The Use of Autologous Protein Solution (Pro-Stride^®) and Leukocyte-Rich Platelet-Rich Plasma (Restigen^®) in Canine Medicine

Figures & data

Figure 1 The timing and cell types involved in wound repair demonstrating the role of WBC (including neutrophils, macrophages, and lymphocytes) in successful healing.

Note: Adapted from King W, Toler K, Woodell-May J. Role of White Blood Cells in Blood-and Bone Marrow-Based Autologous Therapies. BioMed Res Int. 2018;2018. Creative Commons license and disclaimer available from: http://creativecommons.org/licenses/by/4.0/ legalcode.²²

Table 1 Growth Factors and Their Proposed Functions in PRP

Name	Abbreviation	Proposed Function in PRP
Transforming Growth Factor-Beta	TGF-β	Combined with PDGF and IGF: Chemotaxis for stem cells and osteoblasts, angiogenesis, bone matrix formation, and collagen synthesis^Citation64
Vascular Endothelial Growth Factor	VEGF	Endothelial cell proliferation^Citation65
Hepatocyte Growth Factor	HGF	Mediates anti-inflammatory effects^Citation66
Fibroblast Growth Factor	FGF	Prolonged endothelial cell growth^Citation65
Epidermal Growth Factor	EGF	Enhances angiogenesis^Citation67
Platelet-Derived Growth Factor	PDGF	Alone, chemotactic for macrophages. Combined with TGF-β assists in bone mineralization^Citation64
Insulin-like Growth Factor	IGF	Combined with PDGF and TGF-β: Chemotaxis for stem cells and osteoblasts, angiogenesis, bone matrix formation, and collagen synthesis^Citation64

Table 2 Survey for Canine Owners at Beginning of Lameness Evaluations

Step	Lameness Evaluation Owner Questions
1	What limb is the patient favoring?
2	How did the owner perceive that the injury/lameness occurred?
3	When did the injury/lameness occur?
4	Has the owner noticed any swelling, heat, sores, or wounds on the limbs?
5	Is the lameness getting worse or improving?
6	What % of the time is the patient using the leg?
7	Is the owner administering any type of pain or anti-inflammatory medications?
7a	What medication(s) are they using, what dose are they giving and how frequent are they administering the medication(s), and how long have they been administering the medication(s). (incl. CBD oil, essential oils, and other supplements)
8	Has the patient been seen previously for this injury/lameness in the past?
8a	If so, how long ago did the injury/lameness occur?
8b	Did the treatment plan work at that time?
8c	If so, for how long did the patient stay comfortable?
8d	Were any diagnostics performed at this previous injury/lameness evaluation?

Table 3 Physical/Neurologic Examination Steps

Step	Physical/Neurologic Examination Steps
1	Physical exams started with a technician taking down notes on weight, temperature, and the diet that that was being fed to the patient. The technician also recorded medical history from the owner.
2	The doctor then reviewed the information collected by the technician and then began the rest of the physical exam.
3	Head: On the head we looked at ears, eyes, nose, mouth, and teeth, and any cranial nerve damages. We checked for discharge from the eyes and nose, normal appearance of structures, detailed oral exam to evaluate condition of the patient’s teeth, character of the mucous membranes to assess hydration and blood circulation. Sometimes tools such as otoscopes and ophthalmoscopes were used to get more detailed information for the ears and eyes.
4	Skin and Coat: We took notes on the condition of the coat and skin. Finding ticks, and lumps was important because ticks can transmit diseases that can cause shifting lameness in our patients and lumps can grow in locations that can put pressure on nerves and muscle tissues that could also cause lameness. Dehydration was also accessed using skin tent techniques during this portion of the exam. Looking for wounds, sores, and or swelling was also helpful with diagnosing reasons for injuries/lameness.
5	The Chest: Listening to the heart and lung with a stethoscope helped in diagnosing heart conditions such as murmurs and lung complications. Circulation was also evaluated by checking pulses to make sure they were in sync with the heart rate.
6	Orthopedic: The orthopedic portion of the exam included a variety of steps: Assessing the symmetry (or lack thereof) of the musculature, observing how the pet moved/ambulated, and physically manipulating limbs and their joints. Manipulation limbs and joints allowed us to access the range of motion and laxity of the joints. During this portion of the exam, it was important to be mindful of swelling and heat over joints and long bones as this could indicate reasons for injuries/lameness. The spine was assessed individually, feeling down every intervertebral junction to identify painful spots.
7	The Abdomen: When palpating the abdomen, abdominal masses, abnormal organ sizes, and the condition of the gastrointestinal and urinary tracts were felt for. The patient’s body score condition for obesity were also evaluated.
8	Lymph Nodes: The lymph nodes that are easily palpated such as the submandibular, popliteal, and prescapular for enlargement were checked, as these could indicate disease processes.
9	Neurologic: Reflexes were checked for delayed or absent responses. When there were severe neurologic deficits, the toes were pinched and assessed for withdraw. Tests were also performed on the subjects to check for appropriate proprioception.

Figure 2 Blood draw process from canine patients. (A) Blood draw sites were prepared by clipping area over the jugular vein and then the skin was aseptically cleaned. (B) The needle was inserted into the jugular vein and the syringe was slowly pulled back to check for a flash of blood to confirm needle placement. (C) The blood was slowly drawn while rocking syringe to ensure mixture of blood and anti-coagulant.

Figure 3 Representative pictures of 60mL L-PRP device processing: 1) a 18-gauge needle was attached to a 60mL syringe and 5–8mL of ACD-A was withdrawn. 2) The cap was unscrewed on center port of the L-PRP device and the green packaging post was discarded. Blood was slowly loaded into the center port. The syringe was removed and the tethered cap was attached to its port. 3) The L-PRP device was placed into the centrifuge and balanced with a counterbalance. 4) The L-PRP device was spun in centrifuge at 3200 RPM for 15 minutes. 5) The yellow cap was removed on the side port and a 30mL syringe was connected. The device was tilted at an angle, avoiding inverting to keep top blue vent dry, and all of the platelet-poor plasma (PPP) was removed. The yellow cap was replaced. 6) The red cap was removed on the side port and a 10mL syringe was connected. 2mL of PRP was withdrawn and syringe was left attached. With the 10mL syringe attached, the PRP was suspended by gently shaking L-PRP device for 30 seconds. The remaining PRP suspension was extracted into the attached 10mL syringe.

Figure 4 Representative pictures of APS device processing: 1) The APS Concentrator device was gently shaken to ensure beads were evenly distributed across bottom of top chamber. The yellow cap was unscrewed on the APS Concentrator device and filled with the output of L-PRP device from the 10mL syringe. The 10mL syringe was removed and the tether cap on port was attached. The paddle was spun until the cell solution was fully mixed with beads. 2) The concentrator was placed into the centrifuge. The centrifuge was balanced with a counterbalance. The concentrator device was spun for 2 minutes at 2000 RPM. 3) The APS was gently resuspended in the bottom of the APS Concentrator. The red cap was unscrewed and connected to a sterile 10mL syringe. The APS was extracted.

Table 4 Procedures Used During Canine Joint Injections

Joint	Common Indications	Limb Position	Needle Gauge, Length	Typical Volume (mL)/Product
Stifle	Arthritis, ACL Surgery, TPLO Surgery, Patellar Luxation	Slightly Flexed	22g, 1.0”	1.0 - APS
Elbow	Arthritis	Slightly Flexed	22g, 1.0”	1.0 - APS
Hock	OCD Surgery	Deep Flexion	22g, 1.0”	3.5- PRP
Lumbosacral	Arthritis	Normal Standing	22g, 1.5”	6.0 - PRP
Carpus	Arthritis	Flexed	22g, 1.0”	0.3 – APS
Shoulder	Bicipital tendinitis	Slightly Flexed	22g, 1.5”	1.3 - APS
Hip	Arthritis, FHO Repair	Apply traction and external rotation	22g, 1.5”	1.0 - APS

Table 5 Frequency of L-PRP Applications in Our Practice Including Tendon/Ligament Applications, OA, and Joint Applications

	Disease/Treatment	Cases	Volume (mL)	Injection Locations	Outcomes
					+	±	-	ND
Tendon/Ligament	ACL Surgery	4	3.0 −3.75	IA, SQ	4	0	0	0
	Bicipital Tendonitis	4	4.8–6.0	SQ	0	0	2	2
	Patellar Luxation	7	1.0–6.0	IA, SQ	5	0	0	2
	Capsule Thickening	1	ND	ND	1	0	0	0
	Femoral Head Osteotomy	1	ND	SQ	0	0	0	1
	Achilles Laceration	1	7.0	Tendon Area	1	0	0	0
	OCD Lesion Surgery	3	2.5–7.0	SQ	2	0	1	0
	Torn ACL	1	3.0	IA, SQ	1	0	0	0
Osteoarthritis/Joint	Lumbosacral Pain	7	5.8–6.9	SQ	3	2	2	0
	Shoulder	3	1.0–4	SQ	1	0	2	0
	Elbow	3	2.5–4	IA, SQ	0	1	1	0
	Carpal Pain	2	2.0–3.4	SQ	1	0	2	0
	Stifle	3	0.5–3.0	IA	3	0	0	0
	Hip	2	2.5–3.5	IA, SQ	1	0	1	0
	Tarsus Pain	1	7.0	SQ	0	0	1	0

Notes: Outcomes: + indicates satisfactory clinical outcomes, ± indicates mixed clinical outcomes, and – indicates unsatisfactory clinical outcomes.

Abbreviations: IA, intra-articular; SQ, subcutaneous; ND, no data; ACL, anterior cruciate ligament; OCD, osteochondral dissecans.

Table 6 Description of APS Use in for a Variety of Orthopedic Conditions Including Tendon/Ligament Applications, OA, and Joint Applications

	Disease/Treatment	Cases	Volume Used	Injection Location	Outcomes
					+	±	−	ND
Tendon/Ligament	ACL Surgery	43	0.25–2.8	IA	39	2	1	1
	ACL Tears	6	0.25–1.25	IA	5	0	1	0
	Bilateral ACL Tears	6	1.1 −1.3	IA	6	0	0	0
	ACL Partial Tear	5	0.2–1.2	IA	4	0	0	1
Osteoarthritis/Joint	Hip OA	3	2.1–3.0	IA	0	0	2	1
	Bilateral Hip OA	11	1.0–1.3	IA	6	0	4	1
	Hip Dysplasia	5	1.2–1.5	IA	5	0	0	0
	Stifle OA	5	0.3–4.0	IA	5	0	0	0
	Bilateral Stifle OA	10	0.5–1.7	IA	9	1	0	0
	Elbow OA	2	1.0–2.0	IA	2	0	0	0
	Post-TPLO	6	1.0–3.0	IA and SQ	5	1	0	0
	Bilateral Post-TPLO	2	1.3	IA	2	0	0	0
	Stifle Bursitis	1	1.1	IA	1	0	0	0
	Hip Laxity	1	2	IA	1	0	0	0

Notes: Outcomes: + indicates satisfactory clinical outcomes, ± indicates mixed clinical outcomes, and – indicates unsatisfactory clinical outcomes.

Abbreviations: IA, intra-articular; SQ, subcutaneous; ND, no data; ACL, anterior cruciate ligament; OCD, osteochondral dissecans.

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