status of biotherapeutics in equine & canine...

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


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


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


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


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.


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


Inflammation ACS/PRP ACS

The use of regenerative medicine in the

treatment of:


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


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




of chondrocytes.

Hyaluronic acid (HA)

IA injection no less than 2 million Dalt on

Viscosupplementation

Corticosteroids

(IA injection e.g. Triamcinolone 1mg per joint)


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




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



b) IRAP (IL1-RA)

c) Stem Cells

d) Bone Marrow Concentrate

Ben Anghileri


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?



b) IRAP (IL1-RA)

c) Stem Cells

d) Bone Marrow Concentrate Ben Anghileri


SDFT 2

19yr old eventer

Pulled up during cross country phase

Lesion is the extent on the tendon

Zamar (cold therapy)



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

http://www.nupsala.com

status of biotherapeutics in equine & canine...

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