Bioactive Glass and DBM for Enhanced Bone Healing

Transcription

1 Bioactive Glass and DBM for Enhanced Bone Healing

2 BACKGROUND Bone void fi llers comprised of human demineralized bone matrix (DBM) in a biologically acceptable carrier are an important tool for the orthopedic surgeon. DBM-based bone-void fi llers aid in bone healing, minimize the need for autologous graft material and eliminate donor site complications. NanoFUSE DBM is a new demineralized bone matrix product that combines the osteoinductive capabilities of DBM with the osteopromotive properties of 45S5 bioactive glass. NanoFUSE DBM is formulated to reconstitute into a paste, syringeable gel, or putty upon addition of a sterile fl uid, e.g., water for injection (WFI) or sterile saline. NanoFUSE DBM, provided in single-use 2, 5, and 10cc sizes in a pre-fi lled syringe ready for reconstitution, is both sterile and pyrogen free. The dry product, in single-use syringes, is sterilized post packaging by ionizing radiation (electron beam). At point of use, the surgeon reconstitutes the product with an appropriate sterile solution of his/her choice (e.g. WFI or sterile normal saline). The coated particles rehydrate in less than 30 seconds and do not require mixing to form a uniform paste or putty. The material is then extruded by the surgeon into the appropriate bone voids. Results of studies conducted by both Nanotherapeutics and independent laboratories demonstrate that the osteoinductive capacity of the NanoFUSE DBM with bioactive glass is preferable to other commercial bone void fi ller implant preparations with only DBM. Using histopathological observation of ectopic bone growth, including the appearance of cartilage-forming and bone-forming cells, the presence of new cartilage, new bone, and new bone marrow provides excellent measures for comparison. Additionally, the inclusion of calcium-based 45S5 bioactive glass, the fi rst humanmade material to release calcium and form a direct chemical bond with tissue, NanoFUSE DBM produces an exceedingly strong interfacial bond between the graft and adjacent boney tissue within minutes. The original formulation of 45S5 bioactive glass was invented in 1971 (Hench, et al). The capacity for rapid interfacial bonding, which is the defi ning characteristic of a bioactive material, is the result of chemical reactions that take place on the surface of bioactive glass when it is exposed to bodily fl uids. This rapid bone bonding ensures positional stability of the graft during the critical period immediately following surgery. The ability of bioactive glass to rapidly bond with native tissue and trigger the cellular process of healing has prompted its use in an increasing number of clinical settings. NanoFUSE DBM (K062459), a malleable putty-like bone void fi ller, is indicated for use in general orthopedic procedures (both elective surgeries and trauma injuries). NanoFUSE DBM is comprised of human demineralized bone matrix and synthetic calcium phosphor-silicate particulate material (45S5 bioactive glass) particles, both coated with Type A gelatin (acid treatment, USP) derived from porcine skin. Gelatincoated particles are packaged dry in a polypropylene syringe, double-wrapped in peel-back pouches, and fi nal packaged in a dust cover paperboard carton. The product packaging is in the form of a luer-lock syringe, with a one-way check valve at the end, in which the device is reconstituted. The product is intended for single-patient use only. PERFORMANCE MEASURES Cytotoxicity Cytotoxic effects of NanoFUSE DBM and the empty syringe assembly (vented syringe, luer lug, check valve fl uid path-way) were evaluated with the test protocol ISO MEM Elution Using L-929 Mouse Fibroblast Cells (also described in the USP 29 <87> Biological Reactivity Tests, In Vitro). NanoFUSE DBM and the empty syringe assembly did not induce cytotoxicity and no abnormal events such as ph change or debris were noted in this assay. NanoFUSE DBM is non-cytotoxic. Mutagenicity Mutagenicity was evaluated with the test protocol Bacterial Mutagenicity Test Ames Assay, which evaluates the mutagenicity potential of a test article by measuring its ability to induce back mutations at selected loci of several strains of bacteria in the presence and absence of microsomal enzymes by a method compliant with the requirements specifi ed in ISO :2003. It was also determined that NanoFUSE DBM did not cause increases in point mutation, exchanges or deletions. NanoFUSE DBM is considered non-mutagenic. Pyrogenicity Since NanoFUSE DBM includes a biologic component, information regarding endotoxin level and pyrogenicity nanofuse DBM - 2

3 are included in the release specifi cation for each manufactured lot. Intact, granular samples of NanoFUSE DBM are submitted to an independent test laboratory to perform Limulus Amebocyte Lysate (LAL) testing on a lot by lot basis, using the Kinetic Chromogenic Method. To conform to current USP standards, blood-contacting devices must show endotoxin levels of <20 EU/device or <0.5 EU/mL. NanoFUSE DBM may be considered non-pyrogenic. Sensitization Sensitization of NanoFUSE DBM was evaluated using the test protocol ISO Guinea Pig Maximization Sensitization Test Method for Biomaterial Extracts which evaluates if the material stimulates the immune system to produce an allergic re-sponse. Since the response is usually due to leachable substances in the material, extracts are used for administration to guinea pigs. Extraction of NanoFUSE DBM was performed according to the standard ANSI/AAMI/ISO Sample Preparation and Reference Materials. Freund s Complete Adjuvant (FCA) and sodium lauryl sulfate (SLS) were used to enhance a potential weak sensitizing effect. NanoFUSE DBM showed no sensitization response and was equivalent to negative controls. Systemic Toxicity Systemic toxicity of NanoFUSE DBM was evaluated with the test protocol ISO Acute Systemic Injection Test which was conducted in accordance with the ISO :1993, Biological Evaluation of Medical Devices - Part 11: Tests for Sys-temic Toxicity and USP 29, This test screens for potential toxic effects as a result of a single-dose systemic injection in mice. None of the NanoFUSE DBM treated animals presented adverse clinical signs at any of the observation periods and none of the animals lost in excess of 2 g body weight over the course of the study. NanoFUSE DBM does not elicit systemic toxicity. Intracutaneous Reactivity Intracutaneous reactivity of NanoFUSE DBM was evaluated with the test protocol ISO Intracutaneous Reactivity Test which was conducted in accordance with the ISO :2002, Biological Evaluation of Medical Devices, Part 10: Tests for Irritation and Delayed-type Hypersensitivity. This test evaluates chemicals that may leach out of or that are extracted from the test article are capable of causing local irritation in the dermal tissues of a rabbit. The mean test score both for NanoFUSE DBM cottonseed-oil extract and for control extract was 0 (out of 0 to 4). NanoFUSE DBM is considered a non-irritant. Implantation Intramuscular reactivity of NanoFUSE DBM was evaluated with the test protocol ISO Intramuscular Implant Test 4 Week Duration in Rabbits which was conducted in accordance with the ANSI/AAMI/ISO :1995, Biological Evaluation of Medical Devices, Part 6: Tests for Local Effects after Implantation. This test evaluates local toxic effects of a biomaterial in direct contact with living muscular tissue of the rabbit for four weeks. All animals survived to the scheduled endpoint of the study and no abnormal clinical signs were noted with all implants and implant sites appearing macroscopically normal. Tissue reactions such as granulation tissue with poly- and mononuclear cell infi ltration, fi broblasts, neovascularization, and capsule formation are common and expected in this model. Comparison of NanoFUSE DBM to an inorganic material differential scoring reported NanoFUSE DBM as a moderate irritant. Tissue reaction in the muscle tissue is not unexpected when using an implant made of organic fragmented material such as NanoFUSE DBM. Human demineralized bone matrix (DBM) is xenograft material in a rabbit model. Osteoconductivity: Critical-size Defect in the Rabbit Radius Nanotherapeutics conducted a GLP compliant study at an independent test laboratory to evaluate the proprietary formulation of NanoFUSE DBM for enhancing bone formation and subsequent healing of a criticalsize defect in the rabbit radius. A comparison was made among NanoFUSE DBM, a FDA compliant Predicate Device, and autogenous bone graft, which is the gold standard in clinical practice. The Predicate Device for comparison was a DBM paste with inert porcine collagen carrier. In the comparison study, 54 male, New Zealand White Rabbits were assigned to one of fi ve study arms (n=6 for the con-trol and n=12 animals for each test article arm; Predicate Device, NanoFUSE DBM, NanoFUSE DBM w/o DBM, and NanoFUSE DBM w/o bioactive glass). Each animal had one 1.5 cm defect surgically created in one of its front limbs and each animal was assigned one treatment. Each defect was fi lled with one of the four test articles or autogenous bone as a positive control. The autogenous control performed as expected with 5 out of 5 fusions (for animals completing the full 12 weeks). The Predicate Device had one animal scoring likely fused based on radiographic results and 11 animals without fusion. The most likely explanation for this nanofuse DBM - 3

4 outcome is that human DBM in the rabbit model would elicit an immune response that interfered with complete healing. Each of the remaining test articles had one animal that was scored as fused based on radiographic interpretations. All test articles and the Predicate Device produced less callus than the autogenous bone (p<0.001, Student s t test, 2 tail, heteroscedastic). However, NanoFUSE DBM produced more callus than the Predicate Device (p<0.159). The explanted tissue was decalcifi ed and sectioned for histology. Cross sections were taken at the proximal, midline, and distal ends of the explant. These slides were evaluated on the presence or absence of the following characteristics: 1) original DBM, 2) bone callus, 3) estimate % of normal bone involved in callus, 4) chondroblasts/ chondrocytes, 5) osteoblasts/ osteocytes, 6) cartilage/osteoid, 7) new bone, 8) bone marrow, 9) connective tissue admixed with bone elements, 10) foreign material. The histological fi ndings for NanoFUSE DBM were substantially equivalent to those of the Predicate Device. Studies above were conducted in compliance with U.S. Food and Drug Administration Good Laboratory Practices regulations set forth in 21 CFR part 58. Nanotherapeutics contracted an independent test laboratory to oversee the long-term implantation study. Radiography and radiographic interpretations were performed by this facility. Histology preparations and histopathology readings were performed at another independent test laboratory. Scanning and digital analysis of radiographs were performed by Nanotherapeutics. Osteoinductivity: Rodent Ectopic Implant Assay The rodent ectopic (RE) bone assay is considered the gold standard of assays for osteoinductivity measurement (versus in vitro cell-based assays or antibodybased assays for bone morphogeneic proteins, McKay, et al. 2006, p. 10; ASTM F04.4 Division 2003). In brief, test materials are surgically implanted into intramuscular pouches. After 28 days, the implant site is excised and prepared for histolopathological examination for evidence of new bone formation. The ability of materials to induce bone formation in non-boney sites is characterized as osteoinduction. The following four materials were submitted for the RE assay: NanoFUSE DBM, DBM raw material used to produce NanoFUSE DBM, Osteofil RT DBM Paste, and Accell DBM100. All test articles were tested either as dry powders to which, after weighting, several drops of saline were added to aid in handling or, in the case of Accell DBM100, tested as received. Athymic nude male mice were implanted with 25 mg of test material in a bluntly-dissected muscle pouch in each hind leg. After 28 days, the explanted tissues were subjected to a thorough histopathological examination. Each explant was scored as either positive or negative for evidence of ectopic ossifi cation. Determination of ectopic ossifi cation was made using the following fi ve criteria: 1) presence of chondroblasts/ chondrocytes; 2) presence of osteoblasts/osteocytes; 3) presence of cartilage/osteoid; 4) presence of new bone; and 5) presence of bone marrow. Each implant site was graded on a scale from 0 to 4 for evidence of ectopic ossifi cation as follows: 0 = no evidence; 1 = 1-25% of fi eld shows evidence; 2 = 26-50% of fi eld shows evidence; 3 = 51-75% of fi eld shows evidence; 4 = % of field shows evidence. Test materials for which at least one implant site scored 1 or higher were considered osteoinductive. Figure 1 depicts the incidence of implantation sites exhibiting evidence of bone marrow cells, of osteoblasts/osteocytes, and of new bone formation. NanoFUSE DBM and Accell DBM100 each had only FIG 1: Results of ectopic rodent assay show that NanFUSE DBM scores for the presence of osteoblasts/ostecytes, new bone, and bone marrow are signifi cantly higher than the other clinically available products. one implant site that scored a 0 on the three criteria exhibited in Figure 1. Notably, Osteofi l RT DBM Paste scored a 0, for all three criteria, at 5 out of the 10 implant sites. Although they were scored similarly, there was a distinct and important difference between the osteoinductive performance of the NanoFUSE DBM and Accell DBM100; NanoFUSE DBM produced more bone cells, bone, and marrow than Accell DBM100. Figures 2 and 3 below are two different magnifi cations of an exemplar histograph for the NanoFUSE DBM study arm. nanofuse DBM - 4

5 Over half of the new bone formation evidence detected for the Accell DBM100 implant sites was related to presence of chondroblasts/chondrocytes and presence of cartilage/osteoid only. If one considers just the scores that indicate the presence of osteoblasts/ osteocytes, the presence of new bone, and the presence of bone marrow, then Accell DBM100 would have scored exactly the same as Osteofi l RT DBM Paste even though much of the new bone formation in Accell DBM100 was non-mineralized. Both of these other two clinically available DBM bone void fillers had a much lower incidence of sites exhibiting key characteristics of osteoinduction, namely the presence of osteoblasts/osteocytes, new bone, and bone marrow when compared to the NanoFUSE DBM study arm. Conclusion Ready to use or ready to mix bone void fi llers are chosen over native DBM because of their superior handling characteristics. The presence of a carrier, such as porcine gel, makes the fi ller a putty rather than a clump of DBM particles with little cohesion. The results of these studies of NanoFUSE DBM with bioactive glass demonstrate superior bone formation to Osteofi l RT DBM Paste or Accell DBM100. NanoFUSE DBM is a bone-void fi ller that combines 45S5 bioactive glass, a known osteoconductive and osteopromotive material, with DBM in a porcine gelatin carrier. As such, NanoFUSE DBM combines the osteoconductive properties of bioactive glass with the osteoinductive properties of DBM, showing superior bone healing results when compared to Osteofi l RT DBM Paste or Accell DBM100. Most notably, the rodent ectopic assay demonstrates that DBM in combination with bioactive glass provides comparatively better osteoinductivity than the DBM-only products, as measured by the actual amount of bone cells, bone, and bone marrow observed. NanoFUSE DBM has been implanted in more than 1200 patients with no adverse events reported (data derived from graft tracking records). More than 270 surgeons have used the device and reported procedures including: shoulder, arm, hand/wrist, leg/hip, knee, and foot/ankle surgeries (both primary and revision; both trauma and elective). FIG 2: NanoFUSE DBM explants at 4 weeks post-implantation; 10X objective, H&E stained, decalcifi ed. FIG 3: NanoFUSE DBM explants at 4 weeks post-implantation; 20X objective, H&E stained, decalcifi ed. nanofuse DBM - 5

6 REFERENCES NanoFUSE DBM: Bioactive Glass and DBM for Enhanced Bone Healing ASTM F04.4 Division. Draft Standard: Standard Guide for the Assessment of Bone Inductive Materials. Wolfi nbarger, Revision Date: 11/18/2003. Hench, L.L., et al. Bonding mechanisms at the interface of ceramic prosthetic materials. J. Biomed. Mater. Res. Symp. 1971;5(6): McKay, W.F., et al. The science of rhbmp-2, Quality Medical Publishing, 2006, pp McKay, W.F., et al. The science of rhbmp-2, Quality Medical Publishing, 2006, p 10. BIBLIOGRAPHY Adkisson, H.A., et al. A rapid quantitative bioassay of osteoinduction. J Orthopedic Res, 2000, 18: Anajarwalla, N.K., et al. Posterior spinal fusion using bone graft substitutes. Intl. Soc. Lumbar Spine Ann. Meeting. Ade-laide, Australia. April, Cunningham, B.W., et al. The use of bioglass for posterolateral spinal arthrodesis and iliac crest donor site repair An In Vivo Sheep Model. Transactions ORS 45th Annual Meeting. 1999;p.357. Elshahat, A., et al. The use of Novabone and Norian in cranioplasty: A comparative study. J. Craniofac. Surg. 2004;15(3): Fujishiro, Y., et al. Quantitative rates of in vivo bone generation for Bioglass and hydrosyapatite particles as bone graft substitute. J. Mater. Sci. - Mater. Med. 1997;8: Gaisser, D.M., et al. Particulate bioactive glass in the repair of iliac crest autograft donor sites. Transactions Sixth World Biomaterials Congress. 2000;23:260. Kawanabe, K., et al. Effects of injecting massive amounts of bioactive ceramics in mice. J. Biomed. Mater. Res. 1991;25(1): Kotani, S., et al. Enhancement of bone bonding to bioactive ceramics by demineralized bone powder. Clin. Orthop. Relat. Res. 1992;(278): Oonishi, H. Particulate bioglass compared with hydroxyapatite as a bone graft substitute. Clin. Orthop. Relat. Res. 1997;334: Pajamaki, K.J., et al. Induction of new bone by allogeneic demineralized bone matrix combined to bioactive glass composite in the rat. Ann. Chir. Gynaecol. Suppl. 1993;207: Piétrement, O. and E. Jallot. AFM mechanical mapping at the interface between a bioactive glass coating and bone. Nan-otechnology. 2002;13: Rezwan, K, et al. Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials. 2006;27: Urist M.R. Bone: Formation by autoinduction. Science. 1965;150: Vallet-Regi, M., et al. Bioactive glasses as accelerators of apatite bioactivity. J. Biomed. Mater. Res. 2003;66A: Wheeler D.L., et al. Assessment of resorbable bioactive material for grafting of critical-size cancellous defects. J. Orthop. Res. 2000;18: Wheeler, D.L., et al. Effect of bioactive glass particle size on osseous regeneration of cancellous defects. J. Biomed. Mater. Res. 1998;41(4): Wilson J., et al.. Spinal fusion using titanium spacers with bioglass and autogenous bone: a comparative study in sheep. In Bioceramics ; p L. Sedel and C. Ray (Eds). Elsevier Science. New York. nanofuse DBM - 6

7 Wilson, J., et al. Toxicology and biocompatibility of bioglasses. J. Biomed. Mater. Res. 1981;15(6): Wozney, J.M. The bone morphogenetic protein family and osteogenesis. Mol. Repro. Dev. 1992;32(2): Disclaimer: NanoFUSE DBM is a registered trademark of Nanotherapeutics, Inc. Osteofi l RT DBM Paste is a registered trademark of RTI Biologics, Inc. Accell DBM100 is a registered trademark of Integra Orthobiologics (subsidiary of Integra Lifesciences Corporation). Bioglass is a registered trademark of the University of Florida. nanofuse DBM - 7

8 350 Main Street Malden, MA P: F: