Evidence-based Regenerative Medicine 12/13/12 Evidence-based Regenerative Medicine Vet-Stem Credentialing Course Veterinary Regenerative Medicine 102 Veterinary Regenerative Medicine 102

Evidence-Based Medicine - Outline 12/13/12 Regenerative medicine – the new paradigm In vitro evidence of efficacy Laboratory animal studies Canine / Feline / Equine Clinical Data Veterinary Regenerative Medicine 102

Regenerative Medicine The New Paradigm 12/13/12 Regenerative Medicine The New Paradigm Pain Reduction Inflammation Reduction Tissue Regeneration Quality of Life Extension Veterinary Regenerative Medicine 102

Evidence Based Medicine 12/13/12 “The fundamental tenet of EBM is that the practice of medicine should be based on valid, clinically relevant research data, whenever possible” Evidence-Based Veterinary Medical Association, 2012 Veterinary Regenerative Medicine 102

Evidence Pyramids www.hsl.virginia.edu/collections/ebm/pyramid.cfm 12/13/12 There are a wide variety of “evidence pyramids” in the literature, showing that there is stratification of the various types of data. www.hsl.virginia.edu/collections/ebm/pyramid.cfm Veterinary Regenerative Medicine 102

Evidence-Based Medicine 12/13/12 The Evidenced-based veterinary medicine association proposes this hierarchy. www.ebvma.org, 2012 Veterinary Regenerative Medicine 102

Evidence Based Medicine U.S. Preventative Services Task Force Level I: at least one randomized controlled trial Level IIa: controlled trials without randomization Level IIb: cohort or case-control studies Level IIc: uncontrolled trials – multiple time points Level III: expert opinions But perhaps a more quantitative look comes from this Level I-III evidence proposed by the US Preventive Services Task Force. U.S. Preventive Services Task Force (August 1989). Guide to clinical preventive services: report of the U.S. Preventive Services Task Force. DIANE Publishing. pp. 24–. ISBN 978-1-56806-297-6.

EBM for Clinical Solutions 12/13/12 Education + CE Experience Published Literature Solve Clinical Problem In the field, the clinicial must put together education/CE + experience + published literature to solve a clinical problem. Veterinary Regenerative Medicine 102

Regeneration Via Differentiation 12/13/12 Here is an example of the in vitro data on stem cell differentiation. This demonstrates that a cultured canine adipose-derived stem cell can differentiate into at least three cell lineages – fat, bone and cartilage. This type of data can be found for bone marrow, cord stem cells and others as support for them being a “true” stem cell. Adipogenic Osteogenic Chondrogenic Vieira N, Brandalise V, Secco M, et al. Isolation, characterization and differentiation potential of canine adipose-derived stem cells. Cell Transpl 2010;19(3)279-289. Veterinary Regenerative Medicine 102

Regeneration Via Differentiation 12/13/12 Clinical Relevance: “Equine AT-MSCs represent a suitable cellular source for regenerative treatment of bone or cartilage defects.” Braun et al showeed that the adipose stem cell from the equine can become cartilage in vitro. Braun et al, Am J Vet Res, Oct 2010, 71(10):1228-1236 Veterinary Regenerative Medicine 102

Joint Lubrication 12/13/12 In joint function, lubrication is critically important. In this experiment, Dr. Lee at the UC Davis Medical school demonstrated that a stem cell extracted from the infrapatellar fat pad could not only produce cartilage, but could produce Lubricin, one of the key molecules providing lubrication of the joint. Lee et al, Biochem Biophys Res Comm , 2008, 376:148-153. Veterinary Regenerative Medicine 102

Canine Medial Shoulder Instability Repair 12/13/12 a. Glenoid b. Glenohumeral lig. c. Subscapularis ten. d. Humeral head Sherman Canapp, DVM, Dipl. ACVS; Presented AVMA 2009 Pre – Stem Cell 90 Days Post – Stem Cell Dr. Sherman Canapp (Baltimore Veterinary Orthopedics and Sports Medicine) presented a number cases at the AVMA regenerative medciine forum in 2009 and 2010. This particular case was presented to demonstrate soft tissue repair in the canine shoulder. The first video shows a severely damaged shoulder in an agility dog. This particular dog was treated with intraarticular adispose-stem cells after arthroscopic examination. The second video shows the same joint 90 days post stem cell. You can see considerable regeneration of the subscapularis tendon, a result similar to that found by Nixon et al in his horse tendon injury model. The glenohumeral ligament also shows considerable repair. Veterinary Regenerative Medicine 102

Canine Elbow Coronoid Disease Cartilage Regeneration 12/13/12 Canine Elbow Coronoid Disease Cartilage Regeneration Arthroscopy Timeline Initial – surgical cleanup only Day 90 – no change, lame, Add adipose stem cell Rx Day 180 – cartilage regeneration Sherman Canapp, DVM, Dipl. ACVS; Presented AVMA 2009 Pre – Stem Cell – Day 90 Day 180 (90 Days Post – Stem Cell) This case is also one from Dr. Canapp. This athlete had severe damage to the cartilage in the elbow and severe lameness. The initial arthroscopy was done to clean up the joint in a typical repair surgery. At 90 days post surgery, the dog was still lame and was re-scoped. The missing cartilage looked the same as at the initial surgery, with considerable eburnated bone and a significant crack in the remaining articular cartilage. At this point the only therapy was intraarticular adipose stem cells. The second video shows that the joint has considerably improved in terms of infalmmation and there is new cartilage now in the area where none was before. Dr. Canapp is very careful to point out that this is seen in nly about 20% of cases and many dogs have a return to clinical normality while still having significant cartilage loss. Veterinary Regenerative Medicine 102

Rabbit Cartilage Regeneration 12/13/12 As already show in Module 1, cartilage can be regrown in rabbit models of cartilage defects. At 8 weeks, 12/12 (100%) of defects in treated group healing with hyaline-like cartilage. Only 1/12 (8%) of controls healed. Dragoo J et al, “Healing full-thickness cartilage defects using adipose-derived stem cells” Tiss Eng 2007;13(7):1615-21. (Stanford) Veterinary Regenerative Medicine 102

Cruciate Ligament -ChondroProtection 12/13/12 Sham Surgery Adipose Stem Cell IA Untreated Control Above toluidine blue staining of cartilage surface at 20 weeks after cranial cruciate ligament transection. Only treatment was group B given 1ml of adipose-stem cells by intraarticular injection with no carrier/scaffold. And, again, adipose stem cells can be chondroprotective. Toghraie et al, “Treatment of osteoarthritis with infrapatellar fat pad derived mesenchymal stem cells in rabbit” The Knee 2011;1:71-75. Veterinary Regenerative Medicine 102

Severe Muscle Tears 12/13/12 “Adipose derived stem cell therapy demonstrated promising results in five cases of muscle injury in working German shepherd dogs (semitendinosus). All dogs were able to return to their previous training and occupations as police and Schutzhund dogs with a functional gait. In contrast, the surgeon’s previous experience was that the dog’s careers were typically shortened due to apparent fibrosis/muscle contracture, and continuing muscle disease.” Dr. Gary Brown, a boarded surgeon in northern california, did a series of cases of severe tearing of the semitendenosis muslce group in Germ Shep working dogs. The cells were administered intralesionally with ultrasound follow-up. Brown SG. Stem Cells Disc 2012;2(2):41-44. 16 Veterinary Regenerative Medicine 102

Severe Muscle Tear – 5 Cases 12/13/12 Scores- 0=Normal, 4=Very Severely Affected This table shows the pre and post treatment muscle injury grades and the total cells delivered. Below is a pre and post ultrasound exam by an independent ultrasonographer that shows the repair at 84 days after stem cell therapy. The blockage and resolution of scar tissue is a major benefit of cell therapy for these soft tissue injuries Cris: Pre-treatment 84 Days Post-ASC treatment Brown SG. Stem Cells Disc 2012;2(2):41-44. Veterinary Regenerative Medicine 102

NA Vet Regenerative Medicine Meeting 12/13/12 Marty Gardner, DVM 116 cases proximal susp desmitis Adipose SVF – ultrasounded guided >90% return to full work, approx 6 months Wes Sutter, DVM, dACVS Flexor tendonitis PRP+ Fresh BM or PRP + Adipose SVF Both better than PRP alone – Early better Chris Johnson, DVM, dACVS Fresh BM or Fresh adipose SVF Do not wait 30 days – too much scar Most cases improve over conservative Larry Galuppo, DVM – UCD DDF Lesions - 40 horses – minimum 6 month BMAC, BM cultured, Adipose SVF “Reasonable” success for all treatments Adipose SVF better for severe lesions Scores- 0=Normal, 4=Very Severely Affected These are brief abstracts from presntations at the first north american regenerative medicine meeting. 1st North American Veterinary Regenerative Medicine Meeting, 2010 Veterinary Regenerative Medicine 102

Sport Horse Suspensory Study Sport Horse Study Sport Horse Suspensory Study 84 horses with SL desmitis tx with ASCs All horses tx at single referral center All dx with diagnostic anesthesia, u/s and/or MRI Consistent rehab protocol Stringent outcome measure - in full work for minimum of 1 year Rich, R and Carpenter, E, Outcome of 84 horses with suspensory injuries treated with adipose-derived regenerative cells, Proceedings, NAVRMA, 2012, Savannah, GA

Sport Horse Study 85.7% returned to prior level or higher 13.1% returned to lower level 1.2% did not return to work Rich, R and Carpenter, E. Outcome of 84 horses with suspensory injuries treated with adipose-derived regenerative cells, Proceedings, NAVRMA, 2012, Savannah, GA

Chronic Stifle Osteoarthritis Study 12/13/12 365 Day Pilot Study, 9 dogs Chronic Post-Surgical OA (>3 Mo) Single Surgeon all cases Intra-articular stem cells – 1X Vet and Owner assessments at pre, 30, 90, 180, 365 N=9 for 180 day data This is a study with a single board-certified orthopedic surgeon. All 9 dogs had severe post-surgical OA of the stifle joint. The surgeon had previously done arthroscopy and decided there were no more surgical options. A single intraarticular injection of adipose SVF was given to each dog. Veterinary Regenerative Medicine 102

Chronic Stifle Osteoarthritis Study 12/13/12 In this graph, normal is “1”. All dogs started at a 3-4 level of pain and lameness. You can observe that the v-lame (veterinary lameness score) as well as the range of motion and pain scores were substantially reduced. The owner scores likewise were reduced to a normal-mild level. These were last resort dogs where there were no options for surgery and pain medication was not adequate. Vet and owner total scores statistically improved at 30 and 90 days over baselines (P<0.001) Veterinary Regenerative Medicine 102

Prospective Elbow OA Trial 12/13/12 This study by Black et al tested intraarticular adipose-derived stem cell in clinical cases of chronic elbow arthritis. This was a multicenter study with only boarded specialists recruiting and doing the examinations. The outcomes were compared to baseline data. Veterinary Regenerative Medicine 102

Prospective Elbow OA Trial 12/13/12 Five parameters were measured by the attending specialist: list. The imrpvements ranged from around 15% to around 40% for global disability. Veterinary Regenerative Medicine 102

Randomized Controlled Trial 12/13/12 Veterinary Regenerative Medicine 102

Blinded Hip OA Trial Design 12/13/12 18 DOGS-4 Clinics A: TREATMENT B: CONTROL BASELINE VETERINARY/OWNER EVALUATIONS FAT COLLECTION BOTH GROUPS IA SVF INJECTIONS IA SALINE INJECTIONS 1-11 years, 25 – 110#; clinical cases (pets). 18 dogs were divided evenly into treated or control groups. Exams were performed and fat collection from both groups. Each treated dog received a single intraarticular injection of adipose SVF into each hip and controls received saline. Dogs were evaluated at 0, 30, 60, and 90 days. VETERINARY AND OWNER EVALUATIONS 30, 60, 90 DAYS Black et al. Veterinary Therapeutics Vol.8, No. 4, Winter 2007 Veterinary Regenerative Medicine 102

Study Parameters Evaluated 12/13/12 VETERINARY Lameness walk and trot Pain on manipulation Range of motion Functional disability (stiffness) STATISTICAL - Two-way repeated measures ANOVA - 0.05 level of significance - Two-way RM-ANOVA - Data was pooled for further analysis Black et al. Veterinary Therapeutics Vol.8, No. 4, Winter 2007 Veterinary Regenerative Medicine 102

Bilateral Coxofemoral OA Study 12/13/12 These graphs show the substantial benefit over the control groups in lameness, pain on manipulation, range of motion and composite score. There was a placebo (care-giver) effect. Veterinary Regenerative Medicine 102

Spontaneous OA Model with HA 12/13/12 PBS HA PBS + MSC HA + MSC Take Home: Use HA with stem cells to improve cartilage repair Lower score = reduce evidence of OA Spontaneous OA model in Hartley guinea pig. Inject PBS, hyaluronic acid (HA), PBS + MSC, HA + MSC. MSC source is is human bone marrow. HA + MSC had more robust cartilage growth and protection from OA> Top gross hito shows with india ink staining the much smaller area of erosions on tibial plateau. Sato et al. Arthritis Res Therapy. 2012;14:R31. Veterinary Regenerative Medicine 102

Randomized Controlled Trial 12/13/12 Design: 8 Horses – 4 treated / 4 controls Collagenase induced injury – Rx 10 days later Fully blinded histology Controls were treated with same volume of saline Nixon et al. Am J Vet Res, 2008 To look at an equine orthopedic study, this study was conducted by Dr. Alan Nixon at Cornell University. The tendon defect was created in a standardized model using collagenase to get an approximately 50% cross-sectional lesion. It was a fully blinded study. Veterinary Regenerative Medicine 102

Cornell/Nixon Tendon Study 12/13/12 Saline Control Stem Cell Treated Looking first at the pink slides under H&E stain, it is clear that the saline controls had no normal collagen and massive cellular infiltrate. The treated group had more normal architecture and was statistically improved in cell infiltrate, fiber uniformity and overall healing score. The polarized light slides show the more normal crimping of the tendon in the treated compared to the controls. H&E Stain Polarized Light -reduce infiltrate (P<0.015) -Imp collagen fiber uniformity (P<0.040 -Imp overall healing score (P<0.028). Veterinary Regenerative Medicine 102

Saline Control Stem Cell Treated Cornell/Nixon Tendon Study Saline Control Stem Cell Treated

Prospective Tendon Trial 12/13/12 This is an adipose stem cell trial in Finland in equine patients. Of all the horses, seven (12.1%) had a re-injury in the previously treated leg. No significant adverse effects were reported. Fiber scores improved over time. . Leppanen et al, Regenerative Medicine, November 4(6), Suppl. 2 (2009), World Conference of Regenerative Medicine, Leipzig, Germany 28-30.10.2009, Proceedings S21. Veterinary Regenerative Medicine 102

Summary - Regenerative Medicine 12/13/12 1. In-vitro and laboratory animal data support the mechanisms and the theory of using stem cells in orthopedics. 2. There are uncontrolled and also blinded placebo-control clinical trials that support the validity of use of adipose stem cells in canine and equine medicine. 3. The evidence rises to Level I in support of clinical use in the horse and the dog. Veterinary Regenerative Medicine 102