Name: visualizing scar development using scad assay - an ex-situ skin scarring assay
Uploaded: 2022-04-28T13:40:00.000+00:00
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Description: Watch this Scientific Journal Video about Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay at JoVE.com

We acknowledge all the co-authors of Jiang et al. 2020 for contributing to the development of SCAD methodology13. We thank Dr. Steffen Dietzel and the Bioimaging core facility at the Ludwig-Maximilans-Universit&#228;t for access to the Multiphoton system. Y.R. was supported by the Else-Kr&#246;ner-Fresenius-Stiftung (2016_A21), the European Research Council Consolidator Grant (ERC-CoG 819933), and the LEO Foundation (LF-OC-21-000835).

The model presented below provides a detailed step-by-step description of the generation of SCAD assay as briefly described in Jiang et al., 202013. SCAD sample preparations were performed after sacrificing the animals as per the international and the Government of Upper Bavaria guidelines. Animals were housed at the animal facility of the Helmholtz Centre Munich. The rooms were maintained with optimal humidity and constant temperature with a 12 h light cycle. Animals were supplied with food and water ad libitum.
1. SCAD tissue preparation
<ol>
	<li>Sacrifice newborn pups onpostnatal day 0 or day 1 (P0 or P1).</li>
	<li>Carefully excise at least 1.5 cm x 1.5 cm full-thickness dorsal back skin until the skeletal muscle layer using a sterile surgical scalpel.</li>
	<li>Peel the skin using sterile curved forceps, ensuring that the superficial fascia is intact with the underlying panniculus carnosus muscle.</li>
	<li>Wash the excised tissue with 50-100 mL of cold DMEM/F-12 media to remove contaminating blood.</li>
	<li>Wash once with Hanks&#39; Balanced Salt Solution (HBSS) to maintain tissue and cell viability.</li>
	<li>Place the skin upside down (superficial fascia on top) in a 10 cm Petri dish containing DMEM/F12 media.</li>
	<li>Using a disposable 2 mm biopsy punch, excise full-thickness round skin pieces, ensuring that superficial fascia is intact with underlying panniculus carnosus muscle until the epidermis to generate SCAD tissues.</li>
	<li>Prepare and fill 200 &#181;L of DMEM/F12 (without phenol red) complete media-DMEM/F12 media supplemented with 10% FBS, 1x GlutaMAX, 1x MEM non-essential amino acids, and 1x Penicillin/streptomycin into individual wells of a 96-well plate.</li>
	<li>Using sterile forceps, carefully transfer and fully submerge individual SCAD tissue upside down (fascia facing up) to the wells of a 96-well plate.</li>
	<li>Transfer the plate to a cell culture incubator maintained under standard conditions (37 &#176;C, 21%(v/v) oxygen, 5% (v/v) C02, and 95% humidity).</li>
</ol>
2. SCAD - Tissue culture
<ol>
	<li>Culture SCADs in an incubator for up to 5 days.</li>
	<li>On Day 2 and Day 4 of culture, replace the media with 200&#956;l&#160;of fresh pre-warmed DMEM/F12 complete media to ensure continued cell and tissue viability conditions. Replace treatment compounds during each media change. 
	NOTE: Ensure to leave 10 &#181;L of media in the well to avoid tissues sticking to the wells.</li>
	<li>Prepare SCADs for the following experiments: Live imaging (section 3), and Tissue Harvesting and 2D/3D immunofluorescence staining (section 4).</li>
</ol>
3. Live imaging of SCADs
<ol>
	<li>Prepare a minimum of 30 mL of 2%-3% (w/v) low melting agarose solution in PBS in a glass bottle by heating it in a microwave until boiling.</li>
	<li>Immediately transfer the bottle and cool the liquid agarose solution in a 40 &#176;C water bath.</li>
	<li>Transfer the SCAD tissue with fascia/Scar facing upward onto the center of a 35 mm dish.</li>
	<li>Embed the SCAD at room temperature (RT) by slowly transferring 40 &#176;C liquid agarose onto the 35 mm dish using a Pasteur pipette. Agarose polymerizes within 2 min.</li>
	<li>Add 2 mL of pre-warmed DMEM/F12 complete media (without phenol red indicator).</li>
	<li>Acquire time-lapse Images of day 0 SCADs up to 48 h using a confocal or a multiphoton microscope equipped with a suitable incubation system&#160;set to 37 &#176;C, 21% (v/v) oxygen, 5% (v/v) C02, and 95% humidity.</li>
</ol>
4. Tissue harvesting and 2D/3D immunofluorescence staining
<ol>
	<li>Wash the tissues at relevant time points by replacing the media with sterile PBS.</li>
	<li>Using sterile forceps, carefully transfer individual SCADs to 1.5 mL microcentrifuge tubes containing 500 &#181;L of 2% paraformaldehyde to fix the tissues overnight at 4 &#176;C.</li>
	<li>Wash the tissues three times with PBS and proceed with 2D/3D immunofluorescence staining.</li>
	<li>3D immunofluorescence staining
	<ol>
		<li>Permeabilize the tissues by incubating in a 1.5 mL microcentrifuge tube containing 500 &#181;L of PBS supplemented with 0.2% gelatin, 0.5% Triton-X100, and 0.01% Thimerosal (PBSGT)&#160;at RT for 24 h.</li>
		<li>Prepare an adequate amount of primary antibodies as per the manufacturer&#180;s instructions and incubate SCAD tissues in 150 &#181;L of PBSGT solution for 24 h at RT. 
		NOTE: If the optimal antibody concentration for immune fluorescence is not reported by the manufacturer, prior antibody titration needs to be performed on control tissues using varying concentrations (e.g., 1:50, 1:200, 1:500, 1:1000).</li>
		<li>Gently wash the SCADs three times with PBS to remove the unbound primary antibody.</li>
		<li>Incubate tissue with 1:1000 relevant Alexa Fluor secondary antibodies in PBS overnight at RT.</li>
		<li>Gently wash the tissue for 30 min in PBS three times to remove excess unbound secondary antibodies.</li>
		<li>Transfer the tissue onto a 35 mm glass-bottom dish for confocal or multiphoton imaging. 
		NOTE: When using water immersion objectives, embed the SCADs in 2 % low melting point agarose to immobilize the tissue to prevent drifting during image acquisition</li>
	</ol>
	</li>
	<li>2D immunofluorescence staining
	<ol>
		<li>Transfer the tissue to a cryo-mold and gently fill with optimal cutting temperature (OCT) compound to completely immerse the tissue.</li>
		<li>Gently adjust the orientation of the tissue, ensuring the absence of air bubbles to obtain a cross-section or a vertical section.</li>
		<li>Place the mold on dry ice for 20-30 min and incubate the block at -80 &#176;C overnight.</li>
		<li>Set the blade temperature to -25 &#176;C and specimen block temperature to -17 &#176;C. Prepare 6 &#181;m cryo-section using a cryostat, transfer the sections onto an adhesion slide, and store the slides in -20 &#176;C freezer.</li>
		<li>Rinse the slides three times in PBS and incubate&#160;the slides in 5% Bovine Serum Albumin (BSA) w/v in PBST (PBS supplemented with 0.05% Tween) for 1 h.</li>
		<li>Add an appropriate amount of primary antibody to 150&#956;l PGST, and incubate overnight at 4 &#176;C 
		NOTE: If the optimal antibody concentration for immune fluorescence is not reported by the manufacturer, prior antibody titration needs to be performed on control sections using varying concentrations (e.g., 1:50, 1:200, 1:500, 1:1000).</li>
		<li>Gently wash the sections three times with PBS to remove the unbound primary antibody.</li>
		<li>Incubate tissue with 1:1000 relevant Alexa Fluor secondary antibodies in PBS overnight at RT for 2 h.</li>
		<li>Gently wash the tissue for 5 min in PBS three times to remove excess unbound secondary antibodies.</li>
		<li>Mount the slides with a mounting medium containing DAPI and dry the slides in the dark at RT.</li>
		<li>Image the sections using a fluorescence microscope.</li>
	</ol>
	</li>
</ol>

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All authors declare no competing interests.

Several models have already been developed to understand scar formation after injury. While a lot of advances have been rendered in this regard but, actual mechanisms are still not clear. In contrast to the previous technique, the SCAD model incorporates all cell types and cutaneous layers, thereby maintaining the complexity of native skin18,19. This methodology is capable of generating fundamental datasets that are important in understanding molecular mechanisms...

Wound healing is a process of restoration of breached wounds. Tissue injuries in invertebrates result in partial or complete regeneration. In contrast, mammals respond to deep injury by scarring, a process tailored to quickly seal wounds with dense plugs of matrix fibers that minimize the breached area and at the same time permanently deform the injured site1,2,3. Large skin burns or deep open wounds in mammals result in pathological phenotypes such as hypertrophic or keloid scars4,5. These exuberant scars cause a tremendous burden on clinical and global healthcare systems. In the US alone, scar management costs about $10 billion annually6,7. Therefore, the development of relevant methodologies are required to better understand the fundemental processes and mechanisms involved in scar formation.
In recent years, a wide range of studies in mice has revealed heterogeneous fibroblast populations with distinct functional potencies based on their origins in certain skin locations8,9,10. In back skin, Rinkevich et al., 2015, identified that a specific fibroblast population with an early embryonic expression of Engrailed-1 (En1), termed EPF (Engrailed positive fibroblast) contributes to cutaneous scarring upon wounding. Conversely,&#160; another fibroblast lineage with no history of engrailed expression, Engrailed negative fibroblast (ENF), does not contribute to scar formation8. Fate mapping of these En1 lineages using Cre-driven transgenic mouse lines crossed to fluorescence reporter mouse lines such as R26mTmG (En1Cre x R26mTmG) allows visualization of EPF and ENF populations.
Studying fibroblast migration in vivo over several days is limited by ethical and technical constraints. Furthermore, compound, viral and neutralizing antibody library screens to modulate pathways involved in scarring is technically challenging. Previously used in vitro or ex vivo models lack the ability to visualize fibroblast migration and scar formation in genuine skin microenvironments, uniformity in scar development, as well as tissue complexity that emulates in vivo skin environments11,12. To overcome the above limitations, we developed an ex vivo scarring assay termed SCAD (SCar-like tissue in A Dish)13,14. This simple assay can be performed by excising 2 mm full-thickness skin containing the epidermis, the dermis, and the subcutaneous fascia regions and culturing them in serum-supplemented DMSO media for up to 5 days. Scars generated from SCAD reliably replicate transcriptomic and proteomic hallmarks of in vivo scars. In addition, SCADs generated from relevant transgenic mouse lines (e.g., En1 mice) crossed with fluorescent reporter mouse lines allow the visualization of fibroblast migration dynamics and scar development at an unprecedented resolution. Furthermore, this model can be easily adapted for any high throughput applications (e.g., compound library, antibody library, or viral screening)13,14. In this article, we describe an optimized protocol to generate SCADs and subsequent downstream processing applications to study cellular and matrix dynamics in scar development.

<table><tbody><tr><td>10% Tween 20, Nonionic Detergent</td><td>Biorad Laboratories</td><td>1610781</td><td></td></tr><tr><td>Bovine serum albumin, Cold ethanol fract</td><td>Sigma</td><td>A4503-50G</td><td></td></tr><tr><td>DMEM/F-12, HEPES, no phenol red-500 mL</td><td>LIFE Technologies</td><td>11039021</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Gibco</td><td>14190169</td><td></td></tr><tr><td>Epredia Cryostar NX70 Cryostat</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Epredia SuperFrost Plus Adhesion slides</td><td>Fisher scientific</td><td>J1800AMNZ</td><td>Adhesion slides</td></tr><tr><td>Fetal Bovine Serum, qualified, heat inactivated, E.U.-approved, South America Origin-500 mL</td><td>LIFE Technologies</td><td>&amp;nbsp;10500064</td><td></td></tr><tr><td>Fluoromount-G with DAPI</td><td>Life Technologies</td><td>00 4959 52</td><td>Mounting medium with DAPI</td></tr><tr><td>Forceps curved with fine points with guidepinstainless steel(tweezers)125 mm length</td><td>Fisher Scientific</td><td>12381369</td><td></td></tr><tr><td>Gelatin from porcine skin</td><td>Sigma</td><td>G2500-100G</td><td></td></tr><tr><td>GlutaMAX Supplement-100 mL</td><td>LIFE Technologies</td><td>35050038</td><td></td></tr><tr><td>HBSS, calcium, magnesium, no phenol red-500 mL</td><td>LIFE Technologies</td><td>14025092</td><td></td></tr><tr><td>Ibidi Gas incubation system for CO&lt;sub&gt;2&lt;/sub&gt; and O&lt;sub&gt;2&lt;/sub&gt;</td><td>Ibidi</td><td>11922</td><td></td></tr><tr><td>Ibidi Heating system</td><td>Ibidi</td><td>10915</td><td></td></tr><tr><td>Leica SP8 upright microscope - Multiphoton excitation 680&amp;ndash;1300 nm</td><td>Leica</td><td></td><td>Equipped with a 25x water-dipping objective (HC IRAPO L 25x/1.00&amp;thinsp;W) in combination with a tunable laser (Spectra-Physics, InSight DS&amp;thinsp;+&amp;thinsp;Single)</td></tr><tr><td>Non Essential Amino Acids</td><td>LIFE Technologies</td><td>11140035</td><td></td></tr><tr><td>NuSieve GTG Agarose ,25 g</td><td>Biozym /Lonza</td><td>859081</td><td></td></tr><tr><td>OCT Embedding Matrix</td><td>Carlroth</td><td>6478.1</td><td></td></tr><tr><td>Paraformaldehyde, 16% W/V AQ. 10 x10 mL</td><td>VWR International</td><td>43368.9M</td><td></td></tr><tr><td>Pen-Strep</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Stiefel Biopsy-Punch 2 mm</td><td>Stiefel</td><td>270130</td><td></td></tr><tr><td>Straight Sharp/Sharp Dissecting Scissors 11.4 cm</td><td>Fisher Scientific</td><td>15654444</td><td></td></tr><tr><td>Thimerosal Bioxtra, 97%&amp;ndash;101%</td><td>Sigma-Aldrich</td><td>T8784-1G</td><td></td></tr><tr><td>Zeiss Axioimager M2 upright microscope</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

visualizing scar development using scad assay - an ex-situ skin scarring assay

The mammalian global response to sealing deep tissue wounds is through scar formation and tissue contraction, mediated by specialized fascia fibroblasts. Despite the clinical significance of scar formation and impaired wound healing, our understanding of fascia fibroblast dynamics in wound healing is cursory due to the lack of relevant assays that enable direct visualization of fibroblast choreography and dynamics in complex environments such as in skin wounds. This paper presents a protocol to generate ex- situ skin scars using SCAD or &#34;SCar-like tissue in A Dish&#34; that emulate the complex environment of skin wounds. In this assay, 2 mm full-thickness skin is excised and cultured upside down in media for 5 days, during which scars and skin contractures develop uniformly. This methodology, coupled with fibroblast-lineage specific transgenic mouse models, enables visualization of individual fibroblast lineages across the entire wound repair process. Overall, this protocol aids&#160;researchers in understanding fundamental processes and mechanisms of wound repair,&#160;directly exploring the effects of modulators on wound healing outcomes.

Generation of SCADs can be separated into three essential steps: Harvesting&#160;back skin from P0-P1 mice, generating of full-thickness biopsy punches, and subsequent culture of individual scads up to 5 days in 96-well plates. As a readout, this assay can further be applied to analyze the spatial and temporal aspects of scarring. The spatial analysis utilizes 2D and 3D immune-labeling of tissues to study spatial localization of cellular and matrix components within developing scar tissue. Spatiotemporal studies allow vi...

Watch this Scientific Journal Video about Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay at JoVE.com

Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay

This protocol describes the generation of a skin-fascia explant termed "SCar like tissue in A Dish" or SCAD. This model allows unprecedented visualization of single fibroblasts during scar formation.

Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay

The mammalian global response to sealing deep tissue wounds is through scar formation and tissue contraction, mediated by specialized fascia fibroblasts. ...

visualizing-scar-development-using-scad-assay-an-ex-situ-skin

Research

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Biology

2.8K Views.  Helmholtz Zentrum München. Eerder gebruikte in vitro of ex vivo modellen missen dermale celcomponenten en complexiteit van huidweefsel. Deze ex situ SCAD-test overwint een bepaalde beperking en maakt het mogelijk om de ontwikkeling van littekens buiten het gewonde dier te bestuderen. De SCAD-test maakt visualisatie van fibroblastmigratie en littekenvorming in micro-omgevingen van de huid mogelijk.Het stelt screeningsbibliotheken van activatoren of remmers in staat om de mechanistische basis van littekenvorming t...

Video: Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay

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. ...

Medicine

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into changes in clinical standards used for treating wounds and decreasing morbidity and mortality for patients. Wound healing is a complex process that requires strategic cell and tissue interaction and function. One of the many critically important functions of wound healing is individual and collective cellular migration. Upon injury, various cells from the blood, surrounding connective, and epithelial tissues rapidly migrate to the wound site by way of chemical and/or physical stimuli. This migration response can largely dictate the outcomes and success of a healing wound. Understanding this specific cellular function is important for translational medicine that can lead to improved wound healing outcomes. Here, we describe a protocol used to better understand cellular migration as it pertains to wound healing, and how changes to the cellular environment can significantly alter this process. In this example study, dermal fibroblasts were grown in media supplemented with fetal bovine serum (FBS) as monolayer cultures in tissue culture flasks. Cells were aseptically transferred into tissue culture treated 12-well plates and grown to 100% confluence. Upon reaching confluence, the cells in the monolayer were vertically scratched using a p200 pipet tip. Arsenic diluted in culture media supplemented with FBS was added to individual wells at environmentally relevant doses ranging 0.1-10 &#956;M. Images were captured every 4 hours (h) over a 24 h period using an inverted light microscope to observe cellular migration (wound closure). Images were individually analyzed using image analysis software, and percent wound closure was calculated. Results demonstrate that arsenic slows down wound healing. This technique provides a rapid and inexpensive first screen for evaluation of the effects of contaminants on wound healing.

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

This study focuses on an in vitro model of wound healing (scratch assay) as a mechanism for determining how environmental contaminants such as arsenic influence cellular migration. The results demonstrate that this in vitro assay provides rapid and early indications of changes to cellular migration prior to in vivo experimentation.

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into ...

Environment

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Bioengineering

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol applies a macromolecular crowding (MMC) technique to mimic this physiological crowding through the addition of neutral polymers (crowders) to cell cultures in vitro. Previous studies using Ficoll or dextran as crowders demonstrate that the expression of collagen I and fibronectin in WI38 and WS-1 cell lines are significantly enhanced using the MMC technique. However, this technique has not been validated in primary hypertrophic scar-derived human skin fibroblasts (hHSFs). As hypertrophic scarring arises from the excessive deposition of collagen, this protocol aims to construct a collagen-rich in vitro hypertrophic scar model by applying the MMC technique with hHSFs. This optimized MMC model has been shown to possess more similarities with in vivo scar tissue compared to traditional 2-dimensional (2-D) cell culture systems. In addition, it is cost-effective, time-efficient, and ethically desirable compared to animal models. Therefore, the optimized model reported here offers an advanced &#8220;in vivo-like&#8221; model for hypertrophic scar-related studies.

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.

Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment.

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, ...

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

A Mouse Model of Mechanotransduction-driven, Human-like Hypertrophic Scarring

Hypertrophic scarring (HTS) is an abnormal process of wound healing that results in excessive scar tissue formation. Over the past decade, we have demonstrated that mechanotransduction&#8212;the conversion of mechanical stimuli into cellular responses&#8212;drives excessive fibrotic scar healing. A mouse model to assess human-like hypertrophic scarring would be an essential tool for examining various therapeutics and their ability to reduce scarring and improve healing. Specifically, our laboratory has developed a murine wound model that increases mechanical strain to promote human-like HTS. This&#160;protocol utilizes biomechanical loading devices, made from modified 13 mm palatal expanders, whose arms are placed on either side of the incision and distracted incrementally apart in order to apply continuous tension across the wound bed during healing. Over nearly two decades of use, this model has been significantly advanced to improve efficacy and reproducibility. Using the murine HTS model, significant dermal fibrotic scars can be induced to be histologically comparable to human hypertrophic scars. This murine model provides an environment to develop biologics involved in the treatment of HTS and mechanotransduction-related conditions such as foreign body response.

This protocol will explain how to establish a hypertrophic scarring murine model that increases mechanotransduction signaling to simulate human-like scarring. This method involves increasing mechanical tension across a healing incision in a mouse and using a specialized device to create reproducible, excessive scar tissue for detailed histological and bioinformatic analyses.

Hypertrophic scarring (HTS) is an abnormal process of wound healing that results in excessive scar tissue formation. Over the past decade, we have ...

Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers

In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. Here, we describe a protocol to generate a two-dimensional (2D) monolayer from three-dimensional (3D) non-human primate enteroids derived from intestinal crypts of the terminal ileum. These enteroid-derived monolayers were then utilized in an in vitro scratch wound assay to test the ability of hyaluronan 35 kDa (HA35), a human milk HA mimic, to promote cell migration and proliferation along the epithelial wound edge. After the monolayers were grown to confluency, they were manually scratched and treated with HA35 (50 &#181;g/mL, 100 &#181;g/mL, 200 &#181;g/mL) or control (PBS). Cell migration and proliferation into the gap were imaged using a transmitted-light microscope equipped for live-cell imaging. Wound closure was quantified as percent wound healing using the Wound Healing Size Plugin in ImageJ.&#160;The scratch area and rate of cell migration and the percentage of wound closure were measured over 24 h. HA35 in vitro accelerates wound healing in small intestinal enteroid monolayers, likely through a combination of cell proliferation at the wound edge and migration to the wound area. These methods can potentially be used as a model to explore intestinal regeneration in the preterm human small intestine.

Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers

This protocol describes a method to establish and perform a scratch wound assay on two-dimensional (2D) monolayers derived from three-dimensional (3D) enteroids isolated from non-human primate ileum.

In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. ...

Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay

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