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PRP and Ozone Injection Protocol

How studies combined ozone with platelet-rich plasma (PRP) injections: order of injection, ozone concentration and volume, and a general PRP preparation.

What are the basics of PRP and ozone injection?

• PRP is made from the patient's own blood. It is spun in a centrifuge to concentrate platelets, which release growth factors. It is injected into joints and soft tissue. Some practitioners add ozone in the same visit.
• Madrid gives no injection dose for ozone combined with PRP. In its dental section 7.2 it suggests activating PRP in the lab with ozone at 40 µg/mL for 30 seconds. At that dose the growth factors kept their properties. High doses upset fibrin clotting and damaged the growth factors.
• A lab study of plasma rich in growth factors found the way you mix matters. Mixing ozone and plasma in a syringe (20, 40 or 80 µg/mL) left the fibrin and growth factors unchanged. Bubbling ozone through the plasma in a continuous flow weakened the fibrin and cut the growth factors sharply. Mix by syringe and do not bubble.
• Studies did it two ways. Most injected the PRP first and then the ozone gas through the same needle straight away. One mixed the ozone gas with the PRP in a syringe before injecting.
• Dr. Ann Auburn, in a tutorial on regenerative injections, describes injecting ozone into the same spots right after the PRP. In her practice PRP is repeated about monthly. She draws about 60 mL of blood, and her machine concentrates platelets 4 to 6 times (this depends on the system you use).
• Madrid's general joint table gives 5 to 20 mL at 5 to 20 µg/mL for ozone alone. Most studies below used 10 to 20 µg/mL. One (Martínez 2026) used 40 µg/mL, twice the top of Madrid's joint range.

Sources: Madrid Declaration on Ozone Therapy, 4th edition (ISCO3, 2025), dental section 7.2 (PRP activated with ozone in the lab) and Table 1A; studies in the list below; a tutorial by Dr. Ann Auburn. Paraphrased.

What dose and technique are used for PRP and ozone injection?

These are the methods the studies used. They are not a Madrid dose, and they differ from each other.

All of these were knee studies. Do not read them as dosing for other joints or tissues.

What are the best tips when using PRP and ozone injection?

• Use siliconized, ozone-resistant syringes and put a bacterial filter on the gas line (Yeprem 2018).
• If you mix PRP and ozone in a syringe, shake gently and keep air bubbles out, then activate the PRP as your kit directs.
• Do not bubble ozone through the plasma. The lab study showed it weakens the fibrin and cuts the growth factors.
• One study had patients stop NSAIDs 7 days before the PRP injection and allowed acetaminophen for pain.
• PRP schedules in the studies (weekly to every 3 weeks) are shorter than the monthly spacing Dr. Auburn describes. Follow the protocol you were trained in.

What are the precautions for PRP and ozone injection, and who should avoid it?

• Never breathe ozone gas. Keep the room ventilated.
• Sterile technique every time. Madrid says most serious side effects trace back to malpractice, including technique, asepsis and antisepsis. Its reported serious events include septic arthritis of the knee after intra-articular infiltration and abscess after paravertebral injection. It also reports one death from sepsis after a paravertebral infiltration done without proper asepsis (Madrid Declaration on Ozone Therapy, 4th edition (ISCO3, 2025), section 2.5).
• Clean the skin with an antiseptic and let it dry before the needle goes through. Use sterile, single-use needles and syringes, and ozone-resistant syringes for the gas.
• Check with your licensing board that this procedure, and anything you inject along with the ozone, is within your scope of practice.
• Never inject ozone gas into a vein or an artery. Madrid strongly discourages both because of the risk of air embolism, and lists embolic events after paravertebral and intradiscal injections among its serious adverse events.
• PRP is a blood product. Check that your licensing board allows you to draw blood and inject a blood-derived product, and follow your PRP system's instructions for sterile handling.
• Platelets under 50,000 are on Madrid's contraindication list, so check a platelet count if the history raises any doubt.
• Septic arthritis of the knee after intra-articular infiltration is on Madrid's list of serious events. Aseptic technique at the joint is the main safeguard.

Contraindications

The Madrid Declaration lists these contraindications for systemic ozone (IV and blood). It gives no separate list for PRP plus ozone injection, but many practitioners use the same screening before treating:

• Direct inhalation of ozone gas. The fluid lining the lungs has no antioxidants to buffer it, so inhaled ozone is toxic.
• Glucose-6-phosphate dehydrogenase (G6PD) deficiency (favism, acute hemolytic anemia). Madrid lists it as a contraindication for systemic ozone, but whether it is absolute is debated. MediSkill protocols start low and go slow; see the contraindications article.
• Toxic hyperthyroidism (severe Graves' disease)
• Thrombocytopenia (platelets under 50,000) and severe coagulation disorders
• Severe cardiovascular instability
• Acute alcohol intoxication
• Recent acute myocardial infarction
• Acute massive hemorrhage
• Convulsive states
• Hemochromatosis
• Patients receiving intravenous copper or iron therapy

Pregnancy. Madrid advises avoiding ozone therapy in the first trimester (weeks 0 to 13) and reserves its use in pregnancy for specialists in gynecology and obstetrics.

More detail

Steps used in the studies

1. Draw blood from a vein under sterile technique (10 mL in Yeprem 2018; larger amounts with a kit).
2. Prepare the PRP with your centrifuge or kit, following its instructions.
3. Clean the skin over the joint with an antiseptic and let it dry.
4. Inject the PRP into the joint (lateral approach in Yeprem 2018).
5. Leaving the needle in place, attach an ozone-resistant syringe of ozone-oxygen gas (5 mL at 10 µg/mL in Yeprem 2018) and inject it.
6. Or mix equal parts PRP and ozone in a syringe, shake gently for 30 seconds without bubbles, and inject the mixture (Martínez 2026).

Equipment

• Centrifuge or PRP kit with its tubes
• Ozone generator with an ozone-resistant gas path (quartz glass, titanium, silicone)
• Medical oxygen tank and regulator
• Siliconized, ozone-resistant syringes and a bacterial filter
• Sodium citrate or kit anticoagulant, and calcium chloride if your kit activates PRP
• Antiseptic and sterile supplies

What the studies used

Study doses are listed as printed in each paper and are not Madrid dosing.

• In 120 people with knee osteoarthritis, ozone given right after PRP (5 mL at 10 µg/mL, weekly for 3 weeks) improved pain by 81% on the VAS (a 10-point pain scale), against 72% for PRP alone and 66% for ozone alone (p<0.01 for the combination). Ozone alone was given as 12 injections, the combination as 3 (Yeprem 2018).
• In 80 people with early knee osteoarthritis, PRP plus ozone gave about the same overall result as PRP alone, with lower pain at day 10 and less inflammation at the injection site (Dernek 2019, a retrospective comparison).
• In 49 adults with knee osteoarthritis, ozone-PRP (about 3 mL at 20 µg/mL, every 3 weeks for 3 injections) lowered pain less than concentrated growth factor (VAS fall 3.32 vs 4.32, p=0.028). Final pain and WOMAC (a knee pain and function questionnaire) scores did not differ (Eurasian Journal of Medicine 2026).
• In young people with kneecap cartilage pain, ozonated PRP (10 mL of ozone at 40 µg/mL mixed with 10 mL of PRP, plus a smaller amount into the kneecap bone; bone injection is outside this sheet) on days 0, 7 and 14 improved nearly every WOMAC item at 6 months. There was no comparison group (Martínez 2026).

Evidence is thin and mixed: one randomized trial, one comparison that favored a different product, and an uncontrolled cohort, all in the knee. Doses differ between studies and none compared ozone doses with each other.

Learn more

Background, benefits and research on injections: Ozone injections, full article. Knee technique: Knee ozone injection protocol.

Related protocols: Knee Ozone Injection Protocol, Hip Ozone Injection Protocol, Shoulder Ozone Injection Protocol.

Other injection protocols

For licensed practitioners. Educational reference only, not medical advice. Follow your scope of practice and local regulations.