CONFORM FLEX TECHNICAL MONOGRAPH

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1 CONFORM FLE TECHNICAL MONOGRAPH

2 CONFORM Flex INTRODUCTION MTF is a nonprofit organization founded in 1987 by academic orthopaedic surgeons dedicated to providing tissue of high quality and safety for transplantation. MTF has distributed over 6 million allografts since their inception, and has never reported a confirmed case of viral disease transmission. MTF s exemplary safety record is directly attributed to their commitment to the donor families and to the tissue recipients served. This tremendous commitment provides customers with the assurance that this gift of human tissue is safe and that it comes from a trustworthy source. MTF also thinks beyond safety. While safety governs every decision made, quality and efficacy also matter. Current techniques used by some tissue banks to clean, process, and sterilize demineralized allografts have been shown to be detrimental to the quality of the tissue. These methods vary widely from bank to bank, because the industry donor criteria and processing standards are open to interpretation. Demineralized allograft tissue of less-than-optimal quality may yield a graft that does not perform its intended function, which could lead to a less-than-optimal clinical outcome. PRINCIPLES OF BONE HEALING AND GRAFT INCORPORATION 1 Four components are necessary for bone healing and/or bone graft incorporation: the presence of host cells, a signal to trigger differentiation of the host cells to bone-forming cells, a scaffold or matrix on which the new bone can form, and an adequate blood supply. When bone fracture or injury occurs, there is loss of mechanical integrity of the bone and disruption to the blood supply. The healing cascade begins immediately. The 3 phases are: inflammation, repair, and remodeling. Inflammation is the process by which host cells remove debris from the injured site, prepare the local matrix into a site which can support cell growth, and enable new bone to be formed. Revascularization, which is required for new bone to grow, begins in the inflammation phase. Repair includes the recruitment and differentiation of host cells into osteoblasts, which in turn produce new bone at the injured site. Lastly, remodeling is the resorption of immature or extraneous bone coupled with reorientation of bone along the direction of mechanical loading to provide adequate structural support. These phases (Figure 1) are regulated by the release of local cytokines. DePuy Synthes Trauma CONFORM Flex Technical Monograph

3 Figure 1: Example of the healing cascade in fracture repair. The three phases of fracture repair include A) the inflammatory phase, B) the reparative phase, and C) the remodeling phase. 1 A Monocytes Mesenchymal cells Macrophages PDGF Neovascularization Recruitment TGF-ß etc... Cartilage B Intramembranous ossification Fibroblasts Osteoblasts Chondrocytes C Osteoclast/osteoblast remodeling occurs on the surface and via osteons New bone formation and bone healing are influenced by several factors in the bone graft material, some of which can be controlled, such as: Bone-forming potential Porosity ph Host factors are not as easily controlled: Age Systemic disease Vascularity Presence of infection Quantity and quality of host cells Use of anti-inflammatory drugs CONFORM Flex Technical Monograph DePuy Synthes Trauma 1

4 Bone grafts are often used as bone void fillers to assist with bone healing. Grafts can provide support and/or cell signals. During the healing process, the bone grafts are incorporated into the host bone by remodeling and/or creeping substitution. Bone graft materials used as bone void fillers can be described as osteogenic, osteoinductive, and/or osteoconductive. Osteogenic tissues are capable of forming new bone from living cells. Osteoprogenitor cells proliferate and differentiate into osteoblasts (bone-building cells) and eventually into osteocytes (mature bone cells). These cells represent the osteogenic potential of the graft. 1 Osteoinductive tissues are ones which promote chemotaxis, mitogenesis, and formation of osteoprogenitor cells that have osteogenic capacity (as described above). 2 Osteoinductive materials will form bone when implanted into tissues which would not otherwise form new bone. 3 Osteoconductive tissues allow for fibrovascular tissue development and osteoprogenitor cell invasion of a porous structure. This material then acts as a temporary scaffold which will be replaced with newly formed bone. 2 RATIONALE FOR CLINICAL USE OF DEMINERALIZED BONE Autograft bone provides all three components necessary for bone healing. It has been widely used during bone grafting procedures due to availability of donor graft sites and good incorporation upon transplantation. However, the use of autograft bone requires a second surgical site (to procure bone) which has associated morbidity risks. Allogeneic demineralized bone eliminates the need for a second surgical site, and its demineralized state results in bone that is osteoconductive and has osteoinductive potential. HOW DEMINERALIZED BONE WORKS The exact mechanism of the osteoinductive potential of demineralized bone has not been very well defined. However, it is thought that the removal of the mineral component of bone exposes the active bone morphogenetic proteins (BMPs) present in the demineralized bone while retaining the inherent osteoconductive properties of the bone. When implanted, the active BMPs are thought to signal the host mesenchymal cells thus causing the cells to proliferate and differentiate into chondroblasts, which in turn will form a cartilage matrix. Ultimately, the cartilage matrix is converted into a calcified extracellular matrix. This calcified matrix will become vascularized, and osteoprogenitor cells will form new bone on this matrix. This will be followed by the formation of bone marrow and marrow elements. 3 2 DePuy Synthes Trauma CONFORM Flex Technical Monograph

5 Figure 2: Schematic showing differentiation of a stem cell into an active osteoblast, and demonstrating the influence of growth factors. 2 STEM CELL BMP S COMMITTED OSTEOBLASTIC STEM CELL CHONDROCYTE IGFs TGF-ß BMPs MUSCLE FIBROBLAST ADIPOCYTE IGFs TGF-ß BMPs OSTEOBLAST WHAT CONSTITUTES GOOD DEMINERALIZED BONE MTF has developed and validated a procedure for cancellous bone demineralization. This procedure provides safe, high-quality demineralized bone allograft and was developed through rigorous testing to ensure that the osteoinductive potential of the tissue was not compromised. Many factors contribute to the high quality of MTF demineralized cancellous bone including: Stringent donor selection Careful processing Quality control metrics QUALITY TISSUE MTF s quality and safety standards consistently meet or exceed the requirements of the American Association of Tissue Banks (AATB) as well as the guidelines for screening and testing of tissue donors set forth by the U.S. Food and Drug Administration (FDA). The AATB and FDA set only minimal guidelines to ensure safety of tissue. Potential MTF donors must pass through an extensive quality assurance process. CONFORM Flex Technical Monograph DePuy Synthes Trauma 3

6 Screening begins at the site of recovery with a comprehensive medical and social history that includes the cause of death. Tissue and blood samples are tested for infectious diseases, including hepatitis, HIV, and syphilis. A team of medical/ technical specialists from the infectious disease and tissue banking fields evaluates all information, including test results, before the donor is released for processing. Figure 3 lists those conditions for which MTF voluntarily defers donors for safety or quality reasons even when not required by the FDA or AATB. Figure 3: Tissue bank screening criteria. Note: MTF voluntarily exceeds both AATB and FDA screening criteria. SCREENING CRITERIA MTF AATB FDA Hepatitis B virus Hepatitis C virus HIV 1/2 Malaria Sepsis Syphilis Transmission spongiform encephalopathy (TSE) Vaccinia West Nile virus (WNV) Clinically significant metabolic bone disease Leprosy (Hansen s disease) Polyarteritis nodosa Rabies Rheumatoid arthritis Sarcoidosis Systemic lupus erythematosus Systemic mycosis Tuberculosis Active genital herpes Ankylosing spondylitis Antiphospholipid syndrome Autoimmune hemolytic anemia Autoimmune lymphoproliferative syndrome Autoimmune thrombocytopenic purpura Autoimmune vasculitis Cancer Chagas disease Clinically active Epstein Barr virus (mononucleosis) Clinically active gonorrhea Clostridium difficile infection Cold agglutinin disease Encephalitis Endocarditis Guillain-Barre syndrome Illicit drug use Meningitis Methicillin-resistant Staphylococcus aureus (MRSA) Mixed connective tissue disease Multiple sclerosis Myasthenia gravis Peritonitis Poliomyelitis Pyelonephritis Reactive arthritis (Reiter s syndrome) Rheumatic fever Vancomycin-resistant Enterococcus (VRE) Varicella zoster Wegener s granulomatosis Any acute infectious/septic illness 4 DePuy Synthes Trauma CONFORM Flex Technical Monograph

7 CAREFUL PROCESSING To maintain biological integrity, MTF processes all tissue using aseptic techniques in ISO Class 4 (certified) clean rooms. MTF s use of these clean rooms is designed to prevent any environmental contamination of the tissue and thus eliminates the need for terminal sterilization by high-dose gamma radiation, which has been shown to compromise the biological and biomechanical integrity of allograft tissue (Figure 4). 4,5 Figure 4: Gamma radiation decreases osteoinductive potential of demineralized bone powder in a dosedependent manner, as measured using an in vitro alkaline phosphatase (ALP) assay. 5 ALP mmol/mg protein/min Demineralized Bone Matrix lot n 0 kgy n 12 kgy n 18 kgy n 25 kgy MTF s demineralized bone matrices are produced from both cancellous and cortical bone which are subjected to controlled cleaning processes (including hydrogen peroxide and ethanol), followed by demineralization with hydrochloric acid. Extended exposure to harsh chemicals such as hydrogen peroxide has been shown to have a negative effect on the osteoinductive properties of tissues as shown in Figure 5. 6 MTF s aseptic processes have been designed to minimize exposure to harsh chemicals and preserve the biological integrity of the tissue. Figure 5: Osteoinductivity versus soaking time (in hours) of tissue in 3% hydrogen peroxide the negative effect of peroxide on bone osteoinductivity gets more pronounced as soak times increase. Osteoinductivity Effect of hydrogen peroxide time on osteoinductivity y= x R 2 = Soaking Time (in hours) CONFORM Flex Technical Monograph DePuy Synthes Trauma 5

8 PACKAGING CONFORM Demineralized Cancellous Tissues are provided in a hydrated form (Q-PACK Zero Rehydration Technology). Lyophilized cancellous tissues take as long as 5 20 min to rehydrate prior to use. The Q-PACK Technology ready to use packaging is a way to provide grafts in a hydrated state directly from the packaging. The Q-PACK Technology: Allows tissue to be stored in a fully hydrated state at ambient temperature Saves valuable operating room time for the surgeon/hospital Allows for tissue to be used for emergency purposes, when zero preparation is essential BMP-2 CONTENT Bone induction is a sequential, multistep cascade which involves various growth factors. Bone morphogenetic proteins (and other intrinsic growth factors) in bone are exposed by the demineralization process. However, BMP-2 has been shown to be the best single predictor of osteoinductive potential based on statistical analysis of the correlation between in vitro levels of various growth factors and in vivo osteoinductivity. 7,8 BMP-2 levels in demineralized cancellous tissues as measured in vitro via enzyme-linked immunosorbent assay (ELISA) are shown in Figure 6. Figure 6: BMP-2 levels in CONFORM Demineralized Cancellous Tissue compared to OsteoSponge (Bacterin). 9 BMP-2 concentration (pg/g bone) CONFORM Demineralized Cancellous Tissue n CONFORM Demineralized Cancellous Tissue n OsteoSponge OsteoSponge 6 DePuy Synthes Trauma CONFORM Flex Technical Monograph

9 CELL COMPATIBILITY/BIOCOMPATIBILITY 10 Cancellous bone is naturally a highly porous, three-dimensional biologic scaffold that is inherently osteoconductive. After processing the bone using a specific demineralization and ph restoration recipe, an optimal cell friendly environment that also possesses exposed bone morphogenetic proteins is created for CONFORM Demineralized Cancellous Allografts. In the clinical setting, bone marrow aspiration is often used to recover autologous osteogenic cells and aspirates can easily be combined with CONFORM Demineralized Cancellous Tissue scaffolds. This technique provides cells capable of new bone formation and additional biological growth factors that assist in the healing and remodeling of bone. In order to evaluate the ability of CONFORM Demineralized Cancellous Tissue grafts to support cellular adhesion and osteogenic differentiation, an in vitro study was performed where scaffolds were seeded with human mesenchymal stem cells (hmscs) and then cultured in osteogenic medium. Cells were first observed to attach to the demineralized cancellous bone and populate the scaffolds during the first day following cell seeding (Figure 7). Subsequently, these cells remained adhered to and spread out over the bone surfaces during longer term culture (Figure 8A). When exposed to osteogenic medium, hmscs exhibited osteogenic behavior on CONFORM Demineralized Cancellous Tissue scaffolds as evidence of mineralization was observed at 6 weeks in culture (Figure 8B and 8C). Collectively, these findings indicate that CONFORM Demineralized Cancellous Allografts are highly biocompatible scaffolds for hmscs and are capable of supporting osteogenic differentiation and bone matrix deposition. Figure 7: Time sequential images of hmscs seeded onto CONFORM Demineralized Cancellous Tissue scaffolds that were obtained during the first day of culture. The cells were fluorescently stained green using CellTracker TM Green CMFDA while the scaffolds were stained red using Alexa Fluor minutes 3 hours 24 hours CellTracker is a trademark of Thermo Fisher Scientific Inc. CONFORM Flex Technical Monograph DePuy Synthes Trauma 7

10 Figure 8: (A) Histological image of hmscs seeded onto fully demineralized cancellous bone scaffolds at 14 days after cell seeding. Sections were stained with hemotoxylin and eosin. (B, C) Histological images that represent either (B) von Kossa staining of sections (black color) or (C) Alizarin Red staining (dark red color) and depict evidence of mineralization through the deposition of calcium phosphate by hmscs on demineralized cancellous bone scaffolds at 6 weeks after cell seeding. MSCs were exposed to osteogenic medium during culture to elicit osteogenic differentiation. 8 DePuy Synthes Trauma CONFORM Flex Technical Monograph

11 IN VIVO OI POTENTIAL 11 To assess the in vivo performance and osteoinductive potential of cancellous tissues when combined with bone marrow aspirate (BMA), an athymic rat muscle pouch model was used. Multiple lots of the tissue were mixed with BMA and implanted bilaterally in the hamstring muscles of athymic rats. Animals were sacrificed at 4 weeks post-implantation. Decalcified histology was then performed on the explanted samples, with one histological section prepared for each sample. Slides were stained with hematoxylin and eosin and evaluated for osteoinductivity. A semi-quantitative scoring system was utilized to assess osteoinduction. Osteoinductive scores were based on the degree to which new bone, bone cells, osteoid, calcified cartilage remnants, and marrow elements were present. To be consistent with proposed standards in the industry, 12 the scoring system in Table 1 was utilized. 13 Table 1: Osteoinductivity Scoring Scale and Criteria Score Criteria 0 No evidence of new bone formation % of the section is covered by new bone % of the section is covered by new bone % of the section is covered by new bone 4 >75% of the section is covered by new bone Cancellous tissue combined with BMA was consistently osteoinductive in this model; 100% of the samples were osteoinductive, with an average osteoinduction score (pooling data from 3 donors) of 3.94 ± 0.23 (Table 2). Figures 9 and 10 show the representative histological response to Cancellous + BMA, with robust new bone formation including bone marrow. Table 2: Summary statistics, number of samples that could be histologically evaluated, and number of osteoinductive samples for each group. Number of osteoinductive samples is divided by the number of evaluated samples to give the % of osteoinductive samples for each group. Summary Statistics Cancellous + BMA Osteoinduction Score (0-4 Scale) # Ranked Samples Mean Std Dev /36 (100%) # Osteoinductive Samples (Percentages) CONFORM Flex Technical Monograph DePuy Synthes Trauma 9

12 Figure 9: Cancellous + BMA demonstrating the presence of bone marrow and new bone formation (arrows). H&E stain; 100 magnification; BAR = 100 µm. Figure 10: Cancellous + BMA demonstrating residual CONFORM Demineralized Cancellous Tissue, the presence of bone marrow and new bone formation (arrows). H&E stain; 100 magnification; BAR = 100 µm. 11 DePuy Synthes Trauma CONFORM Flex Technical Monograph

13 SUMMARY MTF s processes for bone demineralization and creation of DBM tissue forms have been designed and validated to ensure the safety of the allografts without adversely affecting their biological performance. The processes and studies described here demonstrate that MTF s decontamination and demineralization procedures preserve the endogenous proteins and osteoinductive potential of the tissues and create allografts that support cell infiltration and function. CONFORM Flex Technical Monograph DePuy Synthes Trauma 11

14 REFERENCES 1. Mehta S, Collings C. Orthobiologics: Improving Fracture Care Through Science: Lippincott Williams & Wilkins; Mouch CS, Einhorn TA. Bone Morphogenetic Proteins and Other Growth Factors to Enhance Fracture Healing and Treatment of Nonunions. In: Lieberman JR, Friedlander GE, editors. Bone Regeneration and Repair: Biology and Clinical Applications: Humana Press; p Boyan BB, McMillan J, Lohman CH, Ranly DM, Schwartz Z. Bone Graft Substitutes: Basic Information for Successful Clinical Use with Special Focus on Synthetic Graft Substitutes. In: Laurencin CT, editor. Bone Graft Substitutes: ASTM; p Vangsness CT, Jr., Wagner PP, Moore TM, Roberts MR. Overview of Safety Issues Concerning the Preparation and Processing of Soft-Tissue Allografts. Arthroscopy. 2006;22(12): Han B, Yang Z, Nimni M. Effects of Gamma Irradiation on Osteoinduction Associated with Demineralized Bone Matrix. J Orthop Res. 2008;26(1): DePaula CA, Truncale KG, Gertzman AA, et al. Effects of hydrogen peroxide cleaning procedures on bone graft osteoinductivity and mechanical properties. Cell Tissue Bank. 2005;6(4): Blum B, Moseley J, Miller L, et al. Measurement of Bone Morphogenetic Proteins and Other Growth Factors in Demineralized Bone Matrix. Orthopedics. 2004;27(1 Suppl):s Murray SS, Brochmann EJ, Harker JO, King E, Lollis RJ, Khaliq SA. A Statistical Model to Allow the Phasing out of the Animal Testing of Demineralised Bone Matrix Products. Altern Lab Anim. 2007;35(4): Osteosponge. Bacterin. Available from: Accessed on October 1, CONFORM with Q-PACK : An evaluation of a Fully Demineralized Scaffold with Q-PACK Technology Seeded with Human Mesenchymal Stem Cells. 11. Osteoinductivity of MTF CONFORM TM Cube with Bone Marrow Aspirate in the Athymic Rat Model (White paper.) 12. Draft Standard: Standard Guide for the Assessment of Bone Inductive Materials, ASTM F04.4 Division, Draft by Barbara Boyan, Univ. of Texas Health Science Center at San Antonio, downloaded from ASTM website Edwards JT, Diegmann MH, Scarborough NL. Osteoinduction of human demineralized bone: characterization in a rat model. Clin Orthop Relat Res. 1998;(357): DePuy Synthes Trauma CONFORM Flex Technical Monograph

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16 Indications CONFORM Demineralized Cancellous Tissue is regulated by the FDA as an HCT/P (Human Cells, Tissues, and Cellular and Tissue-Based Product) Limited Warranty and Disclaimer: DePuy Synthes Trauma products are sold with a limited warranty to the original purchaser against defects in workmanship and materials. Any other express or implied warranties, including warranties of merchantability or fitness, are hereby disclaimed. WARNING: In the USA, this product has labeling limitations. See package insert for complete information. CAUTION: USA Law restricts these devices to sale by or on the order of a physician. Not all products are currently available in all markets. Some devices listed in this technique guide may not have been licensed in accordance with Canadian law and may not be for sale in Canada. Please contact your sales consultant for items approved for sale in Canada. Manufactured or Distributed by:: Synthes USA Products, LLC 1302 Wrights Lane East West Chester, PA Telephone: (610) To order: (800) Fax: (610) Processed by Musculoskeletal Transplant Foundation 125 May Street Edison, NJ T. +1 (732) Fax. +1 (732) DePuy Synthes All rights reserved. DSUS/MOC/0914/0108 7/15 DV CONFORM, and Q-PACK are registered trademarks of The Musculoskeletal Transplant Foundation.