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Status of Biotherapeutics in Equine &
Canine Medicine
Osteoarthritis
It is a common cause of
chronic pain and the common
type of arthritis in dogs as it
has been estimated that about
20% of adult dogs suffer from it
(Allen 2007; Innes 2010)
LIFE LONG - LIFE SYLE
DISEASE
The joint is composed of bone, articular
cartilage, and periarticular soft tissues
Changes in the joint
Grading OA
Bone
Cartilage
Synovium
1 2 3 4 5 OA Phases
Equine Tendon Disease
Introduction
SDFT functions as elastic energy store for energy-efficient locomotion
16% strain at gallop
High loads - 1 tonne peak load; 1cm2 CSA
Tendon operating close to its functional limit
Low tolerance (in vitro rupture 12-20%)
Cause of injury
Sudden excessive over-extension
Preceding tendon degeneration with superimposed sudden over-extension
Tendon ageing
MMP mediated
Equine Tendon Disease
Outcome most related to
severity of the initial injury
Need for regeneration rather
than repair
Severity Time out of
training*
Prognosis
(return to
racing)*
Mild 10 months 63%
Moderate 11 months 30%
Severe 18 months 23%
* National Hunt and point-point racehorses
(Marr et al. 1993)
7
Need
Tendon & ligament injuries repair with scar tissue
Scar limits efficiency and increases incidence of re-injury
Goals:
Therapy which regenerates tendon/ligament tissue
Therapy which is clinically applicable / affordable Scanning EM SCARRED tendon
Scanning EM NORMAL Tendon
Introduction
Orthopedic disorders treated with regenerative medicine
Tendon & ligament injuries
Meniscal injuries
Fracture repair
Traumatic osteochondral fragmentation
Traumatic arthritis
Degenerative joint disease
OCD
Common issues – predisposed to reinjury, poor healing & long time to recovery.
Therapeutics
ACS Interleukin-1 Receptor Antagonist
PRP / PET Platelet Rich Plasma / Platelet Enrichment Therapy
BMAC Bone Marrow Aspirate Concentrate
Stem Cells Autologous (Bone Marrow, Adipose & Cord Blood)
PAAG Arthramid-Vet
Commonly used therapeutics in treatment/ management of
degenerative conditions
ACS (IL1-Ra)
ACS – Basic Principle
IL1-Ra is an anti-inflammatory protein that
counteracts the destructive effects of
inflammatory proteins Interleukin-1 within
the inflamed join
IL-4, IL-10, IL-13 & IL1-Ra have been shown
to be spontaneously elaborated by synovial
membrane and cartilage
Getting the balance back to normal !
ACS – Indications (Equine & Canine)
Use in equine joint disease include
a well defined synovitis/capsulitis, particularly those that do not respond well to conventional anti-inflammatory joint medication
Horses and dogs that have had arthroscopic surgery and have been found to have focal cartilage diseases.
Where it is NOT recommended
Use in tendon sheaths or bursae
in joints where there are bone fragments
Fractures,
meniscal or ligamentous injury unless it has been successfully treated arthroscopically
bone cysts,
advanced osteoarthritis
ACS vs ACP
ACS in Canine OA
Tips, Tricks & Answers
Treatment timings ?
Equine patients 7-10 days apart
Canine patients every 7 days
Can HA and/or antibiotics be used during treatment?
Do NOT add anything to the ACS
Be very gentle when with syringe because we want to minimize the amount of cell lysis that will occur to the red blood cells
After drawing the blood, how long do I have before it needs to be placed in the incubator?
Maximum 2 hours. Blood may be transported in a portable incubator as needed. Transport should be gentle and should be avoided if possible.
What happens if you incubate longer than 24(equine) 8(canine) hours?
If you incubate for more than 24 hours, you will need to discard the syringe and start over. Increase in IL-1
PRP – Platelet Rich Plasma
Indications
Suspensory/ check ligament
Tendinitis/ Tendinosis
Articular pathology (Canine & Human)
PRP in Canine Osteoarthritis
J Am Vet Med Assoc 2013;243:1291–1297
Randomized controlled trial of the efficacy of autologous platelet therapy for the treatment of
osteoarthritis in dogs
Fahie M. Ortolano G. Schaffer J. Au J. Hettlich B. Phillips T. Allen M. Bertone A.
Study indicated significant reduction in
osteoarthritic pain and lameness after 12
weeks for dogs injected with C-PET
• For control dogs, lameness scores, pain
scores, and PVF at week 12 were not
significantly different from pretreatment
values
• dogs that received platelet injections,
lameness scores (55% decrease in median
score), pain scores (53% decrease in median
score)
Platelet Rich Plasma - Basic Concept
Growth Factors
PDGF* - regulates secretion and collagen synthesis
TGF-β* Stimulates the proliferation of undifferentiated mesenchymal cells and stimulates endothelial cell chemotaxis and angiogenesis
VEGF – increases vascular permeability and angiogensis
FGF – promotes the growth and differentiation of chondrocytes and osteoblasts, and stimulates mitogenesis of mesenchymal cells, chondrocytes and osteoblasts
These factors signal the local mesenchymal, epithelial and endothelial cells to migrate divide, and increase collagen and matrix synthesis
PRP as a scaffold
Platelets were isolated from horse blood
and activated with thrombin, a process
known to induce growth factor release.
This produced a platelet gel composed
of platelet-rich plasma (PRP)
• Wounds treated with PRP gel exhibit more
rapid epithelial differentiation and
enhanced organization of dermal collagen
• Mixed with bone autograft in the filling of
Subchondral bone cysts
• Mixed with osteoconductive scaffolds for
filling periodontal pockets
Not all PRP’s are equal !
• Centrifugal forces can activate platelets
• Filtration vs. centrifugation??
• Specialist systems offer more bespoke
solutions
• PR(PLAMSA) in the joint?
• As many Pro’s as there are Con’s
• Increased chemotaxis and chrondogenic
activity. Abrams GD, et al Platelet-rich Plasma in orthopedic
applications : evidence-based recommendations for treatment j Am Acad
Orthop Surg 2013; 21:739-748
• Once injected PRP will be activated and
may form fibrin clots that may cause
problems. Smiley ST, King JA, Hancock WW. Fibrinogen Stimulates
Macrophage Chemokine Secretion Through Toll-Like Receptor 4. The Journal
o Immunology, 2001
Comparison
Tips, Tricks & Answers
Do I need to worry about platelet concentration ?
Above x4 increase is considered therapeutic
Concentrations differ from patient to patient and timing
Not all PRP’s are equal!!
Do I need to worry about white cells?
Try to avoid white cell contamination but most systems will have white cells present in the final preparation
Can I store what I don’t use?
Yes PRP can be frozen. Store in 1ml vials and use once thawed. Freezing will cause lysis of the white cells.
Is one injection all that is necessary?
There are no long term follow up studies using PRP to indicate a single injection is all that is required, regular review points are necessary
Are WBCs and RBCs deleterious
Synoviocyte cell death is linked to RBC contact
LR-PRP can lead to proinflammatory mediator production
BMAC – Bone Marrow Aspirate
Concentrate (BMMNCS)
Indications
Suspensory ?check ligament
Tendinitis/ Tendinosis
Modulation rather than just stimulation
Basic Principle
Equine ABM, PRP, and
serum contain anabolic factors that promote
matrix synthesis
BMAC/ BMMNCS contains
cells know to be
modulatory
3 months post injection
PRP vs. BMAC
Cranial Cruciate Ligament Tears <50%
• 36 dogs with a partial cranial cruciate ligament tear treated with BMAC or ADPC and PRP
• Stifle arthroscopy finding at 90 days post-treatment on 13 dogs – 9 dogs had fully intact CCL with marked neovascularization and normal fiber pattern. 1 dog showed improvement and received a further treatment. The remaining had a >50% tear and a TPLO performed
Stem cells in dogs
Front Vet Sci. 2016 Sep 9;3:61. doi: 10.3389/fvets.2016.00061. eCollection 2016.
The Use of Adipose-Derived Progenitor Cells and Platelet-Rich Plasma Combination for the Treatment of Supraspinatus
Tendinopathy in 55 Dogs: A Retrospective Study
Canapp S, Canapp D, Ibrahim V, Carr B, Cox C, Barrett J
Prior to treatment 61.8% had previously failed to respond to NSAIDs or rehabilitation.
PRP alone had no improvement
Retrospective analysis of 55 dogs treated for supraspinatus tendinopathy with ADPC-PRP
At 90 days post treatment 25 dogs underwent gait analysis. 88% of cases had no significant difference in total
pressure index of the injured limb to the contralateral. Remaining 12% had significant improvement.
Ultrasound of the treated tendon at 90 days showed 82% of cases CSA reducing to normal.
All cases showed improvement of fiber pattern at 90 days
88% of cases undergoing gait analysis returned to normal loading by 90 days.
82% of cases examined ultrasonographically showed CSA of treated tendon
returning to normal by 90 days
Supraspinatus Tendinopathy
Tips, Tricks & Answers
Sternum vs. Tuba Coxae
One site is sufficient – 5th sternebrae
How much do I inject
Inject until you meet resistance
Air bubbles in the injection will show location of the implant.
Scoring the needle with a scalpel blade will make it easier to locate on ultrasound
14th Year anniversary
Stem Cell TreatmentStem cell process
Stem cell process
Adipose collection from dogs for
stem cells culture
10g of adipose tissue (2x thumb
size) subcutaneous vascular fat –
midline or behind the scapular.
Serum blood tubes for
suspending the cells x 15ml
10-14 days later cells are ready
c20 million (2x1ml)
Or SVF 48hrs later
Reinjury Statistics
% Re-injury Dose Response
Dose
range
No.
horses
No.
horses
re-
injured
% Re-
injury
1-9m 35 7 20
10-19m 217 25 12
20-29m 52 5 10
30-39m 12 1 8
40-49m 6 0 0
50-59m 6 0 0
Retrospective analysis of dose response
Tips, Tricks & Answers
Sternum vs. Tuba Coxae
One site is sufficient – 5th sternebrae
How much do I inject
Inject until you meet resistance
Air bubbles in the injection will show location of the implant.
Scoring the needle with a scalpel blade will make it easier to locate on ultrasound
What’s new
Conclusions: Both IA and IV RLP of the distal limb result in MSC persistence in perfused tissues.
The IA perfusion resulted in more reliable cell distribution to the pastern and foot area.
Potential relevance: Regional limb perfusion of MSCs might be used in cases where intralesional injection is not possible or in order to avoid iatrogenic needle damage. Further work is needed to assess the safety of IA RLP before its clinical us
A new OA approach:
2.5% crosslinked Polyacrylamide hydrogel
Arthramid Vet®
Polyacrylamide Hydrogel of Arthramid
Vet
38
Confidential
Injectable hydrogel consisting of 97.5% water
and 2.5% cross-linked polyacrylamide
Homogeneous (no micro-particles),
biocompatible, non-degradable and does not
migrate
The hydrogel integrates into the soft tissue
with vessel bearing delicate fibrous network
Exchanges water with host tissue, permeable
for salts and organic molecules
Because the hydrogel is non-absorbable, it
provides augmentation that lasts for years
Excellent safety profile validated through 10
years of use in humans for various indications
Computer generated image of the
proprietary polyacrylamide hydrogel
Electron microscopy images of the
hydrogel
39
Confidential
3D Soft tissue scaffold
40
Confidential
Joint elastance
CONFIDENTIAL DRAFT REPORT
Figure 1: The elastance of a joint capsule sample measured by the Instron machine (N/m2).
Results The values in the charts below correspond to the elastance of the joint capsule. A higher value corresponds to joint capsule with higher elastance, a better elasticity-resistance without disruption. Indicative averages are for information only. Abbreviations: RL = Right lateral RM: Right medial LL: Left lateral LM: Left medial
CONFIDENTIAL DRAFT REPORT
1. Comparison of RL and LL from control and non-injected goats Figure 2: Comparison of the lateral side of the joint capsule from the right and left stifle joint. Goat D and goat F are non-injected goats
In figure 2 the values of the lateral joint capsule from the right and left stifle joint from each goat have been shown. When comparing the lateral sides from the right and left leg, the biggest difference in elastance is seen in goats D and F with a 3.2-fold and 2.7-fold difference in elastance, respectively, the lateral side of the joint capsule from the right leg having less elastance than the left leg. Goats B and E have a 1.9-fold and 2.5-fold difference in elastance, respectively, the lateral side of the joint capsule from the left leg being having less elastance than the right leg. There is no difference in elastance of the joint capsule from goats A and C.
2. Comparison of RL and LL from control and injected goats Figure 2: Comparison of the lateral side of the joint capsule from the right and left stifle joint. Goat D and goat F are non-treated goats
In figure 2 the values of the lateral joint capsule from the right and left stifle joint from each goat have been shown. When comparing the lateral sides from the right and left leg, the biggest difference in elastance is seen in goats D and F with a 3.2-fold and 2.7-fold difference in elastance, respectively, the lateral side of the joint capsule from the right leg having less elastance than the left leg. Goats B and E have a 1.9-fold and 2.5-fold difference in elastance, respectively, the lateral side of the joint capsule from the left leg being having less elastance than the right leg. There is small no difference in elastance of the joint capsule from goats A and C.
CONFIDENTIAL DRAFT REPORT
3. Comparison of LL and LM from control and injected goats Figure 3: Comparison of the elastance of the lateral and medial sides of the joint capsule from the left stifle joint. Goat D and goat F are non-treated goats
In figure 3, the elastance values of the lateral and medial sides of the left stifle joint from each goat have been shown. When comparing the left leg (injected joint), the biggest difference is seen in goats A and B with 3.3-fold and 3-fold difference, respectively, the joint capsule of the lateral side having less elastance. In all the other goats the difference from the lateral and medial sides are 2-fold or less. Surgery was made on the medial side with scar tissue, it could explain the difference between medial and lateral sides.
Conclusion This goat study [5] has provided a lot of information regarding the effect of the PAAG when injected into an OA joint. Some of the data, especially from the pathology, and the joint capsule elastance suggest that this biomaterial might act mainly on the joint soft tissue and especially the synovial membrane. Osteoarthritic joints typically show joint stiffness, the joint capsule elastance investigation has shown that the joint capsule and synovial membrane of joints injected with PAAG had a better elastance when compared to the joint capsule and synovial membrane of the control joint, hence less stiffness. Osteaorthritic joints typically show joint stiffness which is a major source of pain in OA. This is supported by a recent study on human knee joint stiffness, which showed that the stiffness co-efficient was higher in individuals with painful OA compared to those with normal knees [6]. By increasing the joint capsule elastance, and thus decreasing the joint stiffness, the PAAG might relief pain in the o - .
References
The use of regenerative medicine in the treatment of:
Joint Tendon Ligament
Joint Tendon Ligament
Inflammation ACS/PRP ACS
The use of regenerative medicine in the
treatment of:
Joint Tendon Ligament
Inflammation ACS/ PRP ACS
RegenerativePRP / Stem
Cell/BMAC
PRP / Stem
Cells/ BMAC
PRP / Stem
Cells/ BMAC
The use of regenerative medicine in the
treatment of:
Clinical and Imaging
Observations
Joint Pathology Treatment Options Strategy
Gra
de 1 History of damage, dysplasia or surgery
Intermittent lameness
Little visible on radiographs
Early cartliage pathology with some surface disruption
Early synovitis
NSAIDs Inhibition of prostaglandins. Limit to 3 months and re-
view after 7 days of non use
Corticosteroids
IA injection e.g. Triamcinolone 1mg per joint
Inhibition of phospholipase, in ammatory cytokines and
enzymes
ACS-IRAP
IA injection 7 days apart for 3 or 4 treatments
Target in ammatory cytokines (IL-1)
Gra
de 2 Intermittent or mild lameness
Some discomfort on manipulation
Mild e usion
Early marginal osteophytes
Early subchondral sclerosis
Synovial uid less viscous
Cartilage focal ssuring
Established mild patchy synovitis
ACS-IRAP
IA injection 7 days apart for 3 or 4 treatments
Target in ammatory cytokines (IL-1)
Platelet rich plasma (PRP) Through growth factors released from the platelet: regu-
late collagen synthesis, stimulate cell proliferation, cell
chemotaxis and angiogenesis. Growth and di erentiation
of chondrocytes.
Hyaluronic acid (HA)
IA injection no less than 2 million Dalt on
Viscosupplementation
Corticosteroids
IA injection e.g. Triamcinolone 1mg per joint
Inhibition of phospholipase, in ammatory cytokines and
enzymes
Gra
de 3 Sti ness
Some reduction of joint movement
Crepitus / some pain
Sclerosis and osteophytes X-Ray
Synovial uid physical changes
Cartilage with some focal erosions
Chronic synovitis
Osteophytes obvious at margins
Some brous hypertrophy of joint capsule
Platelet rich plasma (PRP) Through growth factors released from the platelet: regu-
late collagen synthesis, stimulate cell proliferation, cell
chemotaxis and angiogenesis. Growth and di erentiation
of chondrocytes.
Hyaluronic acid (HA)
IA injection no less than 2 million Dalt on
Viscosupplementation
Corticosteroids
(IA injection e.g. Triamcinolone 1mg per joint)
Inhibition of phospholipase, in ammatory cytokines and
enzymes
Gra
de 4 Obvious thickening and restriction of joint
Crepitus and pain
Marked bony changes visible on X-Ray
E usion and synovial uid changes pos-
sible
Cartilage loss and erosion
Established chronic synovitis with thickened brotic syn-
ovium
Peripheral nociceptor transformation
Osteophytes obvious
Thickening of brous joint capsule
Platelet rich plasma (PRP) Through growth factors released from the platelet: regu-
late collagen synthesis, stimulate cell proliferation, cell
chemotaxis and angiogenesis. Growth and di erentiation
of chondrocytes.
Bone marrow aspirate concentrate (BMAC)
Stromal vascular fraction (SVF)
Stem cells to modulate cell proliferation and di erenti-
ating young cells through the continuous synthesis of
growth factors
Arthramid Vet A visco-elastic implant with tissue integration to protect
synovial tissue and provide joint lubrication
Gra
de 5 Obvious pain on manipulation
Reduced movement in enlarged joint
Crepitus obvious
Limited response to pain medication
Extensive cartilage loss and disruption
Bone remodeling under cartilage with deformation of joint
Chronic synovitis with extensive brosis
Peripheral and central neuroperception increase with central
plasticity
Extensive marginal osteophytes
Very thickened joint capsule
Bone marrow aspirate concentrate (BMAC)
Stromal vascular fraction (SVF)
Stem cells to modulate cell proliferation and di erenti-
ating young cells through the continuous synthesis of
growth factors
Arthramid Vet A visco-elastic implant with tissue integration to protect
synovial tissue and provide joint lubrication
Grading & Treatment of Canine OA - A guideline for clinicians
Your partner in
regenerative medicine Contact us for more info: [email protected] +44 (0)1865 922227
©NupsalaVeterinaryServices2016
The fight against scar tissue
% le
sio
n
optimal (C.Type I)
Scar Tissue (C. Type III)
50%
60%
40%
12 months
Inflammatory
Phase
Reparative Phase
Opportunity for using PRP/Stem Cells
% le
sio
n
optimal (C.Type I)
Scar tissue(C. Type III)
50%
60%
40%
12
Inflammatory
Phase
Reparative Phase
Treatment strategy
% le
sio
n
optimal (C.Type I)
Scar tissue (C. Type III)
50%
80%
20%
11 semaines
Reparative PhaseInflammatory
Phase
When to use what
Degree of damage
D0Post-inflammation
D7 D14 D21 D28 D35 +++
15%
PRP
PRP /BMAC PRP / BMAC
Case studies
Lameness 1
Lameness 2
SDFT 1
SDFT 2
All cases received a biological product following diagnosis
Lameness 1
12 yr Eventer
Initial Presentation: 1/10 lame left fore (LF) straight line, 2/10 LF on hard circle left rein, Soft Lunge: sound. Negative to hoof testers.
Positive to palmer digital nerve block, Positive to intra articular coffin joint anaesthesia. Radiographs NAD
Differential diagnosis: Coffin joint osteoarthritis
Coffin joint synovitis
Subchondral bone pain
Deep digital flexor tendon lesion
Navicular syndrome
Side bone formation
Initial treatment: Coffin joint medicated with 10mg triamcinolone
Sound for 2 wks then represented lame
Lameness 1: Diagnosis
MRI: T1W 3D FRO LF MRI: T1W 3D SAG LF
Lameness 1: Medial Palmer P2 cystic lesion
Which biological would you use:
a) Pallet Rich Plasma
b) IRAP (IL1-RA)
c) Stem Cells
d) Bone Marrow Concentrate
Ben Anghileri
Oakham Equine Hospital – Leicestershire
Lameness 2 8 yr Eventer
Initial Presentation: 1/10 lame right fore (RF) straight line, 3/10 RF hard circle left rein, 2/10 RF hard circle right rein.
Palmer digital nerve block: 75%, Abaxial sesamoid nerve block: 100%
Navicular bursa anaesthesia: No change after 5 mins.
Intra articular coffin joint anaesthesia: 100% improvement
Radiographs NAD
Differential diagnosis: Coffin joint osteoarthritis
Coffin joint synovitis
Subchondral bone pain
Deep digital flexor tendon lesion
Side bone formation
Collateral ligament desmitis
Joint medicated with 10mg triamcinolone
Sound for 2 wks then represented lame
Lameness 2: Diagnosis
MRI: T2W FSE TRA RF MRI: PDW SE TRA RF
Lameness 2: Collateral Ligament Desmitis
Which biological would you use:
a) Pallet Rich Plasma
b) IRAP (IL1-RA)
c) Stem Cells
d) Bone Marrow Concentrate
Ben Anghileri
Oakham Equine Hospital – Leicestershire
SDFT 1
History: 12yro Advanced eventer.
1/10 lame right fore 3 days post event with swelling to proximal
palmer cannon. Ultrasound examination NAD
Restricted exercise and cold therapy treatment for 1 week. No
improvement
2 weeks post injury: Effusion spread down palmer aspect of
cannon. Repeat ultrasound examination NAD.
SDFT 1
3 week post initial presentation.
Ultrasound: hypoechoic area to medial
aspect SDFT level 1B.
Aetiology: traumatic injury?
Complications: communication with
the carpal sheath?
Which biological would you use:
a) Pallet Rich Plasma
b) IRAP (IL1-RA)
c) Stem Cells
d) Bone Marrow Concentrate Ben Anghileri
Oakham Equine Hospital – Leicestershire
SDFT 2
19yr old eventer
Pulled up during cross country phase
Lesion is the extent on the tendon
Zamar (cold therapy)
Which biological would you use:
a) Pallet Rich Plasma
b) IRAP (IL1-RA)
c) Stem Cells
d) Bone Marrow Concentrate Andy Bathe
Rossdales - Newmarket
Take home message
Biologicals - they are not all the same
Timing is critical
Get the balance right – catabolic vs. anabolic
Variations exist from patient to patient
Greg McGarrell
Tel. +44 7745399843
www.nupsala.com