Name: preparation, procedures and evaluation of platelet-rich plasma injection in the treatment of knee osteoarthritis
Uploaded: 2019-01-04T13:00:00
Description: Watch this Scientific Journal Video about Preparation, Procedures and Evaluation of Platelet-Rich Plasma Injection in the Treatment of Knee Osteoarthritis at JoVE.com

This study was supported by the National Natural Science Foundation of China (81371991) and the Major Program of Science and Technology of Guangdong (2015B020225007).

The methods described were approved by the Ethics Committee of Guangdong General Hospital.
1. Obtain PRP by Manual Centrifugation
<ol>
	<li>Prepare the patient in a supine position in a sterile laminar flow operating room with a comfortable room temperature and humidity: room temperature is 22 &#176;C and room humidity is 60%.</li>
	<li>Use a 1-mL syringe to draw 0.2 mL of heparin sodium (2 mL = 12,500 U), and then moisten a 50-mL syringe. 
	NOTE: 3 mL of sodium citrate is also typical ...

<ol>
	<li>Loeser, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+the+musculoskeletal+system+and+the+development+of+osteoarthritis.">Age-related changes in the musculoskeletal system and the development of osteoarthritis.</a> Clinics in Geriatric Medicine. 26 (3), 371-386 (2010).</li><li>Bhatia, A., Peng, P., Cohen, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiofrequency+procedures+to+relieve+chronic+knee+pain:+an+evidence-based+narrative+review.">Radiofrequency procedures to relieve chronic knee pain: an evidence-based narrative review.</a> Reg Anesth Pain Med. 43 (1), 72 (2018).</li><li>S&#225;nchez, 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=A+randomized+clinical+trial+evaluating+plasma+rich+in+growth+factors+(PRGF-endoret)+versus+hyaluronic+acid+in+the+short-term+treatment+of+symptomatic+knee+osteoarthritis.">A randomized clinical trial evaluating plasma rich in growth factors (PRGF-endoret) versus hyaluronic acid in the short-term treatment of symptomatic knee osteoarthritis.</a> Arthroscopy the Journal of Arthroscopic &amp; Related Surgery. 28 (8), 1070-1078 (2012).</li><li>LaPrade, R. 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=Biologic+treatments+for+sports+injuries+II+think+tank-current+concepts,+future+research,+and+barriers+to+advancement,+part+1:+biologics+overview,+ligament+injury,+tendinopathy.">Biologic treatments for sports injuries II think tank-current concepts, future research, and barriers to advancement, part 1: biologics overview, ligament injury, tendinopathy.</a> Am J Sports Med. 44 (12), 3270-3283 (2016).</li><li>Ornetti, P., 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=Does+platelet-rich+plasma+have+a+role+in+the+treatment+of+osteoarthritis?.">Does platelet-rich plasma have a role in the treatment of osteoarthritis?.</a> Joint Bone Spine. 83 (1), 31-36 (2016).</li><li>Knop, E., Paula, L. E. D., Fuller, R. <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+for+osteoarthritis+treatment.">Platelet-rich plasma for osteoarthritis treatment.</a> Revista Brasileira de Reumatologia (English Edition). 56 (2), 152-164 (2016).</li><li>Weibrich, G., Kleis, W. K., Hafner, G., Hitzler, W. E. <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+levels+in+platelet-rich+plasma+and+correlations+with+donor+age,+sex,+and+platelet+count.">Growth factor levels in platelet-rich plasma and correlations with donor age, sex, and platelet count.</a> J Craniomaxillofac Surg. 30 (2), 97-102 (2002).</li><li>Landesberg, R., Roy, M., Glickman, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+growth+factor+levels+using+a+simplified+method+of+platelet-rich+plasma+gel+preparation.">Quantification of growth factor levels using a simplified method of platelet-rich plasma gel preparation.</a> Journal of Oral &amp; Maxillofacial Surgery. 58 (3), 297-300 (2000).</li><li>Wasterlain, A. S., Braun, H. J., Dragoo, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contents+and+formulations+of+platelet-rich+plasma.">Contents and formulations of platelet-rich plasma.</a> Operative Techniques in Orthopaedics. 22 (1), 33-42 (2012).</li><li>Gobbi, G., Vitale, M. <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+for+biological+therapy:+applications+and+limits.">Platelet rich plasma for biological therapy: applications and limits.</a> Operative Techniques in Orthopaedics. 22 (1), 10-15 (2012).</li><li>Chahla, J., 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=A+call+for+standardization+in+platelet-rich+plasma+preparation+protocols+and+composition+reporting:+a+systematic+review+of+the+clinical+orthopaedic+literature.">A call for standardization in platelet-rich plasma preparation protocols and composition reporting: a systematic review of the clinical orthopaedic literature.</a> Journal of Bone &amp; Joint Surgery-american. 99 (20), 1769-1779 (2017).</li><li>Delong, J. M., Russell, R. P., Mazzocca, A. D. <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:+the+PAW+classification+system.">Platelet-rich plasma: the PAW classification system.</a> Arthroscopy the Journal of Arthroscopic &amp; Related Surgery. 28 (7), 998-1009 (2012).</li><li>Yazigi Junior, J. A., 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=Quantification+of+platelets+obtained+by+different+centrifugation+protocols+in+SHR+rats.">Quantification of platelets obtained by different centrifugation protocols in SHR rats.</a> Revista Brasileira de Ortopedia (English Edition). 50 (6), 729-738 (2015).</li><li>Moojen, D. 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=Antimicrobial+activity+of+platelet-leukocyte+gel+against+Staphylococcus+aureus.">Antimicrobial activity of platelet-leukocyte gel against Staphylococcus aureus.</a> Journal of Orthopaedic Research. 26 (3), 404-410 (2008).</li><li>Tinsley, B. A., Ferreira, J. V., Dukas, A. G., Mazzocca, A. D. <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+nonoperative+injection+therapy-A+review+of+indications+and+evidence.">Platelet-rich plasma nonoperative injection therapy-A review of indications and evidence.</a> Operative Techniques in Sports Medicine. 20 (2), 192-200 (2012).</li><li>Arora, S., Doda, V., Kotwal, U., Dogra, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+platelets+and+platelet+derived+growth+factors+from+platelet-rich-plasma+(PRP)+prepared+at+different+centrifugal+force+(g)+and+time.">Quantification of platelets and platelet derived growth factors from platelet-rich-plasma (PRP) prepared at different centrifugal force (g) and time.</a> Transfusion &amp; Apheresis Science. 54 (1), 103-110 (2016).</li><li>Cohn, C. S., Lockhart, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+platelet-rich+plasma:+evidence+for+clinical+use.">Autologous platelet-rich plasma: evidence for clinical use.</a> Current Opinion in Hematology. 22 (6), 527-532 (2015).</li></ol>

The concentration of blood platelets in normal human blood is between 150,000/&#181;L and 350,000/&#181;L, and it is widely believed that blood platelet concentration of PRP should reach 1,000,000/&#181;L, which is 3 to 5 times normal concentrations9. According to the PAW hierarchy system, it is believed that PRP has no obvious effect when the blood platelet concentration is less than three times the normal concentration, while PRP has an inhibiting effect when its blood platelet concentration is ...

Knee osteoarthritis (KOA) is one of the most frequently seen diseases in the orthopedic department; 30%-50% of people over the age of 65 years experience this disease1. At present, the conservative management of KOA mainly includes oral administration of non-steroidal anti-inflammatory drugs and cartilage nutrient drugs, intra-articular injection of sodium hyaluronate, and physiotherapy. However, these methods cannot stop the process of knee joint degeneration2. Articular cartilage defects can cause articular surface wear, joint instability, and metabolic changes, which are part of the pathogenesis of KOA3. However, because of the absence of blood vessels, nerves and lymphoid tissue in articular cartilage, recovery after damage is difficult. An effective method of repairing cartilage is especially important for the treatment of KOA. The treatment of osteoclasia is also a key focus in KOA treatment.
Platelet-rich plasma (PRP) is an autologous bioactive substance, and the application of PRP to bone and joint problems is being increasingly studied. The biological rationale for the clinical use of PRP includes its effect on the local delivery of growth factors and modification of the inflammatory response and its positive effects on cell proliferation and differentiation4. After activation following intra-articular injection, PRP releases &#945;-granule through degranulation and secretes various growth factors, including the platelet-derived growth factor, the transforming growth factor-&#946;, the insulin-like growth factor, the epidermal growth factor, the vascular endothelial growth factor, and the fibroblast growth factor. These promote osteoblast and chondrocyte proliferation, inhibit cartilage degeneration, strengthen the stability of cartilage and subchondral bone, regulate the gene expression tissue inhibitor of metalloproteinase and maintain the balance of synthesis and degradation of proteoglycans5,6. Therefore, PRP can repair cartilage injury and accelerate bone regeneration.
The outcome of PRP injection is influenced by various factors, including the sampling site7, the type of centrifuge preparation method8, and the use of anticoagulants9 and activators10. There are roughly 3 types centrifugal methods to prepare PRP. Manual centrifugation, equipment-based centrifugation, or plasma filtration techniques are available, although manual and equipment-based methods are the most commonly used. The manual method requires the least equipment, is convenient, is low cost, and is simple to perform (Figure 1). PRP is prepared by performance of manual centrifugation twice. Mixed peripheral blood and anticoagulant are centrifuged to separate hemocytes from plasma and blood platelets. After discarding the red blood cells on the bottom layer, the supernatant liquid is centrifuged for re-separation, dividing it into supernatant platelet-poor plasma, middle PRP, and subnatant residual red blood cells. The middle layer is used for knee joint cavity injection (if the quantity is insufficient, part of the supernatant can be drawn). Evaluations of the method include assessments of blood platelet concentration and clinical outcomes. 
The reporting of PRP preparation protocols in clinical studies is highly inconsistent, and the majority of studies do not provide sufficient information to allow the protocol to be reproduced11. Here, we describe a reproducible method of preparing PRP and treatment of KOA with PRP intra-articular injection, with evaluation of the outcome. Inclusion criteria were patients with knee osteoarthritis who have poor pain relief for simple analgesic medication (such as acetaminophen) and conservative treatment. Exclusion criteria included patients with venous return or lymphatic drainage disorder; patients with knee joint infections; patients with a dermatosis or infection in the injection area; patients with fever; patients with coagulant function abnormality; patients with serious cardiovascular disease. The whole treatment procedure takes less than 30 minutes, and the blood platelet concentration of PRP is proven to reach a standardized measurement. Its effectiveness is demonstrated by evaluating the outcomes during close follow-up.

<table border="0" cellpadding="0" cellspacing="0" style="width:1245px;" width="1245">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:360px;">Centrifuge</td>
			<td style="width:383px;">Eppendorf</td>
			<td style="width:129px;">5702</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge tube</td>
			<td>CORNING</td>
			<td>430828</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Horizontal rotor</td>
			<td>Eppendorf</td>
			<td>LL080</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anerdian III</td>
			<td>Shanghai Likang Disinfectant HiTech Co., LTD</td>
			<td>310173</td>
			<td>Disinfect the skin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1ml Syringe</td>
			<td>KDL&#160; Medical Equipment Co., LTD</td>
			<td>0.4*13 RWLB</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10ml Syringe</td>
			<td>KDL&#160; Medical Equipment Co., LTD</td>
			<td>1.2*38 TWSB</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50ml Syringe</td>
			<td>KDL&#160; Medical Equipment Co., LTD</td>
			<td>0.7*32 TWLB</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Medical Tourniquet</td>
			<td>Changzhou Jinli Latex Products Co., LTD</td>
			<td>0087-2011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Single-use sterile rubber surgical gloves</td>
			<td>Shanghai jinxiang Latex Products Co., LTD</td>
			<td>17060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Disposable Draw Blood Needle</td>
			<td>KDL&#160; Medical Equipment Co., LTD</td>
			<td>0.55*20 L(II) RWLB</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin Sodium Injection</td>
			<td>SPH No.1 Biochemical &#38; Pharmaceutical Co., LTD</td>
			<td>1706101</td>
			<td>2ml:12500U</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Jifro Hand Antiseptic Rinse Free Gel</td>
			<td>Shanghai Likang Disinfectant HiTech Co., LTD</td>
			<td>311793</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Medical Cotton Swab</td>
			<td>Foshan Kangzheng Medical Supplies Co., LTD</td>
			<td>KZ3-12</td>
			<td>Disinfect the skin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10ml Normal Saline&#160;</td>
			<td>Jiangxi Shuangshi Pharmecutical Co., LTD</td>
			<td>140211458</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Automatic Blood Cell Analyzer</td>
			<td>Beckman Coulter</td>
			<td>LH-750</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hole-towe</td>
			<td></td>
			<td></td>
			<td>Sterile</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anticoagulation Tube(Blood Collection Tubes, K2E 3.6mg)</td>
			<td>Becton, Dickinson and Company</td>
			<td>CNL17-COO56</td>
			<td>Store in a cool dry place within 4 to 25 degrees Celcius</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tweezers</td>
			<td>RWD LIFE SCIENCE</td>
			<td>F12006-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile Gauze</td>
			<td>Guangdong Ze Chang Trade Co., LTD</td>
			<td>170915</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adhesive Plaster</td>
			<td>3M Transpore</td>
			<td>1527C-0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Skin Marker Pen</td>
			<td>Guangzhou Mingjia Medical Device Manufacturing Co., LTD</td>
			<td>10110</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation, procedures and evaluation of platelet-rich plasma injection in the treatment of knee osteoarthritis

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited effect on the repair of cartilage and on bone regeneration. Platelet-rich plasma (PRP) is an autologous bioactive substance that can repair cartilage injury and accelerate bone regeneration effectively. However, reporting of PRP preparation protocols in clinical studies is highly inconsistent, with the majority of studies providing insufficient information to allow the protocol to be reproduced. We describe a repeatable method of preparing PRP visually, the treatment of KOA using PRP intra-articular injection, and methods of evaluating the outcome. PRP was prepared using manual double centrifugation. The PRP layer was extracted from peripheral blood and used for knee joint cavity injection. Evaluations included assessments of blood platelet concentrations and clinical outcomes. Preparation of PRP by manual centrifugation requires less apparatus and is less costly than plasma filtration or centrifugation using equipment. The centrifugation time of our double centrifugation method was 6 and 5 minutes for the respective centrifugations at forces of 800 and 1400 x g, respectively, to allow for the consistent preparation of standardized PRP. However, a manual method is susceptible to operator error, and PRP batch preparation is not available. Intra-articular injection of PRP proved to be an effective treatment for knee osteoarthritis. The entire treatment procedure took less than 30 minutes, the blood platelet concentration of PRP could be standardized, and treatment was proven to be effective when evaluated by follow-up.

As a result, the platelet count of the PRP reached a standard concentration level of 1121 x 103/&#181;L. We conducted the 15 follow-up surveys described in the protocol on a 55-year-old male patient with early KOA. It was obvious that early clinical outcome was satisfactory after the intra-articular administration of PRP (Figure 2). However, medium-term efficacy was slightly inferior. A markedly significant analgesic effect was observed (<strong cl...

Watch this Scientific Journal Video about Preparation, Procedures and Evaluation of Platelet-Rich Plasma Injection in the Treatment of Knee Osteoarthritis at JoVE.com

Preparation, Procedures and Evaluation of Platelet-Rich Plasma Injection in the Treatment of Knee Osteoarthritis

Knee osteoarthritis is frequently seen in the orthopedic department. We introduce in detail the entire knee osteoarthritis treatment process with platelet-rich plasma injection, including preparation, procedures, and evaluation.

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited ...

Knee osteoarthritis is frequently seen in the orthopedic department. We introduce in detail the entire knee osteoarthritis treatment process with platelet-rich plasma injection. Including preparation, procedures, and evaluation.Knee osteoarthritis, KOA, is one of the most frequently seen diseases in the orthopedic department. 30 to 50%of people over the age of 65 years experience this disease, however existing non-surgical treatments have a limited effect on the repair of cartilage and on bone regeneration. Platelet-rich plasma, PRP, is an autologous bioactive substance that can repair cartilage injury and accelerate bone regeneration effectively.And the application of PRP to bone and joint problems is being increasingly studied. The biological rational for the clinical use of PRP includes its effect on the local delivery of growth factors and modification of the inflammatory response and its positive effects on cell proliferation and differentiation. However, the reporting of PRP preparation protocols in clinical studies is highly inconsistent.And the majority of studies do not provide sufficient information to allow the protocol to be reproduced. The manual method requires the least equipment, is convenient, is low-cost, and is simple to perform. First of all we prepare PRP by performance of manual double centrifugation.Extract PRP layer from peripheral blood. Next use this layer to perform knee joint cavity injection. At last evaluations of the method include assessments of blood platelet concentration and clinical outcomes.Here we describe a reproducible method of preparing PRP and treatment of KOA with PRP intra-articular injection. With evaluation of the outcome, the whole treatment procedure takes less than 30 minutes and the blood platelet concentration of PRP is proven to reach a standardized measurement. Its effectiveness is demonstrated by evaluating the outcomes during close follow-up.Prepare the patient in a supine position in a sterile, laminar flow operating room with a comfortable room temperature and humidity. Draw point two milliliters heparin sodium. Moisten a 50 mil syringe.Rig a tourniquet. Sterilize the elbow. Draw 30 mil peripheral blood from the elbow vein.Perform the first centrifugation. Divide peripheral blood equally into two 50 mil sterile centrifuge tubes. Put two tubes in horizontal rotors and then in the centrifuge under aseptic conditions.Centrifuge for six minutes with 800 G centrifugal force. Take the horizontal rotors out. Wear sterile gloves.Then take centrifuge tubes out. Observe the stratifications to make sure the peripheral blood is stratified into two layers. Collect the supernatent liquid from one centrifuge tube into a clean centrifuge tube.Repeat this step for another centrifuge tube. Add the supernatent layer liquid to the same centrifuge tube. Perform the second centrifugation.Add an equivalent amount of sterile water or normal saline into another clean centrifuge tube. Put the tubes in the horizontal rotors. Mark the one with the supernatent layer liquid by adhesive plaster.Centrifuge for five minutes at 1400 G centrifugal force. Take the horizontal rotors out. Choose the marked tube.And by observing the stratifications, check that the liquid of the marked tube is divided into three layers. Use a 10 mil syringe to draw four mil liquid from the middle granular cell layer which is the PRP layer and part of the bottom layer of supernatent. If the middle layer quantity is sufficient, just draw four mil from that.Put point four mil of the liquid in a sterile anticoagulation tube for evaluation, leaving 3.6 mil of liquid remaining in the syringe. Let the patient lie supine on the operating table and bend the knee to 90 degrees. Locate the puncture site at the interior margin of the patella and one centimeter from the lateral patellar ligament.Wear sterile gloves, perform skin sterilization on the puncture site three times with anerdian three. Cover with an aseptic hole towel. Place the syringe parallel to the tibial plateau, then perform the puncture at an angle of 45 degrees.The needle should completely insert into the skin. Inject the 3.6 mil PRP from the syringe into the knee joint cavity. Cover the puncture position with sterile gauze and fix it with adhesive plaster.Apply pressure to the wound for 10 minutes. Observe for any severe adverse reaction for 30 minutes. The entire treatment consists of a total of three injections administered at monthly intervals.Evaluate the concentration of blood platelets in the PRP. We use the point four mil PRP from the anticoagulation tube. Analyze the blood platelet concentration of the PRP using an automatic blood cell analyzer.Evaluate the postoperative outcome of the PRP injection. with a consultation one day before each of the three treatments, conduct further patient followup one day after each treatment, three days after each treatment, one week after each treatment, one month after the third treatment, three months after the third treatment, and six months after the third treatment. Use a visual analog scale, The Western Ontario and McMaster Universities Osteoarthritis Index, Knee Society Score, and lysholm knee functional scale to evaluate the postoperative effect.The platelet count of the PRP prepared in the Jove video reached a standard concentration level of 1121 times 10 to the third per microliter. We conducted the 15 followup surveys described in the protocol on a 55 year old male patient with early KAO, it was obvious that early outcome was satisfactory after the intra-articular administration of PRP. However, medium term efficacy was slightly inferior.A markedly significant analgesic effect was observed. KSS knee score was higher than KSS function score which meant the effect of PRP on subjective symptoms was better than on objective symptoms. The lysholm knee functional scale scores indicated that our method had an evidential effect in improving cartilage injury and soft tissue injury symptoms.Overall, the therapeutic effects of our PRP protocol were notable. It's widely believed that blood platelet concentration of PRP should reach about one million per microliter, which is three to five times normal concentrations. Therefore, a minimum requirement for this protocol is the blood platelet concentration of PRP is between these levels.To obtain blood platelets in higher concentrations, we use double centrifugation. The collection rates of blood platelets is related to centrifugal force and time. Through the exploration of our team, we established an optimal centrifugation time for double centrifugation of six and five minutes for the first and second centrifuge.And force is of 800 and 1400 G respectively to prepare the PRP. Finally, for the evaluation of patient outcomes, we used a number of scales. In summary, intra-articular injection of PRP is an effective treatment for knee osteoarthritis.This study report provides the whole treatment procedure in detail, including the preparation of PRP of reliable quality, the introduction of a standard injection procedure, and a scientific and practical evaluation plan.

preparation-procedures-evaluation-platelet-rich-plasma-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

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 lesion in the Achilles tendon; prepare PRP and inject it (or the saline solution); remove the tendon; and perform biomechanical, molecular, and histological evaluations. At each step, all the procedures and methods are described in detail, so they can be reproduced easily.
Achilles tendons have been surgically sectioned (removal of a 5-mm long section). Afterwards, PRP or saline solution was injected to study whether PRP has a positive effect on the healing of the tendon. Three groups of 40 animals (a total of 120 rats were used in this study) were subdivided into 2 subgroups: PRP injection group and a saline injection control group. Rats were sacrificed at increasing time points (Group A: 5 days; Group B: 15 days; Group C: 30 days) and tendons were removed. 90 tendons underwent biomechanical testing before performing transcriptomic analysis and the 30 remaining tendons were submitted to histological analysis.

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

This protocol describes the evaluation process of healing tendons in rats that have been injected with allogeneic platelet rich plasma (PRP) or saline solution after removing part of the Achilles tendon. The progress of tendon healing is evaluated at several time points using different types of analyses.

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 ...

Osteoarthritis Pain Model Induced by Intra-Articular Injection of Mono-Iodoacetate in Rats

The current animal models of osteoarthritis (OA) can be divided into spontaneous models and induced models, both of which aim to simulate the pathophysiological changes of human OA. However, as the main symptom in the late stage of OA, pain affects the patients&#39; daily life, and there are not many available models. The mono-iodoacetate (MIA)-induced model is the most widely used OA pain model, mainly used in rodents. MIA is an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, which causes chondrocyte death, cartilage degeneration, osteophyte, and measurable changes in animal behavior. Besides, expression changes of matrix metalloproteinase (MMP) and pro-inflammatory cytokines (IL1 &#946; and TNF &#945;) can be detected in the MIA-induced model. Those changes are consistent with OA pathophysiological conditions in humans, indicating that MIA can induce a measurable and successful OA pain model. This study aims to describe the methodology of intra-articular injection of MIA in rats and discuss the resulting pain-related behaviors and histopathological changes.

This study describes the method of intra-articular injection of mono-iodoacetate in rats and discusses the resulting pain-related behaviors and histopathological changes, which provide references for future applications.

The current animal models of osteoarthritis (OA) can be divided into spontaneous models and induced models, both of which aim to simulate the ...

Platelet-Rich Plasma Lysate for Treatment of Eye Surface Diseases

Various ocular surface diseases are treated with blood-derived eye drops. Their use has been introduced in clinical practice because of their metabolite and growth factor content, which promotes eye surface regeneration. Blood-based eye drops can be prepared from different sources (i.e., whole blood or platelet apheresis donation), as well as with different protocols (e.g., different dilutions and freeze/thaw cycles). This variability hampers the standardization of clinical protocols and, consequently, the evaluation of their clinical efficacy. Detailing and sharing the methodological procedures may contribute to defining common guidelines. Over the last years, allogenic products have been diffusing as an alternative to the autologous treatments since they guarantee higher efficacy standards; among them, the platelet-rich plasma lysate (PRP-L) eye drops are prepared with simple manufacturing procedures. In the transfusion medicine unit at AUSL-IRCCS di Reggio Emilia, Italy, PRP-L is obtained from platelet-apheresis donation. This product is initially diluted to 0.3 x 109 platelets/mL (starting from an average concentration of 1 x 109 platelets/mL) in 0.9% NaCl. Diluted platelets are frozen/thawed and, subsequently, centrifuged to eliminate debris. The final volume is split into 1.45 mL aliquots and stored at &#8722;80 &#176;C. Before being dispensed to patients, eye drops are tested for sterility. Patients may store platelet lysates at &#8722;15 &#176;C for up to 1 month. The growth factor composition is also assessed from randomly selected aliquots, and the mean values are reported here.

Platelet lysates represent an emerging tool for the treatment of ocular surface diseases. Here, we propose a method for the preparation, dispensation, storage, and characterization of platelet lysate collected from platelet donors.

Various ocular surface diseases are treated with blood-derived eye drops. Their use has been introduced in clinical practice because of their metabolite ...

FSN Therapy for Knee Osteoarthritis Pain Management

Fu's Subcutaneous Needling for Knee Osteoarthritis Pain

Fu's subcutaneous needling (FSN) is a new acupuncture and dry needling technique based on traditional Chinese medicine. It rapidly produces long-lasting effects in soft tissue injuries, particularly in painful musculoskeletal conditions, by providing stimulation primarily in the subcutaneous area. Osteoarthritis (OA) is the most common joint disease in adults worldwide and is often accompanied by a painful syndrome of structural changes in the peripheral joints of the knee. However, the etiology of OA pain is not fully understood, though myofascial trigger points (MTrPs) are commonly found in the lower limb muscles (so-called "tightened muscles") of patients with knee OA. 
FSN has been used in many fields for the treatment of acute pain problems and can relieve muscle contraction from MTrPs, thereby improving the local circulation. This study recruited patients with pain from knee OA into an FSN group or a transcutaneous electrical nerve stimulation (TENS) group with three treatment sessions and a follow-up over the course of 2 weeks. The results showed that FSN was effective in treating soft tissue pain around the knee with OA. This study aimed to establish and visualize three key technical indicators during FSN therapy, including the FSN needle insertion point and layer; the frequency and duration of the swaying movement; and the manipulation of the reperfusion approach. These findings have great potential for future applications in myofascial pain treatment, especially for pain management. Following this protocol could enhance FSN skills.

Author Spotlight: Fu's Subcutaneous Needling for Knee Osteoarthritis Pain  

We present a protocol for using Fu's subcutaneous needling for knee osteoarthritis pain, which combines swaying movement and reperfusion approach techniques. This protocol has great potential for future applications in myofascial pain treatment and could enhance Fu's subcutaneous needling (FSN) manipulation skills.

Fu's subcutaneous needling (FSN) is a new acupuncture and dry needling technique based on traditional Chinese medicine. It rapidly produces long-lasting ...

Treating Knee Osteoarthritis with Tuina in a Rabbit Model

Tuina Intervention in Rabbit Model of Knee Osteoarthritis

Knee osteoarthritis (KOA) is mainly characterized by degenerative changes in the knee joint's cartilage and surrounding soft tissues. The efficacy of Tuina in treating KOA has been confirmed, but the underlying mechanism needs to be investigated. This study aims to establish a scientifically feasible KOA rabbit model treated with Tuina to reveal the underlying mechanisms. For this, 18, 6-month-old normal-grade male New Zealand rabbits were randomly divided into sham, model, and Tuina groups, with 6 rabbits in each group. The KOA model was established by injecting 4% papain solution into the knee joint cavity. The Tuina group was intervened with Tuina combined with the knee joint rotary correction method for 4 weeks. Only the standard grasping and fixation were performed in sham and model groups. At the end of the 1-week intervention, the knee joint range of motion (ROM) was observed, and cartilage hematoxylin-eosin (HE) staining was done. The study shows that Tuina could inhibit chondrocyte apoptosis, repair cartilage tissue, and restore knee joint ROM. In conclusion, this study demonstrates the scientific feasibility of Tuina treatment for KOA model rabbits, highlighting its potential application in the study of KOA and similar knee joint-related conditions.

Author Spotlight: Using a Rabbit Model to Explore the Efficacy of Tuina in Treating Knee Osteoarthritis 

The protocol describes a method for Tuina intervention in a rabbit model of knee osteoarthritis.

Knee osteoarthritis (KOA) is mainly characterized by degenerative changes in the knee joint's cartilage and surrounding soft tissues. The efficacy of ...

Experimental Modeling of Knee Osteoarthritis in Rats

After anesthetizing the rat, use a shaving machine to remove the hair of the right hind limb. Then, place a medical cotton pad between the right ankle and hip joint. Fix the right knee joint at 180 degree extension using five to six layers of wet plaster bandage evenly.Spiral wrap the plaster bandage starting from the ankle and covering one-third of the previous one. Use a hairdryer to dry and harden the plaster. After the plaster bandage dries and hardens, mix the denture base materials to make it sticky.And externally wrap the plaster with denture based materials to fix and prevent it from gnawing. Once the mixture hardens, turn off the anesthesia machine and supervise the blood circulation in the terminal limb. Then, wait for the rat to wake up naturally.After three weeks of continuous immobilization, using surgical scissors, cut off the denture base material outside and plaster bandage. Rinse the lower limb of the rat with saline and dry it with gauze.

Establishment of the KOA Model in Rabbits Using the Modified Videman Method

Application of Acupotomy in a Knee Osteoarthritis Model in Rabbit

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of people with KOA. Among several pathogenic factors, the biomechanical imbalance of the knee joint is one of the main causes of KOA. Acupotomology believes that restoring the mechanical balance of the knee joint is the key to treating KOA. Clinical studies have shown that acupotomy can effectively reduce pain and improve knee mobility by reducing adhesion, contracture of soft tissues, and stress concentration points in muscles and tendons around the knee joint. 
In this protocol, we used the modified Videman method to establish a KOA model by immobilizing the left hindlimb in a straight position. We have outlined the method of operation and the precautions related to acupotomy in detail and evaluated the efficacy of acupotomy in conjunction with the theory of "Modulating Muscles and Tendons to Treat Bone Disorders" through the detection of the mechanical properties of quadriceps femoris and tendon, as well as cartilage mechanics and morphology. The results show that acupotomy has a protective effect on cartilage by adjusting the mechanical properties of the soft tissues around the knee joint, improving the cartilage stress environment, and delaying cartilage degeneration.

Author Spotlight: Investigating the Mechanism of Action of Acupotomy in Treating Knee Osteoarthritis

In this protocol, a knee osteoarthritis model was prepared using the modified Videman method, and the operation procedures and precautions of acupotomy are detailed. The effectiveness of acupotomy has been demonstrated by testing the mechanical properties of quadriceps femoris and tendon and the mechanical and morphological properties of cartilage.

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of ...

Therapeutic Potential of Tuina Massage for Knee Osteoarthritis

Tuina Intervention in Sodium Monoiodoacetate Injection-Induced Rat Model of Knee Osteoarthritis

Knee osteoarthritis (KOA), a common degenerative joint disorder, is characterized by chronic pain and disability, which can progress to irreparable structural damage of the joint. Investigations into the link between articular cartilage, muscles, synovium, and other tissues surrounding the knee joint in KOA are of great importance. Currently, managing KOA includes lifestyle modifications, exercise, medication, and surgical interventions; however, the elucidation of the intricate mechanisms underlying KOA-related pain is still lacking. Consequently, KOA pain remains a key clinical challenge and a therapeutic priority. Tuina has been found to have a regulatory effect on the motor, immune, and endocrine systems, prompting the exploration of whether Tuina could alleviate KOA symptoms, caused by the upregulation of inflammatory factors, and further, if the inflammatory factors in skeletal muscle can augment the progression of KOA.
We randomized 32 male Sprague Dawley (SD) rats (180-220 g) into four groups of eight animals each: antiPD-L1+Tuina (group A), model (group B), Tuina (group C), and sham surgery (group D). For groups A, B, and C, we injected 25 &#181;L of sodium monoiodoacetate (MIA) solution (4 mg MIA diluted in 25 &#181;L of sterile saline solution) into the right knee joint cavity, and for group D, the same amount of sterile physiological saline was injected. All the groups were evaluated using the least to most stressful tests (paw mechanical withdrawal threshold, paw withdrawal thermal latency, swelling of the right knee joint, Lequesne MG score, skin temperature) before injection and 2, 9, and 16 days after injection.

Author Spotlight: Treating Knee Osteoarthritis with Tuina - A New Perspective 

This protocol describes the methods of Tuina intervention in sodium monoiodoacetate injection-induced rat model of knee osteoarthritis (KOA), which provides a reference for the application of Tuina in KOA animal models. This protocol also studies the effective mechanism of Tuina for KOA, and the results will help promote its application.

Knee osteoarthritis (KOA), a common degenerative joint disorder, is characterized by chronic pain and disability, which can progress to irreparable ...

Tuina Intervention in a Sodium Monoiodoacetate Injection-Induced Rat Model of Knee Osteoarthritis

Managing Knee Osteoarthritis Using Ultrasound-Guided Acupotomy

Clinical Effects of Ultrasound-Guided Acupotomy in Knee Osteoarthritis Treatment

The protocol presented here demonstrates the operation method of ultrasound-guided acupotomy for knee osteoarthritis (KOA), including patient recruitment, preoperative preparation, manual operation, and postoperative care. The purpose of this protocol is to relieve pain and improve knee function in patients with KOA. A total of 60 patients with KOA admitted between June 2022 and June 2023 were treated with ultrasound-guided acupotomy. Pathological changes and knee function scores were compared before and after the treatment. After 1 week of treatment, the synovial thickness of the suprapatellar bursae was significantly lesser than before treatment (p &lt; 0.05), the Hospital for Special Surgery Knee Score (HSS) was significantly higher than before treatment (p &lt; 0.05), the Visual analogue scale (VAS) was significantly lower than those of the control group (p &lt; 0.05) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) were significantly lower than those of the control group (p &lt; 0.05). Therefore, ultrasound-guided acupotomy for the treatment of KOA can reduce synovial thickness, relieve pain, improve knee joint function, and have a remarkable curative effect.

Author Spotlight: Minimally Invasive Ultrasound-Guided Acupotomy in Knee Osteoarthritis Treatment

This paper introduces the operation method of ultrasound-guided acupotomy for knee osteoarthritis, which can reduce synovial thickness and improve knee joint function. It has the advantages of precise target, low treatment risk, few complications, and high safety.

The protocol presented here demonstrates the operation method of ultrasound-guided acupotomy for knee osteoarthritis (KOA), including patient recruitment, ...