저자는 국민 건강과 호주의 의학 연구위원회 (NHMRC) (: 632512 프로젝트 부여 ID)의 자금 지원을 인정하고 싶습니다. 루이스 던은 국립 심장 재단 경력 개발 원정대에 의해 NHMRC 초기 경력 친목과 크리스티나 Bursill에 의해 지원되었다.

1. 부목​​ 및 폐색 드레싱의 준비 <ol><li> 0.5 mm 두께의 실리콘 시트 및 사용 가위 나 실리콘 디스크를 만들 수있는 생검 펀치에 10mm 원을 설명합니다. </li><li> 센터 10 밀리미터 원의 중간에 5mm 생검 펀치 부목으로 사용되는 디스크처럼 - &quot;도넛&quot;을 형성하는 구멍을 만들기 위해 단단히 누르십시오. </li><li> 이러한 Opsite 및 원형 드레싱을 만들 때 사용하는 가위로 투명 폐색 드레싱에 10mm 원을 설명합니다. </li></ol> 2. 실험 동물 <ol><li> 수행 할 모든 실험 동물 윤리위원회의 승인을 얻습니다. </li><li> 상업 개종 (예를 들어, 잭슨 연구소)에서 8 주 이전 (22~26g) 남성 C57BL/6J 마우스를 사용합니다. </li><li> 21의 표준 조건에서 쥐를 유지 ° C와 음식과 물을 무료로 액세스 할 수있는 12 시간 빛 어두운주기. </li><li> (선택 사항) : 당뇨병은 알약 복강 인제로 6~7주 오래된 마우스에서 유도 될 수정기적으로 혈당 모니터링 (AccuChek glucometer에)에 의해 확인 된 고혈당과 165 ㎎ / ㎏ 스트렙토 조 토신 (구연산 버퍼, 산도 4.5)의 ction의. 당뇨병 쥐 다뇨가있을 수 있습니다 그래서 그들의 침구 습기를 제거하고 가중치를 면밀히 모니터링해야 더 자주 변경해야 할 수 있습니다. </li></ol> 3. 마취 및 수술 준비 <ol><li> 100 % 산소 isoflurane을 5 %을 (유량 1 L / 분)을 사용하여 전신 마취를 유도하고 isoflurane을 1-3%을 사용하여 마취를 유지합니다. </li><li> 억제 된 마우스의 깊은 페달 반사를 확인하고 발생하기 쉬운 위치에 마우스를 놓습니다. </li><li> 더 뒤로 아래로 두 견갑골 사이 3cm로 목 기지에서 가위로 털을 제거하여 수술 영역을 준비합니다. </li><li> 탈모 크림 라이트 응용 프로그램은 2 분 이상 더 이상 적용 할 수 있습니다. 젖은 거즈 면봉은 모든 크림을 보장 할 수 있으며, 나머지 모피가 제거됩니다. </li><li> 로 피부를 닦아알코올 면봉와 10 %의 두 응용 프로그램 포비돈 요오드 (베타 딘)과 마우스를 주름 잡아 드리운다. </li></ol> 4. 절단 및 상처의 부목 <ol><li> 쥐 상처 치유 모델의 도식 표현은 그림 1에 제시되어있다. </li><li> (그림의 2A-2B 참조) 어깨 높이에서 마우스의 정중선의 양쪽에 상처 두 개의 원형 패턴을 설명하기 위해 멸균 4mm 생검 펀치를 사용합니다. </li><li> 개요 및 panniculus carnosus (그림 2E) 등의 피하 조직 (그림 2C-2D)를 통해 확장 전층 상처를 만들 수 홍채 가위의 중간에 피부를 들어 올려 톱니 모양의 집게를 사용하고, 소비세 원형 조각 조직의. </li><li> 정중선 (그림 2F)의 반대편에 상처 과정을 반복합니다. </li><li> 실리콘 부목의 각 측면에서 플라스틱 보호 코팅을 제거합니다. </li><li> cyanoacryla을 적용테 접착제 (예를 들어, 최고 접착제 또는 크레이지 접착제) 실리콘 부목의 한쪽. </li><li> 센터 상처에 부목 (그림 2G) 및 앵커 위치 (그림 2H)을 보장하기 위해 중단 된 6-0 나일론 봉합사로 부목. </li><li> 다른 상처에 부목 과정을 반복합니다. </li><li> 이 시간 시점에서 필요한 경우, 하나의 상처 (그림 2I), 그리고 다른 차량 제어를 테스트 할 치료 화합물 (100 μL)을 적용합니다. </li><li> 투명 폐색 드레싱 (예를 들어, OpSite) (그림 2J-2K)으로 상처를 덮습니다. </li><li> 통치자는 부목 아래에 배치 현미경이 (2리터 그림) 촬영됩니다. </li></ol> 6. 수술 후 관리 <ol><li> Carprofen은 (5 ㎎ / ㎏) 수술 후 통증 완화를 위해 서브 피부 주사를 통해 매일 한 번 관리됩니다. </li><li> 수술 동물은 개별적으로 갇힌 및 열 미터에서 유지 한 후ATS가 완전히 회복 될 때까지. </li><li> 통증과 체중 감소의 증상에 대한 회 동물을 모니터링 할 수 있습니다. 우리가 통증이나 체중 감소의 더 심한 행동 디스플레이를 관찰합니다. </li></ol> 7. 상처 측정 및 치료 <ol><li> 상처는 매일 측정 할 수 있습니다. </li><li> 5 % isoflurane을 가스 (유량 1 L / 분)을 사용하여 전신 마취를 유도 한 후 마우스의 깊은 감각 반사 보장은 1-3 %의 isoflurane을 사용하여 표시되지 않습니다. </li><li> 집게로 조심스럽게 껍질 폐색 드레싱. </li><li> 상처 직경을 측정하기 위해 수술 캘리퍼스를 사용합니다. 우리는 X, Y 및 Z 축 (그림 2m-2O)뿐만 아니라 나중에 참조 할 수있다 (그림 2 P)에 대한 현미경 따라 3 개의 측정 값의 평균을 가지고 간다. </li><li> 선택 사항 :이 때 동물이 레이저 도플러 영상 (그림 2 분기)를 사용하여 혈액 관류에 대한 평가 할 수 있습니다. </li><li> 치료 화합물 및 차량 제어를 다시 응용 프로그램 perfor 수 있습니다이 시점에서 의대. </li><li> 깨끗하고 투명한 폐쇄성 드레싱 후 다시 적용하고 완전히 회복 될 때까지 동물은 따뜻하게 유지됩니다. </li><li> 참고 : 부목이 제대로 확보되지 않을 경우 상처는 쉽게 계약 할 것이다 (그림 2R). </li></ol> 8. 조직 학적 분석 <ol><li> 마취의 과다 복용 10 일 후에 쥐를 안락사. </li><li> 집게를 사용하고 메스 칼날이 껍질 멀리주의 부목 봉합을 제거합니다. </li><li> 주위 상처 영역에서 폭, 전체 절제술을 만들고 인산 4 % 파라 포름 알데히드의 조직을 배양하기 위해 홍채 가위를 사용하여 4 ° C에서 하룻밤 (PBS) 염분 버퍼. </li><li> ° C -80에서 단층 화합물 및 동결의 조직과 장소에주의 dabbing하여 과량의 용액을 제거 ° C. 4에서 추가로 24 시간 동안 PBS 용액에 17 % 자당에 조직을 전송 </li><li> 헤 마톡 실린과 에오신 염색은 상처 구조와 상피 차이를 시각화하는 데 사용할 수 있습니다. Neovascularisation은 모세 혈관의 수 (폰 빌레 브란트 인자를 사용)를 결정하기 위해 면역 조직 화학 분석에 의해 평가 될 수있다. </li></ol>

<ol>
	<li>Sen, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+skin+wounds:+a+major+and+snowballing+threat+to+public+health+and+the+economy.">Human skin wounds: a major and snowballing threat to public health and the economy.</a> Wound Repair. 17, 763-771 (2009).</li><li>Sen, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+essentials:+let+there+be+oxygen.">Wound healing essentials: let there be oxygen.</a> Wound Repair Regen. 17, 1-18 (2009).</li><li>Gurtner, G. C., Werner, S., Barrandon, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair+and+regeneration.">Wound repair and regeneration.</a> Nature. 453, 314-321 (2008).</li><li>Lindblad, W. 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저자는 공개 아무것도 없어.

이 피부 상처 치유의 실험 쥐 모델입니다. 이 모델의 중요한 특징은 인간의 발생 과정이 유사하고, 재 상피화 및 새로운 조직의 형성이 발생할 수 있도록 상처 수축을 방지하는 실리콘 부목의 사용이다. 이 모델은 다목적 생리적 및 병리 모두 (예를 들면 당뇨병) 설정에서 상처 치유를 평가하는 데 사용할 수 있습니다. 모델은 경제적 인 환경에서 잠재적 인 상처 치유 나 혈관 치료를 평가하?...

손상된 상처 치유 중요한 병적 상태와 세계적으로 사망에 대한 책임이며, 이는 당뇨병 1,2의 환자에 특히 사실이다. 인간, 상처 치유는 3 중복 의미가있는 프로세스의 연속이다. 직후에 남긴 상처, 염증 프로세스가 시작됩니다. 염증 세포는 세포 증식, 이동 및 혈관 신생 과정을 촉진 인자를 놓습니다. 재 상피화 및 새로운 조직의 형성 이후에 같은 콜라겐 매트릭스 단백질의 세포 사멸하고 다시 조직을 모두 수반 개조의 단계가있다. 상처 치유의 복잡성은 현재 체외에서 복제 된이 동물 모델의 사용을 필요로 할 수 없습니다. 지금까지, 상처 치유 연구는 치료 과정은 동일하고 인간에게 비교를 보장하기 위해, 같은 돼지 같은 큰 동물 모델에 한정되어있다. 그러나 큰 애니을 사용하여이러한 연구를위한 LS 집에 어려울 항상 실제 4 수 없습니다 수 있습니다. 실험실 쥐 쉽게 유전자 5-7 역학적 조사 조작 할 수있는 경제적 인 동물 모델을 나타냅니다. 그러나 생쥐 상처 수축 8의 과정 차적으로 인해 인간의 다르게 치료. 이것은 부분적으로 인간에서 크게 결석 panniculus carnosus라는 광범위한 피하 횡문근 층 때문입니다. 마우스,이 근육 층은 피부가 독립적으로 깊은 근육의 이동 할 수 있으며 피부 아래에 입힐 수있는 총알의 급속한 수축에 대한 책임이 있습니다. 이 제한을 극복하기 위해, 쥐 상처 치유는 부목을 사용한다 (그림 1) 8,9에 의해 인간의 상처 치유를 복제하도록 구성 할 수 있습니다. 이 비디오에서 우리는 다시 epithelializa의 인간 프로세스를 상처 수축을 제거하고 더 가깝게 근사 splinted 쥐 상처 모델을 보여TION 및 새로운 조직의 형성. 이 모델에서 panniculus carnosus을 포함 두 개의 전층 절개는 마우스의 정중선의 양쪽에 배부 한에 생성됩니다. 실리콘 부목은 접착제의 도움으로 상처 주위에 배치하고 부목는 중단 된 봉합사로 고정되어 있습니다. 각 마우스함으로써 동물의 수를 줄이고, 하나의 상처를 치료하고 다른 차량 제어를받는과 함께 자신의 제어 역할을합니다. 국소 응용 프로그램에 따라 투명 폐쇄성 드레싱이 적용됩니다. 또한 국소 응용 프로그램 및 / 또는 상처 영역 10,11의 측정을 위해 필요한 경우 드레싱을 제거 할 수 있습니다. 에서 실험 완료, 상처 봉합, 형태 학적 구조와 신생 혈관의 정도는 면역에 의해 평가 될 수있다. 이 경제 모델을 수행하기 쉬운 또한 당뇨병이나 다른 pathophysiologies의 맥락에서 상처 치유를 평가하기 위하여 이용 될 수있다.

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 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Press-to-seal silicone sheeting 0.5 mm thick</td>
 <td>Invitrogen</td>
 <td>P18178</td>
 <td>Cut into "donuts" with external diameter of 1cm external, 0.5 cm internal diameter</td>
 </tr>
 <tr>
 <td>Biopsy punch 5 mm</td>
 <td>Steifel</td>
 <td>BC-B1-0500</td>
 <td>To outline wound area to be excised</td>
 </tr>
 <tr>
 <td>Vannas scissors 8.5 cm curved</td>
 <td>World Precision Instruments </td>
 <td>501232</td>
 <td>For wound incision and excision</td>
 </tr>
 <tr>
 <td>Dumonte #7b forceps, 11 cm</td>
 <td>World Precision Instruments</td>
 <td>501302</td>
 <td>To grip skin when creating incision and excising skin </td>
 </tr>
 <tr>
 <td>Graefe forceps, serrated 10cm</td>
 <td>World Precision Instruments</td>
 <td>14142</td>
 <td>To help attach silicone splint to skin</td>
 </tr>
 <tr>
 <td>Needle holder, smooth jaws, curved, 12.5 cm</td>
 <td>World Precision Instruments</td>
 <td>14132</td>
 <td></td>
 </tr>
 <tr>
 <td>Malis forceps, smooth, straight, 12 cm</td>
 <td>Codman and Shurtleff, Inc (J&amp;J)</td>
 <td>80-1500</td>
 <td>To suture the silicon rings to the skin</td>
 </tr>
 <tr>
 <td>Ruler, 0.5 mm gradation</td>
 <td>n/a</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Calipers 0.25 mm gradation</td>
 <td>Duckworth and Kent</td>
 <td>9-653</td>
 <td>To measure wound area</td>
 </tr>
 <tr>
 <td>Opsite FlexiFix transparent adhesive film. 10 cm x 1 m</td>
 <td>Smith &amp; Nephew</td>
 <td>66030570</td>
 <td></td>
 </tr>
 <tr>
 <td>Rimadyl (Carprofen)</td>
 <td>Pfizer</td>
 <td>462986</td>
 <td></td>
 </tr>
 </tbody>
</table>

murine model of wound healing

상처 치유 및 복구는 인간의 삶에서 발생하는 가장 복잡한 생물학적 과정이다. 부상 후, 여러 생물 학적 경로가 활성화됩니다. 예를 들어, 당뇨병 환자에서 발생하는 손상 상처 치유, 같은 절단 등의 심각한 불리한 결과를 초래할 수 있습니다. 상처 복구를 촉진 새로운 에이전트를 개발하기 위해 증가 자극 그러므로 있습니다. 이러한 테스트는 이러한 종종 비실용적 돼지와 같은 큰 동물 모델에 국한되어 있습니다. 그들은 경제적 인 기계적인 조사를 허용 유전자 조작, 의무이기 때문에 쥐, 이상적인 전임상 모델을 나타냅니다. 그러나, 그것은 주로 수축을 통해 발생할 때 마우스 치유가 인간의 근본적으로 다른 상처. 우리의 생쥐 모델은 상처 주위에 부목을 통합하여이를 극복한다. 상처를 부목으로 복구 프로세스가 긴밀를 반영 상피, 세포 증식 및 혈관 신생, 그때 의존인간의 상처 치유의 생물학적 처리합니다. 일관성과 치료를 필요로하는 반면,이 쥐 모델은 복잡한 수술 기법을 포함, 예를 들어, 혈관 신생을 촉진하거나 염증을 억제 할 수 유망한 에이전트의 강력한 테스트를 할 수 없습니다. 두 개의 상처가 준비되어 더욱이, 각각의 마우스 시험 화합물과 같은 동물에 차량 제어 모두의 응용 프로그램을 사용, 자신의 제어 역할을합니다. 결론적으로, 우리는 인간의 그것과 비교할 상처 치유의 실제, 배우기 쉬운, 강력한 모델을 보여줍니다.

(주 0 직경 / 일 X 지름) - 상처 폐쇄 곡선은 상처의 평균 직경을 계산하여 백분율, 즉 100로 결과를 표현 의해 결정됩니다. 이 실험에서 치료 화합물 (또는 차량 제어)는 상처에 매일 적용되었다. 치료 화합물을 크게 가속 상처 봉합 (그림 3). 그것은 부목의 제거가 빠른 상처의 수축 (그림 2R)으로 이어질 인간의 관찰 상처 치유의 패턴에서 분기되므로 부목은 실험 기간 ...

Watch this Scientific Journal Video about Murine Model of Wound Healing at JoVE.com

Murine Model of Wound Healing

생리와 병리 설정에서 치료 화합물을 평가하는 데 사용할 수있는 피부 상처 치유의 쥐 모델.

상처 치유의 생쥐 모델

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Research

JoVE Journal

Medicine

63.5K 조회수.  The Heart Research Institute. 생리와 병리 설정에서 치료 화합물을 평가하는 데 사용할 수있는 피부 상처 치유의 쥐 모델.

비디오: Murine Model of Wound Healing

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

에 대한 쥐의 내시경<em&gt; 생체</em&gt; 발암 및 소장의 평가의 멀티 모달 이미징 상처 치유 및 염증

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

연구

의학

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.

흉터 상처 수리의 마우스 태아의 피부 모델

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

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

쥐 허혈성 피부 상처 모델의 데모

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The etiology of IBD remains unknown and current therapeutic strategies are based on the unspecific suppression of the immune system. The development of treatments that specifically target intestinal inflammation and epithelial wound healing could significantly improve management of IBD, however this requires accurate detection of inflammatory changes. Currently, potential drug candidates are usually evaluated using animal models in vivo or with cell culture based techniques in vitro. Histological examination usually requires the cells or tissues of interest to be stained, which may alter the sample characteristics and furthermore, the interpretation of findings can vary by investigator expertise. Digital holographic microscopy (DHM), based on the detection of optical path length delay, allows stain-free quantitative phase contrast imaging. This allows the results to be directly correlated with absolute biophysical parameters. We demonstrate how measurement of changes in tissue density with DHM, based on refractive index measurement, can quantify inflammatory alterations, without staining, in different layers of colonic tissue specimens from mice and humans with colitis. Additionally, we demonstrate continuous multimodal label-free monitoring of epithelial wound healing in vitro, possible using DHM through the simple automated determination of the wounded area and simultaneous determination of morphological parameters such as dry mass and layer thickness of migrating cells. In conclusion, DHM represents a valuable, novel and quantitative tool for the assessment of intestinal inflammation with absolute values for parameters possible, simplified quantification of epithelial wound healing in vitro and therefore has high potential for translational diagnostic use.

Accurate assessment of anti-inflammatory effects is of utmost importance for the evaluation of potential new drugs for the treatment of inflammatory bowel disease. Digital holographic microscopy provides assessment of inflammation in murine and human colonic tissue samples as well as automated multimodal evaluation of epithelial wound healing in vitro.

디지털 홀로그램 현미경과 복합 양적 위상 이미징 정확하게 장내 염증과 상피 상처 치유를 평가

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

생성 및 당뇨병 치유 하는 부상을 모델에서 줄기 세포 지방이 많은 파생 (ASC) 시트의 이식

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

치수의의 평가에 대한 직접 펄프 캐핑 모델의 개발 상처 치유와 마우스의 수복 상아질 형성

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

현미경 기반 방법을 생체 외에서 상처 치유 중 상피 세포 이동의 평가 대 한

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Myography 와이어를 사용 하 여 마우스 Mesenteric 동맥의 isometric 수축 측정

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

각 막에 대 한 모델로 눈 버와 각 막 상피 마모 상처 치유

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.