NANOFACTOR FLOW Stem Cell Therapy An Alternative in Managing Knee Arthritis Phil Davidson, MD Park City, UT

Non-Operative Management of Knee OA NSAIDS PT Weight loss Bracing Injection Therapy Corticosteroids Viscosupplementation PRP Autologous MSC’s (BMA derived) Amniotic Tissue Graft-Allogeneic MSC

Legacy Injection Therapies Corticosteroids-good and bad…. Viscosupplementation: unpredictable, often ineffective, only potential to address aching, no reparative capacity, many insurances are not authorizing

CORTICOSTEROID BENEFITS Rapidly reduce pain due to inflammation Last for several weeks to months Joints, Spine radiculopathy, Bursitis, Tendonitis, Neural inflammation (CTS)

CORTICOSTEROID SIDE EFFECTS SHORT TERM USE Atrophy Depigmentation Hyperglycemia Infection Post injection flare Tissue structure weakening, tendon ruptures

PRP in Cartilage Repair Chondrocytes and MSCs exposed to PRP show increased cell proliferation and cartilage extra- cellular matrix synthesis of PG and Type II collagen Synoviocytes cultured in PRP produce more HA- better lubrication and chondroprotective? Better pain scale outcomes with PRP injections versus HA injections for management of knee OA PRP preps highly variable Over 6000 articles on PRP/only 50% of them show an effect-platelets, leucocytes?

BIOLOGICS Knee OA Biologic treatment is interactive KEY ELEMENTS ALL REQUIRED: Growth factors, cells, scaffold Mechanically favorable environment A “Nutrient Rich Soup” includes- Cells (RBC, white cells, MSC) Growth factors (from platelets or plasma) Scaffolds Potential sources of this “Soup”: Bone Marrow Aspirate, Amniotic Fluid

MSC’s in Sports Medicine MSC’s are undifferentiated cells: Capacity for prolonged self-renewal Ability to differentiate into specialized cell types Have shown enhanced cartilage, tendon and meniscus healing These results have increased patient awareness and demand High-profile professional athletes are signing up for relatively untested cell-based therapies

Mesenchymal Stem Cells in Sports Medicine Attractive to patients-they want regeneration technology rather than replacement Harness your own body’s ability to heal Not taking exogenous drugs You are using your cells to heal your tissues Perception that stem cells are reversing the trend not just treating it

Mesenchymal Stem Cells in Sports Medicine MSCs do not recreate tissue Modify the environment to enhance healing Patients feel better-anti-inflammatory effect May have role for Osteoarthritis May be a role for augmenting surgical procedures-ACLR, RCR, cartilage restoration We don’t know the ideal type or number of stem cells in specific indications

Mesenchymal Stem Cells Reparative Promise in Knee OA Increased meniscal volume in post- menisectomy patients who received adult MSC injections into the knee MSC implanted for knee OA showed improved activity levels and cartilage regeneration One-step MSC treatment for large full thickness chondral defects showed improvement and significant cartilage fill

Mesenchymal Stem Cells Reparative Promise in Knee OA Current MSC treatments for OA include BMA derived autogenous stem cells that are immediately injected into the knee Jim Andrews MD at the Andrews Institute believes it controls swelling and inflammation and eases pain in knee OA Results in a pilot study of 31 NFL players, at 10 months, showed efficacy Reported decreased pain up to 45% with scores & improved by 50% from baseline at 6 months

Amniotic Membrane and Fluid Allograft So why use nanofactor FLOW if BMA works? Growth factors, scaffolding, and MSCs First used in 1910 Preservation techniques in 1940 Has been proven in ophthalmology, burns, plastic surgery, foot and ankle, diabetic ulcers Amniotic fluid stem cells have a delayed/more robust differentiation compared to bonemarrow derived MSC in recent studies

Journal Transplantation, April 2015 Prospective Randomized Trial for Knee OA Allograft MSC vs Hyaluronic Acid injected in knee One year follow up MSC patients improved clinical outcomes, including pain vs. HA MSC patients showed actual IMPROVEMENT in cartilage quality and quantity on MRI vs. HA

Attributes of Amniotic Tissue Immunoprivileged (no class 1 or 2 HLA antigens) No rejection reaction Anti Inflammatory Transforming growth factor beta-1 (TGF-1) Insulin-like growth factor I (IGF-I) Anti Microbial- defensing/peptides/ enzymes Cells: MSC’s, Fibroblasts and Keratinocytes +++ Chen EH, et al. J Implant Adv Clin Dent. 2009;2(3):67-75. (2)Bongiovanni cd133+ cell as advanced medicinal product for Myocardial and limb ischemia Stem cells and development vol23, 20, 2014 (3)Karantalis, MSc w CABG circulation research Feb 2014

NANOFACTOR™ FLOW 900,000 cells/mL 44% MSC’s; remainder-kerintinocytes, fibroblasts, epidermal Collagen Types III, IV, V, VII Amino acid precursors – taurine, glutamine Growth factors: Epidermal growth factor, Transforming growth factor alpha & beta-1, Insulin-like growth factor 1, Granulocyte colony stimulating factor (AmnioTechnology LLC, with permission)

Nanofactor Flow: Nanofactor Membrane: MSCs + Growth Factors + Scaffold NanoFx (MSCs) + Growth Factors + Scaffold

PRP 1 Element Tx BMA 2 Element Tx Nanofactor All Elements

Amniotic Membrane

Amniotic Scaffolding Cryofractured Amnion Membrane particle Micronized amnion contained within the allograft was imaged by scanning electron microscopy. Here you can see at a magnification of 20 000x the membrane particles. The membrane particles are approximately 0.5 to 1 um in size. Scanning Electron Microscopic image of cryofractured amniotic membrane particles.

Nanofactor Minimally Manipulated Extracellular Matrix Morcelized Tissue Disrupted 3D Structure CryoFractured Tissue Intact 3D Structure

What makes Nanofactor Flow different? Growth factors Scaffolding Cells and lots of them and immature-pluri- potential-900,000 viable cells/ml, 44% are MSCs in one ml BMA has 1,500 CFU-f/ml, another article stating 2,300 CFU-f/ml (donors<1 yo) to 500 CFU-f/ml (donors>60) BMA cells are senile

Amnio vs BMA: MSC Volume Comparison Amniotic 440,000 MSC’s/1ml Bone Marrow Aspirate 1,600 MSC’s/1ml Jing Li et al: Chin J Cancer Res 23(1): 43-48, 2011

Human MSCs Decline with Age _______ 1,000 1 Estimates obtained by CFU- f assay MSCs per Marrow Cells 1 Bone marrow MSCs decline with age. Bone marrow was obtained, dispersed, placed in a Percoll gradient and the light cell fraction seeded onto culture, After 7-10 days colonies can be visualized. The CFU-F assay is an incomplete and crude estimate of MSCs in marrow samples, but clearly we can see that the number of MSCs in marrow decreases with age. _______ 100,000 _______ 250,000 1 1 _______ 400,000 1 _______ 2,000,000 Newborn Teen 30 50 80 Age (Years) Adopted from: Al Caplan. J Pathol 2009; 217:318-314, 2008

Relative Number of MSC’s by Age Adopted from: Al Caplan. J Pathol 2009; 217:318-314, 2008 MSCs per Marrow Cells 2000 400 250 100 1 Bone marrow MSCs decline with age. Bone marrow was obtained, dispersed, placed in a Percoll gradient and the light cell fraction seeded onto culture, After 7-10 days colonies can be visualized. The CFU-F assay is an incomplete and crude estimate of MSCs in marrow samples, but clearly we can see that the number of MSCs in marrow decreases with age. Newborn Teen 30 50 80 Age (Years)

Amniotic Tissue Allograft Nanofactor FLOW Growth Factors/Peptides Cellular Components Extracellular Matrix So to summarize, the amniotic tissue allograft (Nanofactor flow) contains the same 3 elements present in amniotic tissue and that have been demonstrated to have anti-inflammatory, anti-scarring, anti-microbial, and regenerative capabilities which are the growth factors, the cellular components, and the extracellular matrix/scaffold + +

Amniotic Placental Tissue Collection Scheduled c-section Amniotic fluid (~ 650cc) Amnion membrane (900 cm2) Consent Infectious Disease Testing Shipping to Manufacturing Facility Overnight at 1-10o C Tissue Processing Processed in Class ISO 5 Bioburden Testing USP <61> Environmental Monitoring Micronized Amnion Growth Factors, Cytokines, ECM Proteins Viable Cells 5% DMSO Final Product Lot Release Sterility Testing USP <71> Mycoplasma Testing USP <63> Endotoxin Testing USP < 85> 3-4 weeks Nanofactor Flow is derived from placental tissue donation obtained from consenting donors who are screened in the prenatal unit and donors with a drug or alcohol history and those with communicable diseases such as HIV and hepatitis are excluded from donation. Tissue is procured during a scheduled c-section. Product Packaging and Distribution >65o C

How do the sources compare? “A multipotent stem cell population that is still of fetal origin and may be superior in proliferation and differentiation to cells deriving from adult tissues” Francesco Alviano et al, BMC Developmental Biology

Why do I offer my patients Nanofactor Flow? Patients can benefit from this non surgical treatement modality Many patients want/need to avoid surgery Large demographic of patients wants to “try everything before considering surgery” This is only modality that has the actual ability heal cartilage and tissues (vs steroid/HA) Easier to administer than PRP/BMA Cost is not out of line with PRP/BMA

How do I discuss Nanofactor Flow? Attractive option to include “stem cells” Safe, no rejection, no after-pain New technology without specific indication parameters I discuss the concept of orthobiologic treatment to include the 3 key elements: cells, growth factors and scaffolding I explain that senescent MSC’s make very little sense to me It is not a magic bullet but it does have promise I explain that it is expensive and not covered by insurance

PHASE I Getting Started in the Office + CryoFreezer: If you plan to do any volume Dilutants & Nanofactor

Acceptable Dilutants Flow (Pure) Lidocaine /Marcaine, (not in joints) PRP (Platelet Rich Plasma): PPP (Platelet Poor Plasma): Hyaluronic acid (supartz, eufflexa): Saline: Use Normal Saline: Types of Saline: Normal: Sterile Solution of Sodium chloride in water Bacteriostatic: Sterile Solution with 0.9% benzyl alcohol with no sodium chloride

Unacceptable Dilutants Flow Unacceptable Dilutants Do Not Use Glycerol (eg. Grafton DBM) Glucose Solutions (eg. D5NS) Epinephrine (in or around treatment site) Lidocaine/ Marcaine (with preservative) in joints Do NOT Use Systemically – no IV or intra arterial injections No literature to support this method of administration.

Preparation & Mixing Flow Remove the package from freezer or dry ice container. Cut the package. *Outside of vial is not sterile Defrost vial by holding still in hand for 3-5 minutes. *Do not shake vial Fill syringe with dilutant. (typically 1:1 ratio) Draw Nanofactor™ Flow into syringe with 18g needle. Use a 22 or 23g needle for injection but nothing smaller than a 23g

Recommended Joint Dosing Flow Recommended Joint Dosing Shoulder 1ml Flow Graft, 1:2 up to 1:4 dilution   Elbow 1:1 dilution Wrist, Fingers & Facets (per joint) 0.25ml Flow Graft, Hip 2ml Flow Graft, 1:2 up to 1:3 dilution   Knee (per compartment) Stage 3: 0.5ml Flow Graft, Stage 4: 1ml Flow Graft, 1:2 dilution Ankle 1ml Flow Graft, 1:1 dilution Subtalar & Midfoot Joints 0.5ml Flow Graft, 1:1 dilution   MPJ’s & Toes (per joint) 0.25ml Flow Graft,

PEARLS No anti-inflammatories or ASA (6 weeks pre & post) Increased tissue vascularity improves cellular incorporation Lidocaine 1% preservative free as carrier Full activity (restrictions only if warranted by diagnosis) No issues with icing or polar care machine Not use – systemically, open growth plates, oncologic process, active infection

Summary Nanofactor Flow is an alternative therapy to treat inflammatory and degenerative musculoskeletal conditions. It makes sense given what we know about orthobiologic principles with MSCs, growth factors and scaffolds Patients are asking for this type of treatment The cost is not prohibitive Early observations very encouragin We need more prospective data at all levels Dosing Efficacy Indications/Contraindications

Thank You phildavidsonmd@gmail.com