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 in this step to replace the heparin sodium.</li>
	<li>Rig a tourniquet, sterilize the elbow 2-3 times, and use the moist 50-mL syringe to draw 30 mL of peripheral blood from the elbow vein.</li>
	<li>Perform the first centrifugation.
	<ol>
		<li>Divide peripheral blood equally into two 50 mL sterile centrifuge tubes.</li>
		<li>Put two tubes in horizontal rotors and then in the centrifuge, under aseptic conditions.</li>
		<li>Centrifuge for 6 minutes at 800 x g.</li>
		<li>Take the horizontal rotors out, wear sterile gloves, and take centrifuge tubes out.</li>
		<li>Observe the stratifications to make sure that the peripheral blood is stratified into two layers.</li>
		<li>Use a clean 10-mL syringe to collect the supernatant liquid from these two centrifuge tubes into a clean centrifuge tube.</li>
	</ol>
	</li>
	<li>Perform the second centrifugation.
	<ol>
		<li>Use a clean 10-mL syringe to add an equivalent amount of sterile water or normal saline into another clean centrifuge tube for balance. Put the tubes in the horizontal rotors. Mark the one with the supernatant layer liquid by adhesive plaster.</li>
		<li>Centrifuge for 5 minutes at 1400 x g.</li>
		<li>Take the horizontal rotors out, and by observing the stratifications check that the liquid of the marked tube is divided into three layers.</li>
		<li>Use a 10-mL syringe to draw 4 mL of liquid from the middle granular cell layer (leukocyte-rich, PRP layer) and the bottom layer of supernatant. If the middle layer quantity is sufficient, just draw 4 mL from that.</li>
		<li>Put 0.4 mL of the liquid in a sterile anticoagulation tube (K2EDTA, 3.6 mg) for evaluation, leaving 3.6 mL of liquid remaining in the syringe. 
		NOTE: The protocol can be paused here.</li>
	</ol>
	</li>
</ol>
2. Intra-Articular Administration of PRP
<ol>
	<li>Let the patient lie supine on the operating table and bend the knee to 90 degrees.</li>
	<li>Locate the puncture site at the inferior margin of the patella and 1 cm from the lateral patellar ligament. Use a marker pen to mark the site.</li>
	<li>Perform skin sterilization on the puncture site three times with anerdian III, wearing sterile gloves, and cover with an aseptic hole-towel.</li>
	<li>Place the syringe parallel to the tibial plateau, and then perform the puncture at an angle of 45 degrees. Completely insert the needle into the skin.</li>
	<li>Inject the 3.6 mL of PRP from the syringe into the knee joint cavity.</li>
	<li>Cover the puncture position with sterile gauze and fix it with adhesive plaster.</li>
	<li>Apply pressure to the wound for 10 minutes. Observe for any severe adverse reaction for 30 minutes.</li>
	<li>Administer a total of three injections at monthly intervals. 
	NOTE: The protocol can be paused here.</li>
</ol>
3. Postoperative Evaluation of PRP Injection
<ol>
	<li>Evaluate the concentration of blood platelets in the PRP.
	<ol>
		<li>Use the 0.4 mL of PRP from the anticoagulation tube (K2EDTA, 3.6 mg).</li>
		<li>Analyze the blood platelet concentration of the PRP using an automatic blood cell analyzer.</li>
	</ol>
	</li>
	<li>Evaluate the postoperative outcome of the PRP injection.
	<ol>
		<li>With a consultation 1 day before each of the three treatments, conduct further patient follow-up 1 day after each treatment, 3 days after each treatment, 1 week after each treatment, 1 month after the third treatment, 3 months after the third treatment, and 6 months after the third treatment.</li>
		<li>Use a visual analog scale (VAS), the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Knee Society Score (KSS), and Lysholm knee functional scale to evaluate the postoperative effect.</li>
	</ol>
	</li>
</ol>

<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 authors have nothing to disclose.

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 ...

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

Research

JoVE Journal

Medicine

17.9K Views.  Guangdong General Hospital (Guangdong Academy of Medical Sciences). 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.

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

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

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 ...

Using a Knee Arthrometer to Evaluate Tissue-specific Contributions to Knee Flexion Contracture in the Rat

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ROM is called a joint contracture and results in increased morbidity. Due to the difficulty of reversing established knee contractures, early detection is important, and hence, knowing risk factors for their development is essential. The rat represents a good model with which the effect of an intervention can be studied due to the similarity of rat knee anatomy to that of humans, the rat's ability to tolerate long durations of knee immobilization in flexion, and because mechanical data can be correlated with histologic and biochemical analysis of knee tissue.
Using an automated arthrometer, we demonstrate a validated, precise, reproducible, user-independent method of measuring the extension ROM of the rat knee joint at specific torques. This arthrometer can be used to determine the effects of interventions on knee joint ROM in the rat.

The goal of the protocol is to measure the extension range of motion of the rat knee. The effects of various diseases that increase the stiffness of the knee joint and the effectiveness of treatments can be quantified.

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ...

The Lower Body Positive Pressure Treadmill for Knee Osteoarthritis Rehabilitation

Here, based on a clinician&rsquo;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies.

Here, based on a clinician&#8217;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies.

Here, based on a clinician&rsquo;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in ...

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 ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

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 ...

Standardized Tuina Manipulation for Knee Osteoarthritis in Rats

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

Author Spotlight: Understanding the Mechanism of Tuina Manipulation in Rats 

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...