Regenerative Medicine for Joint Disease. Regenerative Medicine for Joint Disease. Regenerative Medicine for Joint Disease

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1 Regenerative Medicine for Joint Disease Ok, may be more correctly considered disease modulation and not regeneration! Your Tour Guide - Steve Budsberg Regenerative Medicine for Joint Disease Regenerative Therapies Cell Therapy Mature Cells Mesenchymal Stem Cells Tissue Engineering Cell-based Scaffolds Cell-free Scaffolds Biological Products Regenerative Medicine for Joint Disease Cell Therapy The use of multi-potent, pluri-potent or isolated cells that replace or induce the regeneration of damaged tissue Regenerative Medicine researchers look at how to leverage such cellular properties for therapeutic applications

2 Terminology (cells) ES: ips: MSC: SVF: Embryonic Cell induced Pluripotent Cell Mesenchymal Stem/Stromal Cell Stromal Vascular Fraction/ Nucleated Cells Terminology (potency) Totipotent: A single cell to divide and produce all cell in an organism (oocyte/zygote) Pluripotent: Differentiate into any of the 3 germ layers (ES, ips) endoderm-gi tract, lungs mesoderm-muscle, bone, urogential Ectoderm-epidermal tissue, nervous system Multipotent: Capacity to differentiate into cellular phenotypes derived from one germ layer (MSC) Unipotent: Glial Cell or Chondrocyte Properties of Regenerative Cells Differentiate: tendon, bone, cartilage, cardiac, nervous, muscle, vascular, liver Induce repair: secrete VEGF, FGF, TFG-beta Homing: respond and migrate to site of injury/ inflammation Immunomodulation: decrease IL-1, suppress T-cell proliferation Anti-apoptosis Self-renewal

3 Trophic Support Trophic support via growth factors and cytokines Anti-scarring Angiogenic (Vascular endothelial growth factor) Anti-apoptotic (block cell death after injury) Stimulation of resident tissue stem cells Caplan and Dennis, J Cell Biochem, 2006 Immunomodulatory Effects of MSC Yi T and Song SU Arch Pharm Res 2012 Anti-inflammatory Decrease pro-inflammatory mediators Increase anti-inflammatory mediators Is use of the SVF ideal? Tholpady et al, Clin Plastic Surg, 2006

4 Homing, Anti-inflammatory, Trophic, Multipotent to Injury Site Mouse hindlimb ischemia model Ligation of all branches of femoral artery Injection of AT cells 24-hours later 500,000 freshly isolated AT-derived CD34+ or CD34- cells were injected intravenously At 2-wks, blood flow was measured via laser Doppler blood flowmeter Miranville et al., Improvement of postnatal neovascularization by human adipose tissue-derived stem cells. Circulation Jul 20;110(3): What are Adult Stem Cells? Adhere to plastic culture dishes and form fibroblastlike colonies Self-renewing Can differentiate into multiple specialized cell lineages (multipotent): Bone, osteogenic Cartilage, chondrogenic Fat, adipogenic Muscle, myogenic Expression of cell surface markers Cluster of Differentiation (CD) Courtesy, NIH Stem Cell website Factors Secreted by Mesenchymal Stem Cells Interleukins IL-3, IL-6, IL-7, IL-8,IL-11, IL-12, IL-14, IL-15 Neurotrophic Factors NGF, BDNF, GDNF Growth Factors and Cytokines LIF, VEGF, HGF, SDF, SCF, M-CSF, bfgf, IGFBP, Oncostatin M, MIP1-B, TIMP-1, TIMP-2, TGFB-1, TGFB-2, PDGF, EGF, KGF * Rehman et al, Circulation 2004, 109: Nakagami et al, J Atheroscler Thromb 2006, Apr 13(2): 77-81

5 Adipose derived MSC Stromal Vascular Fraction immunomodulatory/ anti-inflammatory Endothelial progenitor cells angiogenesis M2 macrophages immunomodulatory IL-1, IL-10 antagonist T-regulatory cells immunosuppressive Riordan NH J Translat Med 2009 Sick Patient 30-gm falciform fat in saline Minced Collagenase Spin SVF Limitations of Autogenous, Adiposederived Stem Cell (SVF)Therapy Anesthesia and surgery needed to collect cells Cost, morbidity Older and sick patients may not have as viable cells Heterogeneous mixture of stem cells and other cells Still don t know optimal # cells, treatment frequency Need additional clinical studies

6 What s the Future? Allogeneic stem cells (AllSC): Treatment is STEM CELLS that are optimized for patient s disease OA: Chondrogenic potential with increased anti-inflammatory cytokine expression and decreased pro-inflammatory cytokine expression Little patient morbidity Fat harvested from dogs undergoing OHE Few ADE following IV or IA injections (preliminary data) Treatment can be made off the shelf Allogeneic Stem Cells Healthy Dog OHE 30-gm falciform fat in saline Minced Collagenase Spin SVF MSC culture conditions Sick Patient Collected Rinsed 90% confluency 3-passages Characterization of Canine Allogeneic Stem Cells Inflammatory mediators in the treatment are bad can we limit them? Yes, culture of cells in KNAC media nearly eliminates COX-2 and IL-1B. Kiefer K, et al. Influence of culture medium on cellular phenotype of canine adipose derived stem cells. Open Journal of Regenerative Medicine, 2014, 3,

7 FDA Regulated CVM Guidance #218 now FINAL Guidance: Cells are drugs and regulated by FDA Pathway via FDA INAD application Autologous use low enforcement Veterinary liability risk, if not followed SPECIFICALLY NOT EXCLUDED Universities Vets using kits in clinic Why Consider Stem Cells for OA? Hypothetical Reduced risk of adverse events from NSAIDs (other oral medications) Extended duration of action Long-term resolution of symptoms Modification of disease progress Use as early in the disease as possible? Vet Therapeutics 2007 Hip OA n= 18 dogs Prospective, blinded, multi-center, placebo control Improved lameness, range of motion and pain on manipulation Effect seen at 30 days

8 Canine OA Studies using Autogenous MSC Additional Studies Limited in scope Curevo et al. Hip Osteoarthritis in Dogs: A Randomized Study Using Mesenchymal Stem Cells from Adipose Tissue and Plasma Richin Growth Factors. Int. J. Mol. Sci. 2014, 15, Improvements at 1, 3, 6 months via clinical scores No placebo group Vilar et al. Assessment of the effect of intra-articular injection of autologous adipose-derived mesenchymal stem cells in osteoarthritic dogs using a double blinded force platform analysis. BMC Vet Res. 2014; 10: dogs showed improvement in PVF and VI at 30 days but not at 90 or 180. No placebo group. More Studies Coming with Allogeneic Cells Adult Mesenchymal Stem Cells delivered via intraarticular injection to the knee following partial medial meniscectomy. RCT: Level 1 Evidence Group A: 50 million allogeneic cells IA + HA Group B: 150 million allogeneic cells IA + HA Group C: HA alone Outcomes: 2-year follow-up for safety Clinical function MRI of meniscus regeneration (>15% increased needed). Clinical function significantly increased in cell groups compared to control group Meniscus regeneration present significantly increased in cell groups, 0% in control group. JBJS, 2014 Assessment of clinical and MRI outcomes after mesenchymal stem cell implantation in patients with knee osteoarthritis: a prospective study. OBJECTIVE: Investigate the clinical and MRI outcomes of MSC implantation in OA knees and determine the association between clinical and MRI outcomes. DESIGN: 20 patients underwent MSC implantation for knee OA were evaluated at 2-years. Clinical outcomes were evaluated according to functional activity scales, and cartilage repair was assessed according to the MRI Osteoarthritis Knee scores. RESULTS: Clinical outcomes significantly improved. Cartilage lesion grades at follow-up MRI were significantly better than the preoperative values. The clinical outcomes significantly correlated with MRI scores. Osteoarthritis Cartilage

9 Regenerative Medicine for Joint Disease Regenerative Therapies Cell Therapy Tissue Engineering Cell-based Scaffolds Cell-free Scaffolds Biological Products Platelet Rich Plasma (PRP) Cell-based Scaffolds Tissue Engineering -Cell-Based Scaffolds- Involves tissue harvest and cell expansion Cells are pre-seeded on the scaffold Autologous cells are most commonly used Scaffold-associated chondrocyte implantation is used for over 10 years in Europe in humans Regenerative Medicine for Joint Disease Regenerative Therapies Cell Therapy Tissue Engineering Cell-based Scaffolds Cell-free Scaffolds Biological Products Platelet Rich Plasma (PRP)

10 Tissue Engineering -Cell-Free Scaffolds- Developed for one stage procedures Can be implanted alone or combined with biological products such as bone marrow or PRP Scaffolds made of a variety of materials Unclear if these have advantages over Cellbased scaffolds Regenerative Medicine for Joint Disease Regenerative Therapies Cell Therapy Tissue Engineering Cell-based Scaffolds Cell-free Scaffolds Biological Products Platelet Rich Plasma (PRP) What is Platelet Rich Plasma (PRP)? Autologous fluid concentrate composed primarily of platelets and is believed to accelerate healing of tendon, ligament, bone and other tissues. Treatment utilizing PRP is often referred to as orthobiologics and used when the goal of therapy is to enhance the body s own innate ability to repair and regenerate tissue. The premise of therapy comes from the function of platelets which includes not only hemostasis, construction of new connective tissue and revascularization, but also the important role in the local inflammatory response at the site of bone healing.

11 Why should we discuss PRP? Autologous Minimally-invasive collection process Rapid preparation Can be cost-effective and time-saving compared to current stem cell products Can be prepared without specialized equipment, using a standard centrifuge Can be modified into various forms according to whether an open (i.e. surgical) or closed (i.e. percutaneous) application Low risk Platelet Activation Upon platelet activation they promote angiogenesis, recruit mesenchymal cells and release of growth factors. Platelet-derived growth factor a and b (PDGF) Transforming growth factor-β (TGF-β) Fibroblast growth factor (FGF) Vascular Endothelial Growth Factor (VEGF) Connective Tissue growth factor (CTGF) Insulin like Growth factor (ILGF 1 and 2) Platelet factor 4 (PF-4) Interleukin 8 (IL-8) Keratinocyte growth factor (KGF) Basic Fibroblastic Growth Factor (bfgf) Epidermal growth factor (EGF) Platelet Activation Activation of PRP is performed, using thrombin, calcium chloride, glass beads, or a combination Growth factor release

12 PRP Commercialization The use of PRP has grown rapidly, perhaps too fast. Commercial products are marketed that have been developed by concentrating the platelets in suspensions of either plasma or a fibrin matrix; however, great variability exists in the preparation of PRP fold increases in platelet concentrations have been reported with different PRP preparatory techniques. Variations in PRP preparations include: whole blood volume used during preparation the presence or absence of white blood cells (WBC) prior activation of platelets before injection Some PRP preparations contain no platelets (PPP) Biologic Products Platelet/Plasma/Protein Products Platelet Rich Plasma (PRP) GenesisCS-2 Platelet Rich Plasma Kit, Vet Stem (San Diego, CA) Autologous Platelet Therapy (C-Pet) C-Pet Canine Platelet Enhancement Therapy Pall Corp. (Port Washington, N.Y) Autologous Platelet Concentrates (PC) Autologous Conditioned Plasma (ACP) Autologous Conditioned Plasma (ACP TM ) Arthrex Inc. (Naples, FL, USA) Platelet Rich Growth Factors (PRGF) Basic Applied PRP Terminology PRP (platelet-rich plasma) plasma containing resting platelets at increased concentration. PRP may or may not be activated prior to use. PRCR (platelet-rich clot releasate) (Platelet Poor Plasma-PPP) Ex vivo (i.e. test-tube) activation of PRP is performed, using thrombin, calcium chloride, glass beads, or a combination. Liquid serum above that clot is the PRCR, and is the treatment product. PRCR does not contain platelets and the growth factor content is therefore fixed. PRFM (platelet-rich fibrin matrix) Fibrin clot developed slowly (1 hour minimum) from PRP and calcium-chloride in a glass tube. Platelet-rich fibrin clot is then inserted into treatment site. Improved handling, the matrix may provide a construct for tissue regeneration, may slowly degrade and release growth factors. PC (platelet concentrate) Can mean about anything but often used interchangeably with PRP.

13 Importance of PRP Details Preliminary evidence suggests a link between PRP formulation and/or application procedures and clinical effect. PRP Formulation number of platelets, balance between platelet secreted and plasma proteins, mechanism of plasma activation Application Procedures number of doses, volume, activation, and injection procedures PRP Veterinary Commercialization Products utilized in dogs in veterinary medicine (probably a partial list) Autologous Conditioned Plasma (ACP TM ) Arthrex Inc. (Naples, FL, USA) C-Pet Canine Platelet Enhancement Therapy Pall Corp. (Port Washington, N.Y) GenesisCS-2 Platelet Rich Plasma Kit, Vet Stem (San Diego, CA) Questions to Ask Does product include platelets? What is increase in platelet concentration? Are the platelets activated? How are the platelets activated? What supporting documents/studies do you have to support your answers? Were these studies independently performed?

14 Additional Studies -Supportive Pilot Data- RCT of effects of 3 PRP injections (immediately after, and at 2 week intervals post surgery) 6 received txment; 4 controls PRP group had significantly greater PVF and VI compared to control at 90 days Short-term study Control group remained very lame at 90 days 5 dogs/group in blinded, RCT Subjective outcome measures only, no negative control group (placebo) Both groups improved the same amount Concerns about care-giver placebo effect

15 Platelet-Rich Plasma Injection With Arthroscopic Acromioplasty for Chronic Rotator Cuff Tendinopathy: A Randomized Controlled Trial. Am J Sports Med PURPOSE: To investigate both the clinical and tissue effects of the coapplication of PRP injection with arthroscopic acromioplasty (AA) in patients with chronic rotator cuff tendinopathy. DESIGN: Randomized controlled trial; Level of evidence, 1. METHODS: 60 randomized patients with rotator cuff tendinopathy. Patients were randomized to AA alone or in combination with an injection of autologous PRP (AA + PRP). Efficacy was assessed by patient-reported outcomes (Oxford Shoulder Score) and tendon biopsy specimens taken 12 weeks after treatment. RESULTS: There was no significant difference in the OSS between AA alone and AA + PRP at any time point in the study. From 12 weeks onward, there was a significant increase in the OSS for both groups compared with their baseline scores (P <.001). CONCLUSION: Arthroscopic acromioplasty significantly improves long-term clinical outcomes up to 2 years. The coapplication of PRP did not affect clinical outcomes. PRP significantly alters the tissue characteristics in tendons after surgery with reduced cellularity and vascularity and increased levels of apoptosis. Platelet-rich therapies for musculoskeletal soft tissue injuries. Cochrane Database Syst Rev OBJECTIVES: To assess the effects (benefits and harms) of platelet-rich therapies for treating musculoskeletal soft tissue injuries. CONDITIONS STUDIED: rotator cuff tears; shoulder impingement syndrome; elbow epicondylitis; anterior cruciate ligament (ACL) reconstruction, patellar tendinopathy, Achilles tendinopathy and acute Achilles rupture surgical repair. MAIN RESULTS: We included data from 19 trials (1088 participants) that compared PRT to control. Used pooled data for primary outcomes (function, pain, adverse events). The evidence for all primary outcomes was judged as being of very low quality. AUTHORS' CONCLUSIONS: Overall, and for the individual clinical conditions, there is currently insufficient evidence to support the use of PRT for treating musculoskeletal soft tissue injuries. There is need for standardisation of PRP preparation methods.

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