Ease of Use

VET Fiber Matrix was designed to have excellent intraoperative handling properties, not only for ease of use by the surgeon, but also because this influences the product performance. Being able to place the graft where it is needed and have it remain in place is optimized by the DBF™ properties where the fibers create a cohesive mass that is easy to manipulate. Compared with powder-based allografts (i.e. DBM), DBF™ cannot easily flow out of the graft site. This characteristic means that DBF™ does not require the use of any excipients and VET Fiber Matrix is additive free, consisting only of DBF™ (demineralized bone fibers).

Features and Benefits

VET Fiber Matrix has the following features and benefits:

  1. VET Fiber Matrix™ comes pre-hydrated in a physiological buffer and is ready to use
  2. As VET Fiber Matrix™ is 100% allograft bone with no additives it also makes it highly biocompatible and cell friendly
  3. The DBF™ fibers readily mix with autograft, bone marrow aspirate and cells within the defect site to augment bone healing by osteogenesis.
  4. The DBF™ fibers are cohesive and this property prevents migration of graft material from the defect site.
  5. The DBF™ fibers are a very effective vehicle for the delivery of autograft ‐ the DBF™ fibers allow easy handling and incorporation of graft into the defect site.
  6. VET Fiber Matrix™ conforms to the defect site and is completely space filling.
  7. The ability to integrate autograft and cells within the DBF™ ensures continuity of the graft construct enhancing performance by providing a contiguous graft construct.
  8. As it wicks cells into the matrix, it also swells slightly such that the defect is completely filled and a contiguous scaffold is maintained to maximize bone formation by osteoconduction.

Bone Healing Performance

Although safety and ease of use are important features the real critical aspect of bone grafts is their ability to heal defects. DBF™ was developed with bone formation performance as the principal objective. The ‘gold standard’ for bone grafts remains autogenous, or autograft, bone recovered from the patient (at a different location such as iliac crest) and is the standard for comparison of bone graft performance. However autograft has several limitations including additional surgical time, trauma to the patient caused by the harvest procedure, pain, as well as limitations of the quantity available. Beyond these well accepted limitations are other less well appreciated ones which limit the bone healing performance and provide an opportunity for developing bone grafts which outperform autograft. Indeed the objective is for DBF™ to act as an autograft enhancer and/or substitute, not just an extender6 and initial studies indicate this is being achieved. A thorough understanding of bone healing biology and of models to assess graft performance are essential ‘tissue engineering’ tools being utilized to create DBF™ and future Veterinary Engineered Tissue (VET) products.

Mechansisms of Bone Formation
Cells
(autograft, BMA, local)
+ Matrix
(DBF)
+ Signals
(BMP's)
Osteogenesis Osteoconduction Osteoinduction

Bone healing is driven by three mechanisms: osteogenesis, osteoconduction and osteoinduction as is shown in the figure above. Osteogenesis is a cell driven process where osteoblasts produce new matrix that is subsequently mineralized. The cells come from various sources including from the defect site by decorticating bone so it is bleeding, harvesting autogenous bone or from bone marrow aspirate. Each of these approaches has limitations which DBF™ mitigates and thus improves their performance. Decortication releases local bone healing cells, but the cells are likely to not remain within the site where they are needed. DBF™’s ability to wick these cells into its matrix and provide a scaffold for them to adhere to and respond with appropriate bone formation enhances their performance potential. It acts similarly when bone marrow aspirate is added. Autograft, is typically chunks of bone that do not adhere to one another and that have gaps between them, is enhanced by DBF™’s ability to hold the autograft in place and provide a bridge between the pieces so the composite graft has a contiguous scaffold. This makes placement of the graft easy and is critical to healing as bone cells are poor at traversing voids.

This leads to the second mechanism of bone healing: osteoconduction, the ability of the matrix to provide a scaffold for conducting bone formation throughout the defect. As previously mentioned, bone cells are not good at bridging gaps on their own so need a scaffold to guide them to fill the entire defect. The geometry and biocompatibility of the DBF™ fibers provides outstanding osteoconduction properties and the fibers act as a ‘super highway’ for bone cells to migrate along and completely fill defects. This property of bone fibers was demonstrated using a challenging bone healing model 6 developed by Scott Boden, MD, a spine surgeon at Emory University and Nelson Scarborough, PhD, in a study that won the prestigious Volvo Award. The rabbit posterolateral spine fusion model was developed to assess bone graft performance and was elegantly designed such that autograft achieved fusion in about 70% of the sites, equivalent to the success rate of autograft in human clinical use, and allowing the ability to determine whether grafts were equal, better, or worse than autograft. When demineralized bone fibers were compared with demineralized bone matrix (DBM) that was 100-500 micron sized particles, the fibers significantly outperformed the powder5,6. The mechanism for this improved performance was further studied by removing the bone morphogenetic protein (BMP) component from the fibers and powder such that the osteoinduction potential was removed. The inactivated DBF™ fibers were still able to achieve fusion in 30% of the sites while the DBM powder did not achieve fusion in any site. It was concluded that the enhanced performance of the fibers was due to their exceptional osteoconduction properties and demonstrated the importance of a contiguous matrix for optimal bone healing.

The third mechanism of bone healing is osteoinduction which refers to the ability of certain proteins to induce differentiation of cells to a bone forming lineage. This phenomenon was first reported by Marshall Urist in 19657 based on work where he placed demineralized bone powder into heterotopic sites (subcutaneous and intramuscular) where no bone cells are present and discovered than bone formation could be induced by stimulating precursor cells to differentiate into osteoblasts. Subsequent work by numerous investigators, particularly Sampath and Reddi8 revealed this capability to be due to Bone Morphogenetic Proteins (BMP’s) that are naturally present in bone. Importantly, mineralized bone powder is not effective at osteoinduction due to the mineral component masking the interaction of the BMP’s with cells. The fact that the mineral can be removed using dilute acids without inactivating or dissociating the proteins from the matrix collagen is a key factor for the DBF™ technology. Normal bone resorption by osteoclasts is achieved by them secreting hydrochloric acid (HCl) to remove bone minerals. The fact that BMP’s remain active during manufacturing demineralization steps is no surprise as the proteins have been teleologically selected to be resistant to degradation by acidic environments.

The ability to assay the osteoinductive performance of demineralized bone has been reported by various laboratories but had significant limitations prior to the development of an athymic rat intermuscular assay reported by this author and collaborators9. This assay has advantages in its ability to assay materials from various species, importantly humans and canines, without the issue of cross species incompatibility interfering with the response. It also allows for reproducible implantation into the site and for studying larger three dimensional materials. Several other assay systems have been described in the literature, but none have the relevance of the athymic rat model where actual bone formation is assessed rather than surrogate measures as, for example, alkaline phosphatase enzyme up-regulation in cell culture models. Of the surrogate assays reported in the literature, TheraCell believes the best is sandwich ELISA for BMP’s, particularly BMP-2 and BMP-7. This method uses two antibodies that bind to the ends of the BMP molecule such that it has to be intact to be detected.

DBF™ Studies

TheraCell’s DBF™ fibers that are used in VET Fiber Matrix have been investigated in a number of studies. An in vitro study with DBF™ was performed to evaluate the interaction of MG63 cells (an immortalized osteosarcoma cell line) with the matrix. The figure below shows that after 3 days the cells have migrated into the DBF™, have normal morphologic appearance and remain viable, demonstrating the excellent biocompatibility of the DBF™. It also shows scanning electron microscopy (SEM) images of MG63 cells on the surface of DBF™.

MG63 cells cultured in the presence of DBF™ for 3 days.


A. Low Magnification

B. High Magnification

SEM images of MG63 cells spreading and adhering to Fiber Matrix.

DBMF with MG63 Cells for 3 Days
DBMF with MG63 Cells for 3 Days

A nonclinical in vivo study of DBF™ was conducted using a rabbit distal femur defect to evaluate performance characteristics in this critical size defect model10. DBF™ was compared to iliac crest autograft and a synthetic bone void filler used in humans, Vitoss Foam (Orthovita, Inc.). Bilateral 6 mm diameter by 10mm deep drill hole defects were made in the medial cortex of the femoral metaphyses of skeletally mature rabbits and approximately 0.3cc of material implanted. Evaluations of bone healing were made at 2, 4 and 6 weeks using radiography, μCT and histological analysis. The figure below illustrates the response to autograft and DBF™ at 2 and 4 weeks. At 2 weeks new bone formation can be observed along the perimeter of the implants with some residual graft remaining in the center. By 4 weeks the DBF™ implants were fully incorporated with minimal residual matrix, whereas the autograft group had more residual graft remaining. This is illustrative of one of the advantages of DBF™ in that by being fully demineralized the remodeling process is accelerated compared to the mineralized autograft.

Rabbit femoral defect model. Histological images of autograft and DBF™ at 2 and 4 weeks.

Autograft and DBF™ at 2 and 4 weeks
Autograft and DBF™ at 2 and 4 weeks

A higher magnification image below demonstrates excellent new bone formation activity with osteoblasts lining the surfaces of the DBF™ and some osteoclastic activity noted where the matrix is being remodeled. Additional observations are osteocytes present within the lacunae, marrow with normal appearing cells and fat, and areas of angiogenesis with erythrocytes present within the newly for blood vessels. No inflammatory response was observed, again demonstrating the excellent biocompatibility of the DBF™/physiological buffer material.

Histological appearance of DBF™ implant at 4 weeks. The arrow () points to osteoblasts and lightning bolt () identifying an osteoclast.

DBF™ at 4 Weeks
DBF™ implant at 4 weeks

At 6 weeks the DBF™ treated defects had regenerated the cortical wall of the defect as is shown below Neither of the other test articles were able to achieve this.

Image of regenerated cortical wall with DBF™ implant at 6 weeks.

Image of regenerated cortical wall with DBF™ implant at 6 weeks
DBF™ implant at 6 weeks

VET Fiber Matrix K-9, has been evaluated in a clinical case series of canine patients at the University of Florida. This series was done to evaluate DBF™ performance in a range of clinical indications as an initial evaluation of intraoperative handling and bone healing performance. This study was intended to get surgeon feedback to identify any potential user needs not previously identified.

VET Fiber Matrix processed using the DBF™ Technology provides a bone graft that is safe, has excellent handling and bone healing properties. The technology was initially developed for human bone grafting products and is now available for veterinary applications. Initial canine clinical cases have demonstrated that the VET Fiber Matrix K-9 product meets the user needs. Preclinical studies in rabbits have demonstrated excellent bone formation including regeneration of the cortex in a distal femur model. Additional clinical and non-clinical studies are planned and underway to develop a data set to support the use of DBF™ as an autograft enhancer or substitute with the initial results showing very positive results. Further, additional products are in the pipeline to leverage the DBF™ technology in various species (canine, feline, equine) and for procedure specific products to facilitate veterinary procedures by saving operative time and costs and improving patient outcomes.

VET Fiber Matrix™ is packaged by weight. When firmly compressed 1 gram of product corresponds to approximately 1cc volume. The product is prehydrated in a physiologic buffer, such that reconstitution is not required. It readily wicks fluids and may be used with autograft and/or bone marrow aspirate to provide additional biological healing elements, i.e. osteoprogenitor cells, stems cells and associated growth factors. The graft site should be prepared to expose bleeding bone.

Purchase VET Fiber Matrix™ K-9

Allograft Safety

Safety of VET Fiber Matrix is ensured by using a series of evaluations and processing techniques.

  • Canine donors are rigorously screened to ensure they are free from potential communicable diseases beginning with medical history review and physical examination.
  • Blood samples are tested for an extensive panel of diseases using state of the art PCR technology.
  • Bone is recovered by trained veterinary personnel using aseptic surgical procedures.
  • All recovered tissues are tested for microbiological contamination.
  • Independent review of all donor records is performed by an independent Medical Director who is a practicing veterinarian.
  • Manufacturing includes steps that have been shown to have robust viral inactivation properties virtually guaranteeing that no virus could survive.
  • Manufacturing is performed using aseptic techniques in a controlled environment to prevent contamination. After processing the product is tested for microbial bioburden.
  • Sterility is further ensured by terminally sterilizing Fiber Matrix in the final package using Electron Beam irradiation.

Contraindications and Precautions

VET Fiber Matrix™ - K9 is intended for canine recipients only as there is potential for cross species reactivity if used in other species.

VET Fiber Matrix™ is intended for single patient use. Discard any unused graft. The graft must be handled using aseptic technique to avoid contamination. Avoid prolonged exposure to the environment after opening to prevent contamination. If the product becomes dry after opening it can be readily rehydrated using sterile physiological solutions such as saline or bone marrow aspirate, blood or autograft.

Although the donors are rigorously screened and the Fiber Matrix™ Technology process has robust viral inactivation properties, the risk for disease transmission, albeit remote, still exists.

In the event of adverse events associated with VET Fiber Matrix™ contact TheraCell - VET promptly. Provide lot number (from the patient label) and date of surgery.

Storage and Handling

Store at room temperature in a clean dry area. Store the pouch in the shelf carton until ready for use. Avoid exposure to temperatures above 40°C. Ensure the product is sterile by noting the sterilization indicator dot is RED. If not, notify TheraCell - VET promptly, including lot number, and return to TheraCell - VET.

Instructions for Use

VET Fiber Matrix&trad™ is packaged by weight. When firmly compressed 1 gram of product corresponds to approximately 1cc volume. The product is prehydrated in a physiologic buffer, such that reconstitution is not required. It readily wicks fluids and may be used with autograft and/or bone marrow aspirate to provide additional biological healing elements, i.e. osteoprogenitor cells, stems cells and associated growth factors. The graft site should be prepared to expose bleeding bone.

  1. VET Fiber Matrix™ - K9 is intended for canine implant only. Not for human use.
  2. VET Fiber Matrix™ is packaged in a jar inside a foil pouch. Everything inside the foil pouch is considered sterile if the sterilization indicator on the outside is RED.
  3. Inspect the pouch for damage and discard if there is damage as this may compromise sterility.Peel the foil pouch open while taking care not to contaminate contents. In a sterile manner transfer the jar into the sterile field.
  4. VET Fiber Matrix™ is packaged hydrated and is ready for use.
  5. Open the jar and remove the product. It may be implanted directly or mixed with autograft or other graft materials.
  6. For optimal use the graft should completely fill the defect.
  7. Use the provided implant label for documenting use in patient chart.

References

  1. Boyce TM, Edwards JT, Scarborough NL: Allograft Bone: The Influence of Processing on Safety and Performance. Orthopedic Clinics of North America, 30(4):571-581, 1999
  2. Scarborough NL: Current procedures for banking allograft human bone. Orthopedics, 15:10, 1161-1167, 1992.
  3. Scarborough NL, White EM, Hughes JV, Manrique AJ, Poser JW: Allograft Safety: viral inactivation with bone demineralization. Contemporary Orthopaedics, 31(4):257-261, 1995
  4. Swenson CL, Arnoczky SP: Demineralization for inactivation of infectious retrovirus in systemically infected cortical bone: in vitro and in vivo experimental studies. J Bone Joint Surg Am. 2003 Feb;85-A(2):323-32.
  5. Morone MA, Boden SD: Experimental posterolateral lumbar spinal fusion with a demineralized bone matrix gel. Spine, 23(2):159-167, 1998
  6. Martin GL, Boden SD, Titus L, Scarborough NL: New Formulations of Demineralized Bone Matrix as a More Effective Graft Alternative in Experimental Posterolateral Lumbar Spine Arthrodesis. Spine, 24(7):637-645, 1999
  7. Urist MR: Bone: Formation by Autoinduction. Science, 150:893-899, 1965
  8. Reddi AH, Weintroub S, Muthukumaran N: Biological principles of bone induction. Orthop Clin North Am, 18:207-212, 1987
  9. Edwards JT, Diegmann MH, Scarborough NL: Osteoinduction of human demineralized bone: characterization of a rat model. Clinical Orthopaedics and Related Research, 357:219, 1998
  10. In vitro and in vivo evaluation of demineralized bone fibers. Abstract submitted to Veterinary Orthopedic Society, 2016