5. Besides acting as a mechanical barrier to prevent continuing motion and tap pain, the beneficial aspect of fat grafting is rather attributed to the cellular level. Human adipose tissue contains multiple cell types, including preadipocytes, mast cells, pericytes, endothelial cells, vascular smooth muscle cells, fibroblasts, and adipose derived stem cells (ADSCs) along with hematopoietic stem cells6. This heterogeneous cell population is referred to as Adipose-Derived Regeneration Cells (ADRCs) or SVF, and can be obtained from the lipoaspirate by an isolation system (see the Table Of Materials) and applied enzyme reagent (see Table of Materials)7. The exact mechanism of action is still not fully understood; however, SVF has been associated with improvements in angiogenesis, immunomodulation, differentiation, and secretion of extracellular matrix8.
The concentrated cell population of the SVF is added to the fat graft with the purpose to reduce the resorption rate and to increase the regenerative potential of the fat graft, as recent studies revealed pain relapse after 12 months with fat grafting without added SVF5. Moreover, the biological action also provides beneficial effects on the nerve itself by reducing disorganized axonal sprouting of the stump9 and subsequent scar adherences which might result in nerve entrapment.
The first clinical study to describe SVF-enriched fat grafting as a novel surgical option for the treatment of symptomatic end-neuromata of SBRN has been recently published10. In general, the technique can be applied to any neuroma occurring after accidental or iatrogenic nerve injury. The aim of the present subsequent study is to methodically illustrate the technical aspects of this procedure.

<table><tbody><tr><td>Cytori Celution System</td><td>Cytori</td><td></td><td>isolation system</td></tr><tr><td>Celase</td><td>Cytori</td><td></td><td>enyzme reagent</td></tr><tr><td>liposuction system</td><td></td><td></td><td></td></tr><tr><td>tumescent solution (500 ml Lactated Ringers, 20 ml of 1% lidocaine and 1 mg epinephrine)</td><td></td><td></td><td></td></tr><tr><td>14-gauge infiltrator</td><td></td><td></td><td></td></tr><tr><td>3 mm-collecting cannula (15, 26, 32 cm)</td><td></td><td></td><td></td></tr><tr><td>60 mL Tommey syringes</td><td></td><td></td><td></td></tr><tr><td>mosquito clamp</td><td></td><td></td><td></td></tr><tr><td>Toomey syringe stand</td><td></td><td></td><td></td></tr><tr><td>4 cannulas (fat injecting cannulas 1.5 mm, syringe NO.2, 90 mm)</td><td>Chalybs</td><td></td><td></td></tr><tr><td>10 mL syringes</td><td></td><td></td><td></td></tr><tr><td>connecting device for syringes</td><td></td><td></td><td></td></tr><tr><td>#11- and #15-blade scalpel</td><td></td><td></td><td></td></tr><tr><td>stevens scissors</td><td></td><td></td><td></td></tr><tr><td>pins pincette</td><td></td><td></td><td></td></tr><tr><td>bipolar electrocautery</td><td></td><td></td><td></td></tr><tr><td>suture material</td><td></td><td></td><td></td></tr><tr><td>adhesive dressing</td><td></td><td></td><td></td></tr><tr><td>binocular loupes magnifier glasses (2.5-3.5x)</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Statistical Package for Social Sciences, version 22</td><td>IBM</td><td></td><td></td></tr></tbody></table>

stromal vascular fraction-enriched fat grafting for the treatment of symptomatic end-neuromata

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata of peripheral sensory nerves, and in this study, specifically of the superficial branch of the radial nerve (SBRN). Despite a multitude of existing treatments, persistent postoperative pain and common pain relapse are still very common, independent of the procedure assessed. The neuroma is microsurgically excised accordingly to standardized protocol. Instead of the relocation of the regenerating nerve stump in neighboring anatomical structures, such as muscle or bone, a fat graft is applied perifocally and acts as a mechanical barrier. In order to reduce the fat resorption rate and boost the regenerative potential of the graft, the highly concentrated SVF is integrated in the grafting. The SVF is isolated from subcutaneous fat by enzymatic and mechanic separation of the lipoaspirate by a specific commercial isolation system. The SVF-enriched fat graft provides both a mechanical barrier and various biological effects at the cellular level, including improving angiogenesis, inflammation, and fibrosis. Both mechanical and biologic effects help to reduce the disorganized axonal outgrowth of the nerve stump during nerve regeneration and hence prevent the recurrence of painful end-neuromata.

Data were analyzed using statistical software and presented as means &#177; standard deviations. The Kolmogorov-Smirnov test was applied to verify the nonparametric distribution of the study population and confirmed a negative empirical distribution. Based on the nonparametric nature of the study population, the Wilcoxon&#39;s test for related samples was employed to compare preoperative and postoperative outcomes. Statistical significance was regarded at p-values less than 0.05<s...

Watch this Scientific Journal Video about Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata at JoVE.com

Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata

The purpose of the study is to illustrate the technical procedure of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata. This technique provides advantages of both the mechanical barrier and the biological action at the cellular level by the processed and concentrated SVF.

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a ...

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Medicine

7.6K Views.  University Hospital Zurich. The purpose of the study is to illustrate the technical procedure of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata. This technique provides advantages of both the mechanical barrier and the biological action at the cellular level by the processed and concentrated SVF.

Video: Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Developmental Biology

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression.
This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring.
Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research

Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in VCA.

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

A Patient-Derived Xenograft Model for Venous Malformation

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional veins. The purpose of this article is to carefully describe the establishment of a murine xenograft model that mimics human VM and is able to reflect patient heterogeneity. Hyper-activating non-inherited (somatic) TEK (TIE2) and PIK3CA mutations in endothelial cells (EC) have been identified as the main drivers of pathological vessel enlargement in VM. The following protocol describes the isolation, purification and expansion of patient-derived EC expressing mutant TIE2 and/or PIK3CA. These EC are injected subcutaneously into the back of immunodeficient athymic mice to generate ectatic vascular channels. Lesions generated with TIE2 or PIK3CA-mutant EC are visibly vascularized within 7&#8210;9 days of injection and recapitulate histopathological features of VM patient tissue. This VM xenograft model provides a reliable platform to investigate the cellular and molecular mechanisms driving VM formation and expansion. In addition, this model will be instrumental for translational studies testing the efficacy of novel drug candidates in preventing the abnormal vessel enlargement seen in human VM.

We present a detailed protocol to generate a murine xenograft model of venous malformation. This model is based on the subcutaneous injection of patient-derived endothelial cells containing hyper-activating TIE2 and/or PIK3CA gene mutations. Xenograft lesions closely recapitulate the histopathological features of VM patient tissue.

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional ...

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

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

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 engineering.

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Bioengineering

Transplantation of Schwann Cells Inside PVDF-TrFE Conduits to Bridge Transected Rat Spinal Cord Stumps to Promote Axon Regeneration Across the Gap

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord injury. The complete transection model, although not as clinically relevant as the contusion model, is the most rigorous method to evaluate axon regeneration. In the contusion model, it is difficult to distinguish regenerated from sprouted or spared axons due to the presence of remaining tissue post injury. In the complete transection model, a bridging method is necessary to fill the gap and create continuity from the rostral to the caudal stumps in order to evaluate the effectiveness of the treatments. A reliable bridging surgery is essential to test outcome measures by reducing the variability due to the surgical method. The protocols described here are used to prepare Schwann cells (SCs) and conduits prior to transplantation, complete transection of the spinal cord at thoracic level 8 (T8), insert the conduit, and transplant SCs into the conduit. This approach also uses in situ gelling of an injectable basement membrane matrix with SC transplantation that allows improved axon growth across the rostral and caudal interfaces with the host tissue.

This article describes a technique to insert a hollow conduit between the spinal cord stumps after complete transection and fill with Schwann cells (SCs) and injectable basement membrane matrix in order to bridge and promote axon regeneration across the gap.

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord ...

Mechanical Micronization of Lipoaspirates for Regenerative Therapy

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of cell products using collagenase. Here, we present a protocol to generate an injectable mixture of SVF cells and native extracellular matrix from adipose tissue by a purely mechanical process. Lipoaspirates are put into a centrifuge and spun at 1,200 x g for 3 min. The middle layer is collected and separated into two layers (high-density fat at the bottom and low-density fat on the top). The upper layer is directly emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance under the oil layer is collected and defined as the extracellular matrix (ECM)/SVF-gel. The oil on the top layer is collected. Approximately 5 mL of oil is added to 15 mL of high-density fat and emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance is also ECM/SVF-gel. After the transplantation of the ECM/SVF-gel into nude mice, the graft is harvested and assessed by histologic examination. The result shows that this product has the potential to regenerate into normal adipose tissue. This procedure is a simple, effective mechanical dissociation procedure to condense the SVF cells embedded in their natural supportive ECM for regenerative purposes.

Here, we present a protocol to obtain stromal vascular fraction from adipose tissue through a series of mechanical processes, which include emulsification and multiple centrifugations.

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

Mechanical Stromal Vascular Fraction in Fibrin Hydrogel for Regenerative Medicine

The Combination of Mechanically Isolated Stromal Vascular Fraction and Fibrin Hydrogel: A Processing Protocol

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the past, the extraction of these cells from adipose tissue required a multistep enzyme-based process, resulting in a heterogenous cell mix consisting of ACSs and other cells, which are jointly termed the stromal vascular fraction (SVF). More recently introduced mechanical SVF (mSVF) isolation protocols are less time-consuming and bypass regulatory concerns. We recently proposed a protocol that generates mSVF rich in stromal cells based on a combination of emulsification and centrifugation. One current issue in mSVF application for wound therapy application is the lack of a scaffold providing protection from mechanical manipulation and desiccation. Fibrin hydrogels have been shown to be a useful adjunct in cell transfer for wound healing purposes in the past. In the work herein, we delineate the preparation steps of an mSVF-fibrin hydrogel construct as a novel approach for translational research and clinical application.

Author Spotlight: Exploring the Potential of Fat-Derived Stromal Vascular Fraction for Wound Healing

Mechanically isolated stromal vascular fraction (SVF) in combination with a fibrin hydrogel offers an easy and efficient carrier for viable adipose-derived stromal cells for various indications, including tissue engineering and or wound healing purposes. Here, we present the preparation of a mechanical SVF (mSVF)-fibrin hydrogel construct for translational research and clinical application.

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the ...

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. ...