Name: distinctive capillary action by micro-channels in bone-like templates can enhance recruitment of cells for restoration of large bony defect
Uploaded: 2015-09-11T14:00:00
Duration: 9 min 35 s
Description: Watch this Scientific Journal Video about Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect at JoVE.com

The authors have no acknowledgements.

1. Polyurethane (PU) Sponge Preparation as Template
<ol>
	<li>Use PU sponges to produce hydroxyapatite templates containing interconnecting pores. Use each sponge to provide the primary trabeculae for the formation of the template struts as well as the formation of micro-channels within the trabeculae.</li>
	<li>Cut and trim 80 ppi (pores per inch) sponges into 2 bridge-shapes with dimensions of 3.5 cm in height x 5 cm in length x 1.5 cm in width. 
	Note: The dimensions and shapes can be chosen according to the desired primary pore size: 100 ppi, 80 ppi, and 60 ppi.</li>
	<li>Make a 100 ml&#160;of 4% (w/v) NaOH solution using a 150 ml&#160;beaker; then immerse and squeeze until the prepared sponges are completely soaked.</li>
	<li>After soaking, place the beaker with the sponges in the ultrasonicator (42 kHz).</li>
	<li>Ultrasonically pre-treat the PU sponges for 15-20 min&#160;without heat to modify the surface properties.</li>
	<li>Rinse with distilled water for 5-10 min. While rinsing, squeeze the sponges and allow them to expand 5 to 7 times in order to remove the residual NaOH inside the sponges.</li>
	<li>Squeeze the sponges with paper towels in order to remove excess water; then dry them in an oven at 60-80 &#176;C.</li>
</ol>
2. Hydroxyapatite (HA) Slurry Preparation for Coating
<ol>
	<li>Before making the HA slurry, measure the weight of a beaker with a magnetic stir bar. This measurement will be used to calculate the powder/liquid ratio.</li>
	<li>Measure 10 g of the nano-sized HA powder.</li>
	<li>Add 20 ml&#160;of distilled water into the 50 ml&#160;beaker. Heat for 120-140 &#176;C and stir using a hot plate magnetic stirrer.</li>
	<li>Add 0.3 g (3% w/w) of polyvinyl alcohol (PVA) (89,000-98,000 MW) per powder into distilled water while stirring at 300-400 rpm.</li>
	<li>Stir until the PVA has completely dissolved. The solution should be clear after complete dissolution of the PVA.</li>
	<li>Turn off the heat and add 0.1 g (1% w/w) of sodium carboxymethyl cellulose (CMC) (ultra-low viscosity) for powder while stirring at 400-500 rpm. The solution should be clear after complete dissolution of the PVA.</li>
	<li>Stir until the CMC has completely dissolved and cool down to RT.</li>
	<li>Add 0.3 g (3% w/w) of ammonium polyacrylate dispersant per powder while stirring at 300-400 rpm. Stir until completely dissolved.</li>
	<li>Add 0.2 g (2% w/w) of glycerin per powder while stirring at 300-400 rpm. Stir until completely dissolved.</li>
	<li>Slowly disperse the HA powder into the solution while stirring at 600-900 rpm and keep stirring for 5 min.</li>
	<li>Sonicate for 5 min&#160;using an ultrasonicator to ensure dispersion of any agglomeration of the HA powder.</li>
	<li>Add an extra 5 ml&#160;of distilled water into the mixture while stirring at 600-900 rpm and heat at 90-100 &#176;C.</li>
	<li>Keep stirring the mixture using a magnetic stirrer at 600-800 rpm at 90-100 &#176;C in order to evaporate the water content.</li>
	<li>Measure the whole weight including the beaker and mixture from time to time until a powder/liquid ratio of 1.75-1.8 is obtained.</li>
	<li>Formatting the powder/liquid ratio, divide the weight of the powder by the total weight of the mixture (2.14), including the powder, reagents, and water, minus the weight of the beaker and stirrer (2.1), and minus the HA powder (2.2). 
	Note: For example: If A (entire mixture including powder, reagents, and water) is 49.05 g, B (beaker with stirrer) is 33.5 g, and then C (HA powder) is 10 g. 
	C/(A-B-C) = 10/(49.05-33.5-10) = 1.80</li>
	<li>Allow the slurry to cool down to RT before using for coating.</li>
</ol>
3. HA coating, Drying, and Sintering
<ol>
	<li>Coat the prepared PU sponges with the HA coating slurry using a stainless spatula until the slurry is homogenously distributed throughout the PU sponge onto a glass plate. 
	Note: After removing the excess slurry, some of pores may still be clogged with slurry because of the high slurry viscosity.</li>
	<li>In order to ensure interconnectivity, uniformity, and open pores, slightly blow the HA coated templates using an air compressor. This process ensures that the templates are homogeneously coated both on the inside and outer surfaces of the PU sponge. 
	Note: If a homogeneous coating is not achieved, the HA coated templates will collapse during the sintering process and may also crack while handling due to low mechanical strength. Additionally, the homogeneous coating is critical in creating micro-channels within the trabeculae.</li>
	<li>Dry the HA coated templates for a minimum of 5 hr&#160;under cooling conditions (20-25 &#176;C) with gentle air circulation. However, extend the drying time based on the size of template. 
	Note: After drying, the HA coated templates will typically shrink approximately 8% to 10% in each dimension.</li>
	<li>After the drying process, place the HA coated templates on an alumina crucible. Then, place them in a high temperature furnace and use the following 8 step sintering profile.
	<ol>
		<li>Heat 2 &#176;C/min until 230 &#176;C.</li>
		<li>Heat 1 &#176;C/min until 280 &#176;C.</li>
		<li>Heat 0.5 &#176;C/min until 400 &#176;C.</li>
		<li>Heat 3 &#176;C/min until 600 &#176;C. Keep at 600 &#176;C for 1 hr.</li>
		<li>Heat 5 &#176;C/min until 1230 &#176;C. Keep 1,230 &#176;C for 3 hr.</li>
		<li>Cool 5 &#176;C/min to RT. 
		Note: Sintering will further shrink the HA coated sponge templates by approximately 22% &#8211; 25% in each dimension.</li>
	</ol>
	</li>
</ol>
4. Ingress and Inhabitancy of Cells into Template
<ol>
	<li>Culture pre-osteoblastic MC3T3 cells in a non-osteogenic media consisting of &#945;-MEM, supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics (streptomycin and penicillin) at 37 &#176;C in a humidified atmosphere containing 5% CO2.</li>
	<li>Add 10 ml&#160;of a cell suspension at 2 x 106 cell density into a single well within a 6-well plate.</li>
	<li>Place the 3 cm x 4 cm x 1 cm bridge-shaped template vertically into the 6-well plate. Place one leg of the template into the plate containing the cell suspension, and the other leg into an adjacent empty well.</li>
	<li>Allow the template to absorb the cell suspension for 10 min.</li>
	<li>Add 5 ml&#160;of media thereafter to the well that was originally filled with the cell suspension.</li>
	<li>Replenish the medium in both wells every 2 or 3 days until 7 days have elapsed.</li>
	<li>Determine the efficacy of cell mobility by hematoxylin and eosin staining11.
	<ol>
		<li>Fix the cells and scaffold by immersing in 100% EtOH for 20-30 min.</li>
		<li>Stain with Hematoxylin for 1-2 min.</li>
		<li>Rinse with distilled water for 1-2 min for twice.</li>
		<li>Dehydrate by immersion in 70%, then 95%, then 100% EtOH for 1-2 min each.</li>
		<li>Stain with Eosin for 20-30 sec.</li>
		<li>Rinse with distilled water for 12 min for twice.</li>
		<li>Dehydrate by immersion in 70%, 80%, 90%, and 100% EtOH for 1-2 min each.</li>
		<li>Embed the scaffold in an acrylic resin for sectioning and imaging.</li>
	</ol>
	</li>
	<li>Determine the cell viability with the 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cell viability assay and Live/Dead assay (Live/dead cell staining kit MPTP) at time points of day 3 and 711. 
	Note: The scheme of bone-like template fabrication protocols are represented in the &#8220;Representative Results&#8221; session.</li>
</ol>

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The authors have nothing to disclose.

A multi component template including cells, growth factors, nutrients, etc. is needed for successful bone regeneration and functional restoration of critical-sized large bone defects. Within these factors, anatomically conforming biological properties are essential. To accomplish biological functionality, the template must exhibit biocompatibility, osteoconductivity, mechanical integrity, sufficient surface area, adequate surface texture, and the means for oxygen and nutrient transport. At the cellular level, the following features are especially crucial for functional restoration of massive bone defects: facilitated penetration into template (active recruitment), uniform distribution throughout the template (retention), accelerated proliferation and high viability (habitation). Finally, the subsequent formation of substantial extra cellular matrix and the triggering of gene expression are crucial in essential biological processes such as rapid vascularization and osteogenesis.
Many different types of synthetic substitutes have been proposed to replace auto-/allo- bone grafts. However, the current scaffold organization does not exhibit an internal microenvironment containing micro-channels and nano-pores, and therefore does not actively facilitate cell infiltration, distribution, and inhabitance deep into the synthetic substitutes that are larger than 10 mm. They do not provide physical cues for pioneering cells to efficiently, rapidly, and uniformly migrate deeply into the bone template. Instead, the limited passive recruitment of cells creates an unequally distributed cell populations between the outer and inner areas of the scaffold. This not only exacerbates the initial challenge of the cells reaching the inner core of the template but also hinders nutrient flow and cell communication with the other end of the synthetic substitute. This type of disproportional cell recruitment and inhabitation results in cell death and incomplete bone growth after the scaffold has been implanted into the body14,15.
Thus, we have introduced the concept of capillary action as the primary physical cue to address these obstacles. We have thoroughly engineered micro-channels in the BMT to induce the capillary action that will account for the primary dragging force responsibly for actively recruiting cells deep into the BMT.
The PU sponge coating technique presents several unique properties. First, it allows for an easy preparation of well-controlled porous trabecular structures, which themselves depend on the pre-defined template structures (i.e., 80 pore per inch template for 300-400 &#181;m). This is very important for optimizing pore size for osteoblast infiltration15. Second, the technique enables the construction of interconnected micro-channels, which account for the significant role of initializing cell relocation11. Third, there are almost no limitations when using the PU sponge in terms of creating custom shapes and sizes of the templates. The maker can use a scissor for simple shapes or even computed laser cutting for complex geometries. Using these precisely controlled technologies, we created the BMT. HA was selected as the starting material because of its biocompatibility and osteoconductive capacity17.
In this study, there are several critical steps that need to be highlighted. During the HA slurry preparation, if the temperature is too high and the stirring speed is too low, the HA slurry will become stuck at bottom edges of the beaker and dry up. After the coating process when blowing out the excess HA slurry, too high of an air pressure can induce cracks on the surface of the BMT. It is important to keep the air pressure relatively low to properly air out the excess HA slurry only. Finally, the second and third steps of the sintering process are most crucial (Heat 1 &#176;C/min until 280 &#176;C and Heat 0.5 &#176;C/min until 400 &#176;C). In this temperature range, the PU sponge will completely burn out while the HA becomes dense. If this protocol is not closely followed, the BMT will be collapsed or crumbled after sintering.
The BMT described in this study offers several advantages. First, the inter-connected macro-pores (300-400 &#181;m) mimic those of human trabecular bone and allows for smooth bone marrow flow. Second, the templates are comprised of micro-channels (25-50 &#181;m) within each trabecular septum to accelerate the initial ingress of bone cells via capillary action. As demonstrated using computational simulation13, if the template only had 300 &#181;m pores (primary pores) and no microchannels, the capillary action would be insufficient for the full saturation of the template with bone marrow. This would especially hold true for large size defects that would require large size templates. Micrometer-sized channels exhibit highly effective fluid absorption, and thus we expected the micro-channels to be primarily responsible for the capillary action in our study. Third, our BMTs have strategically placed nano-pores. Data from the literature indicate that cells are especially sensitive to nano-patterns18,19; therefore, we expected the nano-pores on the walls of the micro-channels to play a role in increasing cell attachment. Nano-sized pores (100-400 nm) on the surface of the trabecular septa allowed immobilized cells to anchor. Overall, the combined effects of these three internal structures resulted in enhanced cell mobilization and adhesion throughout the template. However, there are some limitations of the protocol and the critical steps to fabricate the perfect BMT. For example, there is often a large amount of HA slurry prepared due to the difficulty of keeping a homogeneous viscosity while coating. Also there is a limitation in making templates greater than 5 cm3 in volume due to working time while coating. The coating thickness is critical which varies depending on the maker&#8217;s techniques.
The findings of our study suggest that the BMT capable of absorbing and retaining cells will offer potential advantages over conventional alloplastic (or synthetic) scaffolds. A prospective study is being considered to verify the benefits of BMT on osteogenesis and/or angiogenesis along with bone-related growth factors. Therefore, we claim that our unique featured BMT scaffold can address the major barriers of insufficient bone marrow infiltration into the synthetic constructs and incomplete bone regeneration in large defects.
The ultimate goal of this study is to simplify the current paradigm of bioengineering in bone reconstruction and functional restoration in critical-sized bony defects by eliminating the need for time-/labor-intensive bone marrow stromal cells isolation and expansion processes. Finally, we aim to utilize anatomically conforming 3D-constructs with micro-channels and nano-pores, which induce rapid cell absorption, homogenous distribution, and inhabitance for reconstruction of bone.

The ultimate goal of bone tissue engineering with synthetic constructs is the incorporation of the constructs into the host bone, repopulation of the constructs with host cells, and reconstitution of gas and body fluid exchanges to restore normal bone function. Considerable research has been reported over the last decade in the use of polymeric and ceramic biomaterials for producing scaffolds1,2. However, the ideal material and fabrication technique for optimal bone tissue regeneration has yet to be identified. In addition, there is an overall lack of success in bringing these technologies to the clinic, especially for the reconstruction and restoration of large bone defects. Therefore, restoring critical sized bony defects still remains a clinical challenge1-5. 
Ideally, the scaffolds for bone tissue regeneration should exhibit biocompatibility without causing inflammatory responses or foreign body/toxic reactions, have closely matched mechanical properties when compared to those of native bone, and possess a mechanism to allow diffusion and/or transport of ions and nutrients. Strong bonding with the host bone, dynamic bone growth, vascular ingrowth, and biodegradation of the scaffolds are equally desirable. Although the use of biodegradable polymer scaffolds has exhibited progress in terms of tissue ingrowth, there are controversies over their use for bone regeneration. 
Notwithstanding these extensive efforts, the highly organized structural synthetic constructs still have limited potential in overcoming the obstacle of passive cell penetration. Most of these approaches have resulted in the in vitro tissue ingrowth with cross-sections of less than a few &#181;m to several mm from the external surface, an incomplete integration with host bone, and only partial bone regeneration in vivo6,7. The pioneering cells do not migrate deeply into the constructs because of the lack of an initial force that pulls them inside before cell colonization begins. Consequently, cell colonization strictly occurs at the scaffold periphery, becoming an obstruction from the periphery to the center of the scaffold. Thus, the diffusion of oxygen and nutrients into the inner parts of the templates becomes limited8. Therefore, it is clear that the architecture of the scaffolds (pore size, porosity, interconnectivity, and permeability) that affect the transport and diffusion of substances throughout the scaffolds is critical for achieving well-distributed cell proliferation and differentiation9,10. Although calcium phosphates have been used in the past for scaffold fabrication, different processes and procedures have often resulted in calcium phosphate scaffolds with varying architectures. Thus, the selection of the manufacturing process becomes important in dictating the scaffold architecture needed for successful bone tissue regeneration.
In conclusion, there are still two major shortcomings of bone tissue engineering that need to be addressed: the initial cell recruitment into the template prior to cell attachment and colonization and the quality of substance flow both into and out of the template.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>polyurethan sponge</td>
			<td>Plastifoam</td>
			<td>PU-3215</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td align="right">167176</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydroxyapatite Powder</td>
			<td>Ossgen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinyl Alcoho</td>
			<td>Sigma-Aldrich</td>
			<td align="right">341584</td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxymethyl cellulose sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td align="right">360384</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium polyacrylate</td>
			<td>Vanderbilt</td>
			<td>DARVAN 821A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerin</td>
			<td>Sigma-Aldrich</td>
			<td>G2289</td>
			<td></td>
		</tr>
	</tbody>
</table>

distinctive capillary action by micro-channels in bone-like templates can enhance recruitment of cells for restoration of large bony defect

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not occur. With conventional templates, the absence of internal micro-channels results in the lack of cell infiltration, distribution, and inhabitance deep inside the templates. Hence, a highly porous and uniformly interconnected trabecular-bone-like template with micro-channels (biogenic microenvironment template; BMT) has been developed to address these obstacles. The novel BMT was created by innovative concepts (capillary action) and fabricated with a sponge-template coating technique. The BMT consists of several structural components: inter-connected primary-pores (300-400 &#181;m) that mimic pores in trabecular bone, micro-channels (25-70 &#181;m) within each trabecula, and nanopores (100-400 nm) on the surface to allow cells to anchor. Moreover, the BMT has been documented by mechanical test study to have similar mechanical strength properties to those of human trabecular bone (~3.8 MPa)12.
The BMT exhibited high absorption, retention, and habitation of cells throughout the bridge-shaped (&#928;) templates (3 cm height and 4 cm length). The cells that were initially seeded into one end of the templates immediately mobilized to the other end (10 cm distance) by capillary action of the BMT on the cell media. After 4 hr, the cells homogenously occupied the entire BMT and exhibited normal cellular behavior. The capillary action accounted for the infiltration of the cells suspended in the media and the distribution (active migration) throughout the BMT. Having observed these capabilities of the BMT, we project that BMTs will absorb bone marrow cells, growth factors, and nutrients from the periphery under physiological conditions.
The BMT may resolve current limitations via rapid infiltration, homogenous distribution and inhabitance of cells in large, volumetric templates to repair massive skeletal defects.

The overall structure of the BMT exhibits a unique three-dimensional template with trabecular bone-like internal structures. The BMT contains macro-pores, micro-channels, and nano-pores. Clear configurations of fully interconnected macro-pores (average size of 320 &#181;m), micro-channels (average diameter of 50 &#181;m), and nano-pores (average size of 100 nm) were verified with a scanning electron microscope (EVO-40; ZEISS) as well as through micro-tomography.
Figure 1 shows stepwise detailed protocols in creating a BMT. Through precise control of the protocols from the preparation of the PU sponges to the sintering process (P1 &#8211; P7; Figure 1), the following features can be achieved: a highly dense and smooth surface after HA coating and drying; a precisely shaped and sized 3-D template; a fully interconnected porous trabecular network similar to that of trabecular bone; and micro-channels within each trabecula that mimic intra-osseous channels such as Haversian canals and Volkmann&#8217;s canals (Figures 2 &#38; 3). Furthermore, relatively high mechanical strength (~3.8 MPa) similar to that of human trabecular bone was measured by a compressive strength test. Highly similar histomorphometric parameters with those of human lumbar vertebrae trabecular bone were confirmed by micro-CT analysis12. Different magnitudes of capillary action were demonstrated through different capillary diameters in Figure 4 using computational simulation. Through these simulations, we projected that the BMT would exhibit varying absorption rates within the primary-pores (300-400 &#181;m) and micro-channels (25-70 &#181;m) based on the diameters. Smaller capillaries exhibited stronger absorption capacities. This assumption was verified in this experiment as shown in Figure 5.
The BMT exhibited highly effective fluid absorption and retention through the capillary action of the micro-channel structures; stevenel&#8217;s blue stain was used as the fluid medium to easily track the flow (Figure 5). Based on computational simulation, the BMT with these configurations were seen to absorb and retain cell suspensions up to 8.5 cm in total distance within 10 sec. Due to a strong capillary action induced by the internal structures, the stained medium reached the opposite end of a 3 cm (height) x 4 cm (length) x 1 cm (width) bridge-shaped template within 1 min&#160;and 40 sec. Furthermore, active cell mobilization and incorporation into the BMT was observed (Figure 6). Subsequently, the homogenous cell mobilization and attachment resulted in enhanced proliferation and matrix formation in an evenly distributed formation. Moreover, long-distance (~ 10 cm) migration of cells through the BMT was validated immediately after the BMT was saturated with the cell suspension. Seeded cells survived in the template segment that was exposed to the air and not immersed in the culture medium. In this experiment, the culture medium was provided to the cells by exclusively in the wells touching only the legs of the scaffold. The capillary action exhibited by the microchannels then allowed for fresh medium to reach the top, bridge portion of the scaffold. After 3 days of culture, the template became occupied with rapidly proliferating cells. After 7 days of culture, each trabecula was wrapped by extra cellular matrices and embedded with cells13.
<img alt="Figure 1" src="/files/ftp_upload/52947/52947fig1.jpg" /> 
Figure 1. The overall bone-like template fabrication protocol from the pre-treatment of PU sponge (P1) to the final heat treatment (P7). Keeping the precise sintering profile after P7 is crucial in achieving favorable mechanical strength. <a href="https://www.jove.com/files/ftp_upload/52947/52947fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52947/52947fig2.jpg" /> 
Figure 2. Representative stereo microscope (AmScope; SM-2TZ-M) images (x4) of a 80 ppi sized PU sponge (left), HA coated and dried BMT (middle), and sintered BMT (right). (Dimension: 3 cm in height x 4 cm in length x 1 cm in width). <a href="https://www.jove.com/files/ftp_upload/52947/52947fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52947/52947fig3.jpg" /> 
Figure 3. SEM and micro-CT images of a biogenic template: (A) an overall image of a biogenic template, (B, C, D) images for micro-channels. In order to highlight clear micro-channels in the trabeculae, the template was granulated. <a href="https://www.jove.com/files/ftp_upload/52947/52947fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52947/52947fig4.jpg" /> 
Figure 4. Computational calculation of capillary action with different channel diameters. Within the same time period (0.4 ms), while the largest capillary (d=300 &#181;m: refers to primary-pore) absorbed the medium (blue) up to 0.16 mm in height, the smallest capillary (d=30 &#181;m: refers to micro-channel) absorbed the medium up to 0.415 mm in height. <a href="https://www.jove.com/files/ftp_upload/52947/52947fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52947/52947fig5.jpg" /> 
Figure 5. Images showed differences of absorption capabilities of capillary action based on different sizes of primary-pores and micro-channels (primary-pore size refers to the average diameter: 60 ppi &#8776; 470 &#181;m, 80 ppi &#8776; 320 &#181;m, 100 ppi &#8776; 200 &#181;m). The yellow lines represent the capillary action induced by the combination of primary-pores and micro-channels. The red lines represent the capillary action induced by mainly micro-channels exhibited in each trabecula. As shown in (F), the 100 ppi template induced the strongest capillary action, resulting in the complete saturation of the template within 39 sec. The 80 ppi and 60 ppi templates were tested thereafter. (B) 0 sec, (C) 0.5 sec, (D) 1.5 sec, (E) 17.0 sec, (F) 39.0 sec, (G) 50.0 sec, and (H) 1 min&#160;18 sec&#160;after immersion. (Template dimension: 1 cm x 1 cm x 4 cm in height cuboidal). <a href="https://www.jove.com/files/ftp_upload/52947/52947fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52947/52947fig6.jpg" /> 
Figure 6. The ingress and immigration of cells from the seeded wells (part I) to unseeded wells (part V) through the biogenic templates induced by capillary action. The initially seeded cells reached the end of the unseeded leg (part V) immediately after full saturation. After 3 days, the confluence of the cells was evident throughout the entire template. After 7 days, spatiotemporal collagen matrix formation occurred within the cell populations (H&#38;E stain). (Template dimension: 3 cm in height x 4 cm in length x 1 cm in width). <a href="https://www.jove.com/files/ftp_upload/52947/52947fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect at JoVE.com

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels.

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not ...

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Research

JoVE Journal

Bioengineering

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

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 ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product ...

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 ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...