Name: effects of allogeneic platelet-rich plasma (prp) on the healing process of sectioned achilles tendons of rats: a methodological description
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이 연구는 스탕다르 드 리에주에 의해 지원 되었다 고 Lejeune-Lechien 레온 Frédéricq 자금 부여 합니다.

배려 및 처리는 동물의 배려를 위한 가이드에 따라 수행 된 실험 동물의 사용 국립 과학 아카데미의에 의해 준비 하 고 출판에 의해 국립 연구소의 건강 (미국). 유럽 및 국가 입법 신중 하 게 따라 했다.
1. 동물 준비
<ol><li>132 2 개월 된 남성 Sprague-Dawley 쥐 320-450 g (120 쥐 실험에 대 한) 및 12 쥐 혈액 샘플링, 그림 1에 대 한 무게를 사용 합니다. Dell<sup class="...

<ol>
	<li>Kaux, J., Degrave, N., Crielaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+rich+plasma+traitement+des+tendinopathies+chroniques?+Revue+de+la+litt&#233;rature.+Platelet+rich+plasma+treatment+of+chronic+tendinopathies?+Review+of+literature.">Platelet rich plasma traitement des tendinopathies chroniques? Revue de la litt&#233;rature. Platelet rich plasma treatment of chronic tendinopathies? Review of literature.</a> J. Traumatol. du Sport. 24, 99-102 (2007).</li><li>Anitua, E., 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=Autologous+preparations+rich+in+growth+factors+promote+proliferation+and+induce+VEGF+and+HGF+production+by+human+tendon+cells+in+culture.">Autologous preparations rich in growth factors promote proliferation and induce VEGF and HGF production by human tendon cells in culture.</a> J. Orthop. Res. 23, 281-286 (2005).</li><li>Bosch, G., 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=Effects+of+platelet-rich+plasma+on+the+quality+of+repair+of+mechanically+induced+core+lesions+in+equine+superficial+digital+flexor+tendons:+A+placebo-controlled+experimental+study.">Effects of platelet-rich plasma on the quality of repair of mechanically induced core lesions in equine superficial digital flexor tendons: A placebo-controlled experimental study.</a> J. Orthop. Res. 28, 211-217 (2010).</li><li>Kaux, J. F., Drion, P., Croisier, J. L., Crielaard, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tendinopathies+and+platelet-rich+plasma+(PRP):+From+pre-clinical+experiments+to+therapeutic+use.">Tendinopathies and platelet-rich plasma (PRP): From pre-clinical experiments to therapeutic use.</a> J. Stem Cells Regen. Med. 11, P7-P17 (2015).</li><li>Maffulli, N., Wong, J., Almekinders, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Types+and+epidemiology+of+tendinopathy.">Types and epidemiology of tendinopathy.</a> Clin. Sports Med. 22, 675-692 (2003).</li><li>Molloy, T., Wang, Y., Murrell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+growth+factors+in+tendon+and+ligament+healing.">The roles of growth factors in tendon and ligament healing.</a> Sports Med. 33, 381-394 (2003).</li><li>Lyras, D. 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C., 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=Growth+Factor-Rich+Plasma+Increases+Tendon+Cell+Proliferation+and+Matrix+Synthesis+on+a+Synthetic+Scaffold:+An+In+Vitro+Study.">Growth Factor-Rich Plasma Increases Tendon Cell Proliferation and Matrix Synthesis on a Synthetic Scaffold: An In Vitro Study.</a> Tissue Eng. Part A. 16, 1021-1029 (2010).</li><li>Zhang, J., Wang, J. H. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-Rich+Plasma+Releasate+Promotes+Differentiation+of+Tendon+Stem+Cells+Into+Active+Tenocytes.">Platelet-Rich Plasma Releasate Promotes Differentiation of Tendon Stem Cells Into Active Tenocytes.</a> Am. J. Sports Med. 38, 2477-2486 (2010).</li><li>Kajikawa, Y., 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=Platelet-rich+plasma+enhances+the+initial+mobilization+of+circulation-derived+cells+for+tendon+healing.">Platelet-rich plasma enhances the initial mobilization of circulation-derived cells for tendon healing.</a> J. Cell. Physiol. 215, 837-845 (2008).</li><li>Pasternak, B., Aspenberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metalloproteinases+and+their+inhibitors-diagnostic+and+therapeutic+opportunities+in+orthopedics.">Metalloproteinases and their inhibitors-diagnostic and therapeutic opportunities in orthopedics.</a> Acta Orthop. 80, 693-703 (2009).</li><li>Dell, R. B., Holleran, S., Ramakrishnan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+size+determination.">Sample size determination.</a> ILAR J. 43, 207-213 (2002).</li><li>M&#228;hler Convenor, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FELASA+recommendations+for+the+health+monitoring+of+mouse,+rat,+hamster,+guinea+pig+and+rabbit+colonies+in+breeding+and+experimental+units.">FELASA recommendations for the health monitoring of mouse, rat, hamster, guinea pig and rabbit colonies in breeding and experimental units.</a> Lab. Anim. 48, 178-192 (2014).</li><li>Kaux, J. -. F., 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=&#201;tude+comparative+de+cinq+techniques+de+pr&#233;paration+plaquettaire+(platelet-rich+plasma).">&#201;tude comparative de cinq techniques de pr&#233;paration plaquettaire (platelet-rich plasma).</a> Pathol. Biol. 59, 157-160 (2011).</li><li>Wieloch, P., Buchmann, G., Roth, W., Rickert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cryo-jaw+designed+for+in+vitro+tensile+testing+of+the+healing+Achilles+tendons+in+rats.">A cryo-jaw designed for in vitro tensile testing of the healing Achilles tendons in rats.</a> J. Biomech. 37, 1719-1722 (2004).</li><li>Kaux, J. -. F., 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=Vascular+Endothelial+Growth+Factor-111+(VEGF-111)+and+tendon+healing:+preliminary+results+in+a+rat+model+of+tendon+injury.">Vascular Endothelial Growth Factor-111 (VEGF-111) and tendon healing: preliminary results in a rat model of tendon injury.</a> Muscles. Ligaments Tendons J. 4, 24-28 (2014).</li><li>Docheva, D., Hunziker, E. B., F&#228;ssler, R., Brandau, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tenomodulin+is+necessary+for+tenocyte+proliferation+and+tendon+maturation.">Tenomodulin is necessary for tenocyte proliferation and tendon maturation.</a> Mol. Cell. Biol. 25, 699-705 (2005).</li><li>Lambert, C. A., Colige, A. C., Munaut, C., Lapi&#232;re, C. M., Nusgens, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+pathways+in+the+over-expression+of+matrix+metalloproteinases+in+human+fibroblasts+by+relaxation+of+mechanical+tension.">Distinct pathways in the over-expression of matrix metalloproteinases in human fibroblasts by relaxation of mechanical tension.</a> Matrix Biol. 20, 397-408 (2001).</li><li>Nurden, A. T., Nurden, P., Sanchez, M., Andia, I., Anitua, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets+and+wound+healing.">Platelets and wound healing.</a> Front. Biosci. 13, 3532-3548 (2008).</li><li>Woodall, J., Tucci, M., Mishra, A., Benghuzzi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+effects+of+platelet+rich+plasma:+a+study+on+HL-60+macrophage-like+cells.">Cellular effects of platelet rich plasma: a study on HL-60 macrophage-like cells.</a> Biomed. Sci. Instrum. 43, 266-271 (2007).</li><li>Taylor, D. W., Petrera, M., Hendry, M., Theodoropoulos, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+of+the+use+of+platelet-rich+plasma+in+sports+medicine+as+a+new+treatment+for+tendon+and+ligament+injuries.">A systematic review of the use of platelet-rich plasma in sports medicine as a new treatment for tendon and ligament injuries.</a> Clin. J. Sport Med. 21, 344-352 (2011).</li><li>Mazzocca, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+positive+effects+of+different+platelet-rich+plasma+methods+on+human+muscle,+bone,+and+tendon+cells.">The positive effects of different platelet-rich plasma methods on human muscle, bone, and tendon cells.</a> Am. J. Sports Med. 40, 1742-1749 (2012).</li><li>McCarrel, T. M., Minas, T., Fortier, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+leukocyte+concentration+in+platelet-rich+plasma+for+the+treatment+of+tendinopathy.">Optimization of leukocyte concentration in platelet-rich plasma for the treatment of tendinopathy.</a> J. Bone Joint Surg. Am. 94 (1-8), e143 (2012).</li><li>Boswell, S. G., 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=Increasing+platelet+concentrations+in+leukocyte-reduced+platelet-rich+plasma+decrease+collagen+gene+synthesis+in+tendons.">Increasing platelet concentrations in leukocyte-reduced platelet-rich plasma decrease collagen gene synthesis in tendons.</a> Am. J. Sports Med. 42, 42-49 (2014).</li><li>Virchenko, O., Aspenberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+can+one+platelet+injection+after+tendon+injury+lead+to+a+stronger+tendon+after+4+weeks?:+Interplay+between+early+regeneration+and+mechanical+stimulation.">How can one platelet injection after tendon injury lead to a stronger tendon after 4 weeks?: Interplay between early regeneration and mechanical stimulation.</a> Acta Orthop. 77, 806-812 (2006).</li></ol>

혈소판은 프로세스를 치유 하는 힘 줄의 염증 초기 단계에 대 한 필수적입니다. 이러한 혈소판 바인딩 조직이 나 응고 유발 요인에 노출 되는 때 그들은 α과 립에 방류 하는 성장 인자를 발표할 예정 이다. 이 상호 작용으로 인해 세포 외 매트릭스 고분자 synthetized 고 중간 엽 세포 증식. 혈소판에는 또한 신생 및 세포질 감 별 법6,20을 자극, 혈액 순환 조?...

응고, 염증 성 프로세스 및 혈소판의 면역 변조 역할은 잘 알려진1입니다. 최근에, 그들은 또한 회복 속성2,3는 증명 되었습니다. 실제로, 다양 한 cytokines 및 성장 요인 (VEGF, PDGF, TGF B, IGF-난, 그리고 HGF) degranulation 동안 혈소판에 의해 발표. 이러한 성장 요인 홍보 신생, 조직 리 모델링, 그리고 상처 치유 (뼈, 피부, 근육, 힘 줄)2. 격리 방법 높은 혈소판 농도 포함 된 혈소판 리치 플라즈마 (PRP)을 생성 centrifuging 혈액 헌 혈 (3 사이 10 시간 기준 농도 혈액). 실제로, 다양 한 PRP 준비 기술 동일한 최종 제품을 제공할 수 없습니다. 지금 까지는,이 문제에 아무 국제 일반 계약이 되었습니다. 전반적으로, PRP 치료 tendinopathy, 발바닥의 근 막 염, 관절염, 등 없앤4만성 musculoskeletal 조건에 대 한 매력적인 치료 옵션 일 수 있었다. 그것은 개선 하 고 뼈 임 플 란 트 삽입 후 치유를 가속 구강 수술 및 implantology4 에서 처음으로 사용 되었다. 이 연구에서 우리는 동물 실험4에 대 한 PRP의 수집 수 있는 재연 가능한 방법도 설명 합니다.
힘 줄의 병 변을 자주 운동과 육체 노동자에서 관찰 된다, 이후 큰 관심5는 치유 과정와 따라서 복구 시간을 단축. 자주 개발 하는 새로운 치료 방법을 성장 인자를 사용 하 여 포함 하 고 PRP의 내 인 성 성장 요인4의 조화를 제공 하는 간단 하 고 최소한 침략 적 방법 이다.
여러 생체 외에서 또는 동물 연구 생물학 중재자를 방출 하 여 혈소판, 높은 수준의 포함 된 플라즈마의 생물학 중재자6 를 방출 하 여 힘 줄과 인 대 복구를 자극 수 증명 7,,89. 또한, 다른 연구 PRP 유형 I 및 III 콜라겐 합성 힘 줄에9,,1011세포 자극 수 있습니다 나타났습니다. 그것은 또한 건의 되었다 PRP 매트릭스 metalloproteinases (MMPs)의 활성화를 감소 하 고 따라서 매트릭스의 저하를 줄일 수 있습니다. 염증 과정에 관련 된 셀 MMP-9, 조직 리 모델링 (생리와 pathologic) 염증12에 의해 유도 된에서 역할을 생성할 수 있습니다.
이 정보를 바탕으로, 우리가 쥐의 sectioned 아 킬 레 스 힘 줄에 단일 PRP 주사 복구 과정과 복구 조직의 기계적 강도 향상 시킬 수 있을 것을 가정 했다. 이 복구 프로세스 동안 치유 힘 줄의 biomechanical 속성을 측정 하 여 및 새로 형성된 된 조직에서 개장 하는 콜라겐을 평가 하는 조직학 및 분자 분석을 수행 하 여 테스트 됩니다. 연구의 목표는 경우 수용자 PRP의 단일 주사를 영향을 미칠 수 sectioned 아 킬 레 스 힘 줄의 치유를 관찰 했다.

<table>
	<tbody>
		<tr>
			<td>Xylazine (Xyl-M)</td>
			<td>VMD</td>
			<td>none</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Ketamin (J&#233;tamine 1000 CEVA)</td>
			<td>CEVA Sant&#233; Animale</td>
			<td>none</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Buprenorphin (Veterg&#233;sic Multidosis)</td>
			<td>ALSTOE</td>
			<td>none</td>
			<td>Painkiller</td>
		</tr>
		<tr>
			<td>iso-Betadine</td>
			<td>MEDA-Pharma</td>
			<td>none</td>
			<td>Desinfectant</td>
		</tr>
		<tr>
			<td>resorbable yarn Vicryl 6/0</td>
			<td>Johnson &#38; Johnson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nembutal</td>
			<td>CEVA Sant&#233; Animale</td>
			<td>none</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Preserves structure of the tissue</td>
		</tr>
		<tr>
			<td>Isopropanol 100%</td>
			<td>VWR</td>
			<td>20,922,364</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 95%</td>
			<td>VWR</td>
			<td>20,823,362</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR</td>
			<td>28973.363</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>VWR</td>
			<td>LEIC3950.1006</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Millipore</td>
			<td>1.15938.0025</td>
			<td>Colorant</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>Millipore</td>
			<td>1.15935.0100</td>
			<td>Colorant</td>
		</tr>
		<tr>
			<td>Eukitt</td>
			<td>Sigma-Aldrich</td>
			<td>3989</td>
			<td>Mounting Medium</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

effects of allogeneic platelet-rich plasma (prp) on the healing process of sectioned achilles tendons of rats: a methodological description

이 문서에서는 PRP 긍정적으로 영향을 미칠 수 힘 줄 치유를 관찰 하는 데 사용 하는 실험 절차를 설명 합니다. 따라 4 주요 단계가 있다: 킬;에서 병 변 유발 PRP를 준비 하 고 주사 (또는 염 분 해결책); 제거 힘; 그리고 수행 평가 biomechanical, 분자, 그리고 조직학. 각 단계에서 모든 절차와 방법을 설명 세부 사항, 그래서 그들은 쉽게 재생 될 수 있다 합니다.
아 킬 레 스 힘 줄 수술 sectioned 5-m m 긴 섹션의 (제거) 되었습니다. 이후에, PRP 또는 염 류 용액 공부 PRP에 힘 줄의 치료에 긍정적인 영향을 주입 했다. (총 120 쥐가이 연구에 사용 되었다) 40 동물의 3 개 그룹 2 하위 그룹으로 세분화 했다: PRP 주사 그룹 및 식 염 수 주입 제어 그룹. 쥐가 증가 하는 시간 포인트 (그룹 a: 5 일;에 희생 했다 그룹 b: 15 일; 그룹 c: 30 일)와 힘 줄이 제거 되었습니다. 90 힘 줄 수술 biomechanical transcriptomic 분석을 수행 하기 전에 테스트 하 고 30 나머지 힘 줄 조직학 분석을 제출 했다.

결과 뜻의 평균 ± 표준 편차로 표현 하 고 분산 분석 (ANOVA)와 비교 했다. 양방향의 ANOVA와 임시 게시 테스트 데 있어, 파라메트릭 테스트, 사용 되었다.
쥐의 비 부상 아 킬 레 스 힘 줄의 파열을 자극 하는 데 필요한 궁극 장력 강도 (UTS)는 42.0 ± 5.7 N (n = 10). 인장 강도 크게 증가 (p < 0.0001) 5 일 후 두 그룹에서. 두 그룹을 비?...

Watch this Scientific Journal Video about Effects of Allogeneic Platelet-Rich Plasma PRP on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description at JoVE.com

Effects of Allogeneic Platelet-Rich Plasma PRP on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

이 프로토콜 아 킬 레 스 힘 줄의 일부를 제거한 후 수용자 혈소판 풍부 혈장 (PRP) 또는 염 분 해결책 주입 되어 쥐에 있는 힘 줄 치유의 평가 과정을 설명 합니다. 힘 줄 치유의 진행은 분석의 다른 종류를 사용 하 여 여러 개의 시간 지점에서 계산 됩니다.

쥐의 Sectioned 아 킬 레 스 힘 줄의 치유 과정에서 수용자 혈소판이 풍부한 혈장 (PRP)의 효과: 방법론 설명

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a ...

The overall goal of this procedure is to observe the effects of a single injection of Platelet-Rich Plasma, or PRP, on the resistance and healing process of surgically-sectioned rat Achilles tendons at various time points. This method can help answer quick questions on musculoskeletal field about tendon lesions. The main advantage of this technique is that the surgical, biomechanical, and PRP progression procedures are well-standardized.The implications of these techniques extend toward the use of PRP in humans, because these types of lesion and the tendon healing process are similar between rats and human. Though this method can provide insight into PRP preparation for musculoskeletal injuries, this injection technique can also be applied to other systems, such as heart or skin disease. Visual demonstration of the method is very important, because surgical and biomechanical steps require consistent reproducibility in order to obtain optimal results.Begin by weighing and ear-tagging the rat. After confirming the appropriate level of sedation by toe pinch, apply water drops onto the animal's corneas and remove the hair from the left hind limb. Disinfect the exposed skin with a one-to-10 dilution of iso-Betadine solution and place the animal on a 20-degree Celsius heating pad under a dissecting microscope in the lateral decubitus position, with the left hind limb in the superior position.Grasp the left hind limb paw with forceps and make a 20 to 25 millimeter lateral skin incision around the Achilles tendon. Using fine scissors, dissect the fascia to expose the Achilles tendon complex and remove the plantaris tendon. Cut the Achilles tendon transversely, five millimeters proximal to its calcaneal insertion, and remove a five-millimeter long portion.Then use resorbable yarn and continue with sutures to close the fascia and the skin, and place the rat under a heat source with monitoring until full recovery. While the recipient animals are recovering, collect 20 milliliters of whole blood from one donor rat heart per experimental animal into individual tubes containing 3.2%buffered sodium citrate. To obtain the platelet-rich plasma, centrifuge the blood and use a plastic transfer pipette to carefully harvest the upper PRP-containing phase into a second plastic tube.Determine the volume of collected PRP and measure the platelet count on a hematology analyzer. Centrifuge the PRP again and carefully transfer the supernatant into a new plastic tube. The remaining volume in the PRP tube should be around 2/3 of the volume required to achieve 2.5 times 10 to the sixth platelets per microliter.Next, gently re-suspend the platelet pellet with pipetting. Measure the platelet count of this concentrated platelet-rich plasma, adding back the appropriate volume of autologous platelet-poor plasma to reach the target concentration as necessary. Activate the platelets with 50 microliters of calcium chloride per milliliter of PRP for about one hour at room temperature.Then load the platelets into a one-milliliter syringe equipped with a 21-gauge needle and inject 50 microliters of the platelets directly into the suture site of each animal. At the appropriate experimental endpoint, shave the left hind limb of the anesthetized experimental animal, and position the rat under a dissecting microscope as just demonstrated. Holding the paw with fingers, make a 10-millimeter incision in the surgical site to expose the triceps suralis.To remove the Achilles tendon, cut the calcaneal bone transversally, five to 10 millimeters distal to its attachment and dissect a piece of the triceps suralis large enough to fit into the cryo-jaw. Use forceps to place the sample immediately into the upper jaw of the cryo-jaw and close the instrument. Place the cryo-jaw vertically into a universal testing machine, and fix the calcaneal bone between the lower clamps.To calculate the cross-sectional area, place cameras perpendicular to each other. Add liquid nitrogen into both of the upper jaw basins to freeze the muscle to an order of magnitude stiffer than the tendon, so that it does not deform during the tensile test. When the freezing zone reaches the metal clamp border, set the displacement rate of the machine at a constant speed of one millimeter per second until rupture, and begin the tensile test, using a computer to record the ultimate tensile strength in Newtons.To account for differences in the cross-sectional areas of the healing tendons, normalize the ultimate tensile strength to a unit area that represents the mechanical stress experienced by the tissue. Then when the tendon ruptures, snap freeze the ruptured tendon samples in liquid nitrogen and store them at negative 80 degrees Celsius until RNA extraction and RTPCR analysis. In this representative experiment, a greater force was required to induce tendon rupture in the PRP-injected group than in the control saline-treated group at every time point tested, suggesting a more robust healing process or better mechanical properties within the PRP-treated animals.Although the PRP group demonstrated a higher intra-group scatter than did the saline group, the cross-sectional area of the tendons was determined to be larger in the PRP-injected group at days 15 and 30 after lesion induction. Further, Masson's trichrome staining revealed a higher presence fibrillar collagen, a key component in tendon morphology and strength, in PRP-treated animals on days five and 15, with a similar intensity of staining observed in both groups by day 30 after injection. After watching this video, you should have a good understanding of how to induce a rat standard lesions, and to sample the tendon to evaluate their healing process after a PRP injection.

Research

JoVE Journal

Medicine

Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

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