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/pubmed?term=((Age-related+changes+in+the+musculoskeletal+system+and+the+development+of+osteoarthritis%5BTitle%5D)+AND+%22Clinics+in+Geriatric+Medicine%22%5BJournal%5D)">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/pubmed?term=((Radiofrequency+procedures+to+relieve+chronic+knee+pain%3A+an+evidence-based+narrative+review%5BTitle%5D)+AND+%22Reg+Anesth+Pain+Med%22%5BJournal%5D)">Radiofrequency procedures to relieve chronic knee pain: an evidence-based narrative review.</a> Reg Anesth Pain Med. 43 (1), 72(2018).</li>
<li>Sánchez, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+randomized+clinical+trial+evaluating+plasma+rich+in+growth+factors+%28PRGF-endoret%29+versus+hyaluronic+acid+in+the+short-term+treatment+of+symptomatic+knee+osteoarthritis%5BTitle%5D)+AND+%22Arthroscopy+the+Journal+of+Arthroscopic+%26+Related+Surgery%22%5BJournal%5D)">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 & Related Surgery. 28 (8), 1070-1078 (2012).</li>
<li>LaPrade, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biologic+treatments+for+sports+injuries+II+think+tank-current+concepts%2C+future+research%2C+and+barriers+to+advancement%2C+part+1%3A+biologics+overview%2C+ligament+injury%2C+tendinopathy%5BTitle%5D)+AND+%22Am+J+Sports+Med%22%5BJournal%5D)">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/pubmed?term=((Does+platelet-rich+plasma+have+a+role+in+the+treatment+of+osteoarthritis%3F%5BTitle%5D)+AND+%22Joint+Bone+Spine%22%5BJournal%5D)">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/pubmed?term=((Platelet-rich+plasma+for+osteoarthritis+treatment%5BTitle%5D)+AND+%22Revista+Brasileira+de+Reumatologia+%28English+Edition%29%22%5BJournal%5D)">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/pubmed?term=((Growth+factor+levels+in+platelet-rich+plasma+and+correlations+with+donor+age%2C+sex%2C+and+platelet+count%5BTitle%5D)+AND+%22J+Craniomaxillofac+Surg%22%5BJournal%5D)">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/pubmed?term=((Quantification+of+growth+factor+levels+using+a+simplified+method+of+platelet-rich+plasma+gel+preparation%5BTitle%5D)+AND+%22Journal+of+Oral+%26+Maxillofacial+Surgery%22%5BJournal%5D)">Quantification of growth factor levels using a simplified method of platelet-rich plasma gel preparation.</a> Journal of Oral & 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/pubmed?term=((Contents+and+formulations+of+platelet-rich+plasma%5BTitle%5D)+AND+%22Operative+Techniques+in+Orthopaedics%22%5BJournal%5D)">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/pubmed?term=((Platelet+rich+plasma+for+biological+therapy%3A+applications+and+limits%5BTitle%5D)+AND+%22Operative+Techniques+in+Orthopaedics%22%5BJournal%5D)">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/pubmed?term=((A+call+for+standardization+in+platelet-rich+plasma+preparation+protocols+and+composition+reporting%3A+a+systematic+review+of+the+clinical+orthopaedic+literature%5BTitle%5D)+AND+%22Journal+of+Bone+%26+Joint+Surgery-american%22%5BJournal%5D)">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 & 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/pubmed?term=((Platelet-rich+plasma%3A+the+PAW+classification+system%5BTitle%5D)+AND+%22Arthroscopy+the+Journal+of+Arthroscopic+%26+Related+Surgery%22%5BJournal%5D)">Platelet-rich plasma: the PAW classification system.</a> Arthroscopy the Journal of Arthroscopic & 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/pubmed?term=((Quantification+of+platelets+obtained+by+different+centrifugation+protocols+in+SHR+rats%5BTitle%5D)+AND+%22Revista+Brasileira+de+Ortopedia+%28English+Edition%29%22%5BJournal%5D)">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/pubmed?term=((Antimicrobial+activity+of+platelet-leukocyte+gel+against+Staphylococcus+aureus%5BTitle%5D)+AND+%22Journal+of+Orthopaedic+Research%22%5BJournal%5D)">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/pubmed?term=((Platelet-rich+plasma+nonoperative+injection+therapy-A+review+of+indications+and+evidence%5BTitle%5D)+AND+%22Operative+Techniques+in+Sports+Medicine%22%5BJournal%5D)">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/pubmed?term=((Quantification+of+platelets+and+platelet+derived+growth+factors+from+platelet-rich-plasma+%28PRP%29+prepared+at+different+centrifugal+force+%28g%29+and+time%5BTitle%5D)+AND+%22Transfusion+%26+Apheresis+Science%22%5BJournal%5D)">Quantification of platelets and platelet derived growth factors from platelet-rich-plasma (PRP) prepared at different centrifugal force (g) and time.</a> Transfusion & Apheresis Science. 54 (1), 103-110 (2016).</li>
<li>Cohn, C. S., Lockhart, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autologous+platelet-rich+plasma%3A+evidence+for+clinical+use%5BTitle%5D)+AND+%22Current+Opinion+in+Hematology%22%5BJournal%5D)">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><tbody><tr><td>Centrifuge</td><td>Eppendorf</td><td>5702</td><td></td></tr><tr><td>Centrifuge tube</td><td>CORNING</td><td>430828</td><td></td></tr><tr><td>Horizontal rotor</td><td>Eppendorf</td><td>LL080</td><td></td></tr><tr><td>Anerdian III</td><td>Shanghai Likang Disinfectant HiTech Co., LTD</td><td>310173</td><td>Disinfect the skin</td></tr><tr><td>1ml Syringe</td><td>KDL&amp;nbsp; Medical Equipment Co., LTD</td><td>0.4*13 RWLB</td><td></td></tr><tr><td>10ml Syringe</td><td>KDL&amp;nbsp; Medical Equipment Co., LTD</td><td>1.2*38 TWSB</td><td></td></tr><tr><td>50ml Syringe</td><td>KDL&amp;nbsp; Medical Equipment Co., LTD</td><td>0.7*32 TWLB</td><td></td></tr><tr><td>Medical Tourniquet</td><td>Changzhou Jinli Latex Products Co., LTD</td><td>0087-2011</td><td></td></tr><tr><td>Single-use sterile rubber surgical gloves</td><td>Shanghai jinxiang Latex Products Co., LTD</td><td>17060</td><td></td></tr><tr><td>Disposable Draw Blood Needle</td><td>KDL&amp;nbsp; Medical Equipment Co., LTD</td><td>0.55*20 L(II) RWLB</td><td></td></tr><tr><td>Heparin Sodium Injection</td><td>SPH No.1 Biochemical &amp;amp; Pharmaceutical Co., LTD</td><td>1706101</td><td>2ml:12500U</td></tr><tr><td>Jifro Hand Antiseptic Rinse Free Gel</td><td>Shanghai Likang Disinfectant HiTech Co., LTD</td><td>311793</td><td></td></tr><tr><td>Medical Cotton Swab</td><td>Foshan Kangzheng Medical Supplies Co., LTD</td><td>KZ3-12</td><td>Disinfect the skin</td></tr><tr><td>10ml Normal Saline&amp;nbsp;</td><td>Jiangxi Shuangshi Pharmecutical Co., LTD</td><td>140211458</td><td></td></tr><tr><td>Automatic Blood Cell Analyzer</td><td>Beckman Coulter</td><td>LH-750</td><td></td></tr><tr><td>Hole-towe</td><td></td><td></td><td>Sterile</td></tr><tr><td>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><td>Tweezers</td><td>RWD LIFE SCIENCE</td><td>F12006-10</td><td></td></tr><tr><td>Sterile Gauze</td><td>Guangdong Ze Chang Trade Co., LTD</td><td>170915</td><td></td></tr><tr><td>Adhesive Plaster</td><td>3M Transpore</td><td>1527C-0</td><td></td></tr><tr><td>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...

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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 ...

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18.2K 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

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 ...

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

Immunology and Infection

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

Bioengineering

Preparing Activated Platelet-Rich Plasma for Culturing Human Adipose-Derived Stem Cells

Activated platelet-rich plasma (PRP) prepared from whole blood via centrifugation demonstrated a proliferation-stimulating effect in several kinds of cultured cells, implying a possible use in regenerative medicine. Here, a double-spin method was used to prepare PRP from whole blood. PRP was further activated by autologous thrombin. The platelet count was measured in the activated PRP and the proliferation-stimulating effect in human adipose-derived stem cells (hASCs) was examined. The resulting platelet count was 11.5-times higher in PRP than in whole blood plasma. The proliferation of hASCs was markedly enhanced by incubation with 1% PRP. The described method can be used to reproducibly prepare PRP with a high concentration of platelets. PRP prepared by this method markedly promotes proliferation of hASCs.

In this report, we describe a double-spin method for preparing activated platelet-rich plasma (PRP). Using autologous thrombin, human adipose-derived stem cells (hASCs) were cultured. Activated PRP was shown to promote proliferation of hASCs.

Activated platelet-rich plasma (PRP) prepared from whole blood via centrifugation demonstrated a proliferation-stimulating effect in several kinds of ...

Developmental Biology

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 ...

Mechanical and Controlled PRP Injections in Patients Affected by Androgenetic Alopecia

23 patients (18 male and 5 female) aged 21-70 years who displayed male pattern hair loss (MPHL) in Stage 1 to Stage 5 as determined by the Norwood-Hamilton classification scale, and female pattern hair loss (FPHL) in Stage 1 to Stage 2 as determined by the Ludwig classification scale, were treated with non-activated autologous platelet-rich plasma (A-PRP). Autologous blood (55 mL) was harvested using sodium citrate as an anticoagulant. A-PRP (23 mL) was produced for all cases using a closed system according to the transfusion service protocol. Following centrifugation (260 x g for 10 min) the A-PRP was inserted in a laser light selector device, and after the centrifugation, 9 mL of A-PRP was collected.
The scalp of the patients affected by androgenetic alopecia (AGA) was divided into four areas (frontal, parietal, vertex, and occipital); local anesthesia was not performed. Interfollicular A-PRP injections (0.2 mL x cm2) were performed by controlled and mechanical injections scheduled at a depth of 5 mm using a medical injector gun. Treatment sessions were performed with a 30-day interval. For each patient, three treatment sessions were performed.
PRP was injected in the androgen-related areas of scalp affected by hair loss. Placebo (normal saline solution) was loaded in another syringe (10 mL) and injected on the adjacent side in a similar fashion.

This paper aims to describe the interfollicular autologous platelet-rich plasma injections (0.20 mL x cm2) in the scalp of 23 patients affected by androgenetic alopecia at a depth of 5 mm using a medical injector gun equipped with a 30 gauge needle, in three sessions.

23 patients (18 male and 5 female) aged 21-70 years who displayed male pattern hair loss (MPHL) in Stage 1 to Stage 5 as determined by the ...

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of 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 ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...

Biology

Preparation of Platelet-Rich Plasma from Human Peripheral Blood

This video demonstrates the preparation of high-quality Platelet-Rich Plasma (PRP) by screening for platelet count, collecting and centrifuging blood, and isolating platelets. This PRP is used in therapeutic applications for tissue regeneration and healing.

This video demonstrates the preparation of high-quality Platelet-Rich Plasma (PRP) by screening for platelet count, collecting and centrifuging blood, and ...

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

Summary

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Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Preparing Activated Platelet-Rich Plasma for Culturing Human Adipose-Derived Stem Cells

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

Mechanical and Controlled PRP Injections in Patients Affected by Androgenetic Alopecia

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Platelet-Rich Plasma Lysate for Treatment of Eye Surface Diseases

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Preparation of Platelet-Rich Plasma from Human Peripheral Blood