This work was supported by funds from American Heart Association (A.M.), U.S. Department of Defense (BC074986), and Boston Children&#8217;s Hospital Faculty Career Development Fellowship (T.M., A.M.). The authors thank Amanda Jiang and Elisabeth Jiang for technical assistance.

NOTE: The in vivo animal study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was reviewed and approved by the Animal Care and Use Committee of Boston Children&#8217;s Hospital (Protocol Numbers: 13-10-2526R,14-02-2568R).&#160;All drugs used in this protocol are pharmaceutical grade and these drugs are prepared under sterile conditions.&#160;
1. Fibrin Gel Preparatio...

The authors declare that they have no competing financial interests.

This article introduces a new method to implant biomaterials on the lung surface of living adult mouse. With this system, host lung-derived angiogenesis is successfully recapitulated inside the material. This system allows researchers to explore crosstalk between endothelial cells, other cells (e.g., epithelial cells, mesenchymal cells, immune cells) and various ECM components that are required for local angiogenesis 50-53 and alveolar regeneration 24,54. Although conventional in vivo <...

The overall goal of this protocol is to introduce a method to implant fibrin gel on the lung surface of adult mouse, which allows researchers to characterize the molecular mechanisms of lung vascular and alveolar development, and to leverage this knowledge in order to develop biomimetic materials capable of recapitulating physiological lung vascular and alveolar formation to treat various lung diseases.
More than 35 million Americans suffer from chronic lung diseases including chronic obstructive pulmonary disease and pulmonary fibrosis. These patients have long-lasting chronic respiratory symptoms such as shortness of breath, chest tightness, nagging cough, and tiredness, which significantly impair their daily life 1-3. Despite a great amount of effort to develop effective therapies for these lung diseases, currently there is no cure; therefore, quality of life for these patients is poor and economic and human costs are high 4-7. Currently, lung transplantation is the only way to save patients with end-stage chronic lung diseases. However, because of the shortage of transplant donors, high cost, serious complications, and low survival rate 8-11, transplantation is not an optimal approach. Recent rapid progress in tissue engineering techniques has enabled researchers to bioengineer implantable lung by repopulating decellularized whole lung with various types of progenitor cells or induced pluripotent stem (iPS) cells 12,13. However, these bioengineered lungs are functional in host animals only for several hours after implantation 12,14,15. Utilizing biomaterials to regenerate the complex structures and functions of lungs has also been fairly unsuccessful. This may be because key biological processes that govern adult lung regeneration have not been well explored. In the lung, formation of the vascular system is one of the earliest and most important events during development and regeneration 16-21. Newly formed vasculatures in the lung not only deliver oxygen, nutrients and various cell components required for organ formation, but also provide instructive regulatory signals to surrounding cells 22-25. Thus, angiogenesis plays key roles in regenerative alveolarization in adult lungs 24,26,27. In addition, deregulated angiogenesis contributes to chronic lung diseases such as chronic obstructive pulmonary disease (COPD) 28, bronchopulmonary dysplasia (BPD) 21-23, and pulmonary fibrosis 29. Thus, to develop more efficient strategies for engineering lungs or treating chronic lung diseases, it is necessary to understand the fundamental mechanisms of lung-specific angiogenesis.
Each organ displays unique mechanical and chemical properties, which may differ between physiological and pathological conditions 30-33. These organ-specific microenvironments regulate endothelial cell behaviors and orchestrate vascular network formation in an organ-specific manner 24,34-36. Thus, to develop more efficient strategies for lung regeneration, the mechanism underlying lung-specific angiogenesis needs to be understood. While conventional in vivo angiogenesis assays such as subcutaneous hydrogel implantation have been used extensively for angiogenesis research 37-39, those methods do not recapitulate organ-specific angiogenesis. Recently, a novel method to implant Matrigel in an elastic mold on the mouse lung has been developed and shown to successfully recruit blood vessels and lung epithelial cells into the gels 22. This unique approach will allow researchers to explore the mechanism of lung-specific angiogenesis as well as interactions between blood vessels and non-vascular lung cells in physiological and pathological conditions. Since 1) Matrigel is not suitable for clinical application; 2) the elastic mold used to cast the gel may affect interactions between hydrogels and host lung tissue and 3) the elastic mold on the lung potentially causes impairment of lung function and pain during respiration, as a more clinically relevant approach, a 3D fibrin matrix containing angiogenic factors (vascular endothelial growth factor (VEGF)/ basic fibroblast growth factor (bFGF)) has been implanted on the mouse lung without casting in the elastic mold, and has successfully recapitulated host lung-derived angiogenesis. Fibrin gel, polymer fibrils generated from thrombin-cleaved fibrinogen, is known to trap a variety of angiogenic factors such as bFGF and VEGF to accelerate angiogenesis in vivo 40,41. Because of its regenerative ability and biodegradable nature 42, fibrin gel is widely used in the field of tissue engineering.
This article introduces a novel and unique approach to implant fibrin gel on the lung surface of living adult mouse and demonstrates that host lung-derived angiogenesis is recapitulated inside the gels in vivo. This method, which enables researchers to study lung-specific angiogenesis, will likely lead to the development of new therapeutic approaches for various types of lung diseases and significantly advance efforts to successfully regenerate adult lung.

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	<tbody>
		<tr height="48">
			<td height="48" style="height:48px;width:343px;">Name of Material/ Equipment</td>
			<td style="width:177px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:289px;">Comments/Description</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Fibrinogen from human placenta</td>
			<td>Sigma</td>
			<td>F4883</td>
			<td>For fabrication of fibrin gel</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Thrombin from bovine plasma</td>
			<td>Sigma</td>
			<td>T9549</td>
			<td>For fabrication of fibrin gel</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Recombinant mouse VEGF 164</td>
			<td>R&#38;D</td>
			<td>493-MV</td>
			<td>For supplementation to fibrin gel</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Recombinant mouse bFGF</td>
			<td>R&#38;D</td>
			<td>3139-FB</td>
			<td>For supplementation to fibrin gel</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:343px;">Rodent Intubation Stand</td>
			<td style="width:177px;">Braintree Scientific INC</td>
			<td style="width:147px;">RIS 100</td>
			<td>For intubation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Fiber-Optic Light Source</td>
			<td style="width:177px;">Fisher Scientific</td>
			<td>12-565-35</td>
			<td>For intubation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">20G Elastic catheter</td>
			<td>B.Braun</td>
			<td>4251652-02</td>
			<td>For intubation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:343px;">MiniVent Ventilator</td>
			<td style="width:177px;">Harvard Apparatus</td>
			<td style="width:147px;">CGS-8009</td>
			<td>For ventilation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Stemi DV4 Steromicroscope</td>
			<td style="width:177px;">Fisher Scientific</td>
			<td>12-070-515</td>
			<td>For surgey</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Absobable suture</td>
			<td>Ethicon</td>
			<td>PDP304</td>
			<td>Surgical suture</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Antibody against CD31</td>
			<td>BD Biosciences</td>
			<td align="right">553370</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Antibody against AQP5</td>
			<td style="width:177px;">Abcam</td>
			<td>AB78486</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Antibody against SP-B</td>
			<td style="width:177px;">Millipore</td>
			<td>AB40876</td>
			<td>Immunohistochemistry</td>
		</tr>
	</tbody>
</table>

implantation of fibrin gel on mouse lung to study lung-specific angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

To examine whether host lung-derived vascular formation is recapitulated inside the biomaterials implanted on the lung, fibrin gels supplemented with major angiogenic factors VEGF and bFGF (0, 10 and 100 ng/ml each) were implanted on the surface of living mouse lungs as reported using Matrigel 22. Fibrin gels 47 that contain these angiogenic growth factors were fabricated as shown in Figure 1a. After thoracotomy, a small area of the left lung surface was scraped using forceps and th...

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Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

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9.9K Views.  Boston Children's Hospital and Harvard Medical School. Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse. 