Development of a Biomimetic, Collagen-Based Scaffold for the Repair and Regeneration of the Annulus Fibrosus

Annually, over 5.7 million Americans are diagnosed with two IVD-associated pathologies: IVD herniation (IVDH- a mechanical disruption of the concentric fibrous layers of the annulus fibrosus (AF)) and/or degeneration (IVDD- a multifactorial process which initiates within the inner gelatinous core (NP), and results in a biochemical degradation of NP tissue), with over 2.7 million requiring surgical interventions. Although both underlying pathologies are different, quite often they both lead to a decrease in IVD height, impaired mechanical function, and increased pain and disability. These pain symptoms affect approximately 80% of the adult population during their lifetime with estimated expenditures exceeding $85.9 billion. Current surgical procedures for IVDH and IVDD are palliative and suffer from drawbacks. While they are performed to address patient symptoms, they fail to address the underlying pathology of a focal defect remaining within the subsequent outer layers of the AF. It is hypothesized that an effective AF closure/repair device in conjunction with a less aggressive discectomy for IVDH and/or NP arthroplasty for IVDD may result in improved patient outcomes, decreased pain, and provide fewer revision surgeries via lower re-herniation and expulsion rates. Therefore, an intact AF must be re-established to prevent implant expulsion or re-herniation, thus addressing the two major spinal pathologies directly associated with an IVD. Currently, within the medical device market, no tissue engineering biomaterials are available for AF closure/repair. Current market AF closure devices (Intrinsic Barricaid®, Anulex X-Close® Tissue Repair System, and Anulex Inclose® Surgical Mesh System) are synthetic materials focused solely on preserving and reinforcing the native tissue and lack effective strategies for implantation, fixation, and regeneration. Therefore, there has been an increase in tissue engineering and regenerative therapeutic approaches aiming for structural and biological AF repair investigated over the last decade using in vitro and in vivo experimentation. It is proposed that the optimum AF tissue engineering scaffold should reproduce the native AF microarchitecture and native mechanical properties. Recent articles illustrate several novel sutures, sealants, and barrier techniques currently under development, resulting in an increasing attention at scientific workshops and conferences. To develop a tissue engineering biomaterial that is suitable for AF closure we propose it must first meet the following criteria: (1) mimic the structural angle-ply architecture of the native AF, (2) fundamentally demonstrate mechanical properties mimicking the native functional characteristics, and (3) demonstrate cytocompatibility while promoting tissue regeneration. Current biomaterials gaining attention in the tissue engineering academic field, electrospinning, polymers, glue, silk scaffolds, and honeycomb-scaffolds, require complex manufacturing procedures and typically work to address two of the three criteria (mimicking the biological or structural characteristics). Therefore, the use of a decellularized tissue from a xenogeneic source may be ideal due to its advantage of maintaining native extracellular matrix (ECM) while also removing all potential harmful xenogeneic factors. Although, the mechanical advantage of closing annular focal defects to retain NP material seems intuitive, only recently have AF closure devices begun to examined in human cadaveric or animal tissues for their ability to withstand in situ IDP or flexibility testing. We propose to address all three criteria with the development of a biomimetic, collagen-based angle-ply annulus fibrosus repair patch (AFRP) comprised of the decellularized porcine pericardium. The porcine pericardium was chosen due to its innate type I collagen content, mechanical strength, and cytocompatibility. The objectives of this research were to investigate the development of this biomimetic AFRP to biologically augment AF repair by (1) mimicking and characterizing the micro-architecture of the multi-laminate angle-ply AFRP, (2) mechanically evaluating the AFRP’s mechanical properties and attachment strength in situ, (3) evaluating the ability of the AFRPs to support AF tissue regeneration in the context of a healthy and inflammatory environment, and (4) evaluating the in vivo mechanical strength, biocompatibility, and tissue regeneration capacity of the AFRP in a large animal model for intervertebral disc degeneration/herniation

Characterization of a Multi-Laminate Angle-Ply AF Patch for Annulus Fibrosus Repair

Annually, over 5.7 million Americans are diagnosed with two IVD-associated pathologies: IVD herniation (IVDH- a mechanical disruption of the concentric fibrous layers of the annulus fibrosus (AF))) and degeneration (IVDD- a multifactorial process which initiates within the inner gelatinous core (NP), and results in a biochemical degradation of NP tissue), with over 2.7 million requiring surgical inteventions.1,2 Although both underlying pathologies are different, quite often they both lead to a decrease in IVD height, impaired mechanical function, and increased pain and disability. These pain symptoms affect approximately 80% of the adult population during their lifetime with estimated expenditures exceeding $85.9 billion.3,4 Current surgical procedures for IVDH and IVDD are palliative and suffer from drawbacks. While they are performed to address patient symptoms, they fail to address the underlying pathology of a defect remaining within the subsequent layers of the AF. An effective AF closure/repair device in conjunction with a less aggressive discectomy for IVDH and/or NP arthroplasty for IVDD, may result in improved patient outcomes, decreased pain, and provide fewer revision surgeries via lower re-herniation and expulsion rates.5,6 Therefore, an intact AF must be re-established to prevent implant expulsion or re-herniation, thus addressing the two major spinal pathologies directly associated with an IVD. Currently within the medical device market, no tissue engineering biomaterials are available for AF closure/repair. Current market AF closure devices (Intrinsic Barricaid, Anulex X-Close Tissue Repair System, and Anulex Inclose Surgical Mesh System) are synthetic materials focused solely on preserving and reinforcing the native tissue and lack efficient strategies for implantation, fixation, and regeneration. Therefore, there has been an increase in tissue engineering and regenerative therapeutic approaches aiming for structural and biological AF repair investigated over the last decade using in vivo and in vitro experimentation. It is proposed that the optimum AF tissue engineering scaffold should reproduce the architecture, and the mechanical properties of the native human AF tissue.7 Recent articles illustrate several novel suture, seal, and barrier techniques currently under development, resulting in an increasing attention at scientific workshops and conferences.8-15 To develop a tissue engineering biomaterial that is suitable for AF closure it must meet the following criteria: (1) mimic the structural angle-ply architecture of the native AF, (2) withstand static and dynamic mechanical properties mimicking the native functional characteristics, and (3) express cytocompatibility while promoting tissue regeneration. Current biomaterials growing attention in the tissue engineering academic field, electrospinning, polymers, glue, silk scaffolds, and honeycomb-scaffolds, require complex manufacturing procedures and typically work to address two of the three criteria (mimicking the biological and structural characteristics).5 Although the mechanical advantage of closing annular defects to retain NP material seems intuitive, only recently have AF closure devices begun to examined in human cadaveric or animal tissues for their ability to withstand in situ IDP or flexibility testing.16 Therefore, the use of decellularized tissue from a xenogeneic source is ideal due to its advantage of maintaining native extracellular matrix (ECM) while also removing all potential harmful xenogeneic factors. We propose to address all three criteria with the development of a biomimetic angle-ply annulus fibrosus patch comprised of decellularized porcine pericardium. Porcine pericardium was chosen due to its native type I collagen content, mechanical strength, and cytocompatibility. The objectives of this research were to investigate the development of a biomimetic patch, consisting of decellularized porcine pericardium, to biologically augment AF repair by (1) characterizing the micro-architecture of the multi-laminate angle-ply AF patch, (2) evaluating the mechanical properties through static and dynamic tensile loading and impact resistance of IDP, and (3) evaluating the cytocompatibility of the patch using a healthy alternative cell source for AF tissue regeneration

Expression analyses of candidate resistance genes in the Rpp4 Asian Soybean Rust resistance locus.

Asian Soybean Rust (ASR), caused Phakopsora pachyrhizi, is considered the most severe soybean disease around the world. Infection of susceptible genotypes leads to early defoliation, incomplete seed development, and yield losses as high as 80%. Five ASR resistance genes have been identified in soybean: Rpp1, Rpp2, Rpp3, Rpp4 and Rpp5. Of particular interest is Rpp4, which has remained stable and confers resistance against P. pachyrhizi isolates from around the world. Rpp4 was mapped to soybean linkage group G (chromosome 18), 1.9 cM from simple sequence repeat (SSR) marker Satt288. Sequencing of this region in the susceptible genotype Williams 82 identified a cluster of three CC-NBS-LRR resistance genes. Virus Induced Gene Silencing was used to demonstrate that orthologous genes were responsible for resistance. We have now sequenced a >460 kb region of the Rpp4 locus in the resistant mapping parent PI459025B. Eight CC-NBS-LRR resistance genes have been identified in this region. In order to obtain more information about Rpp4 function, we are using real time quantitative PCR (qRT-PCR) to analyze the expression of all eight genes in different plant tissues, in different stages of development and after inoculation with P. pachyrhizi. We have developed a single pair of primers from the NBD domain that monitors the expression of all eight genes. Direct sequencing of the RT-PCR product differentiates between the eight genes. Detailed sequence analyses of the Rpp4 locus suggest that intra- and intergenic duplications and recombination have played an important role in creating genetic diversity. Alternative splicing of intragenic duplications may create additional sequence diversity at an RNA level. We are developing primers that will allow us to monitor alternative splicing events. Sequencing of the RT-PCR products will determine if alternative splicing plays a role in generating additional sequence diversity at the Rpp4 locus.Edição de Poster da 11. Annual National Outreach Scholarship Conference, Raleigh

Assessment of Seed Viability by Laser Speckle Techniques

This work presents a new technique as a potential methodology to analyse seeds. The technology is known as dynamic speckle, or biospeckle, an optical phenomenon produced when active materials, such as biological tissue, are illuminated by laser light. In the present work, the biological activity of seed tissues has been inferred from quantitative and qualitative measurements of their speckle activity. The aim is to show that the biospeckle technique has a potential as a methodology to assess seed viability. One aspect that needs to be investigated is how the water content in the seeds affects bio-speckle activity. An experiment has been performed to determine the effect of humidity in the results. Seed activity for different levels of humidity was determined using quantitative and qualitative methods. Also, in others experiments, viable and non-viable seeds with different specific humidity levels could be classified using the same technique.Facultad de Ingeniería (FI

Assessment of Seed Viability by Laser Speckle Techniques

This work presents a new technique as a potential methodology to analyse seeds. The technology is known as dynamic speckle, or biospeckle, an optical phenomenon produced when active materials, such as biological tissue, are illuminated by laser light. In the present work, the biological activity of seed tissues has been inferred from quantitative and qualitative measurements of their speckle activity. The aim is to show that the biospeckle technique has a potential as a methodology to assess seed viability. One aspect that needs to be investigated is how the water content in the seeds affects bio-speckle activity. An experiment has been performed to determine the effect of humidity in the results. Seed activity for different levels of humidity was determined using quantitative and qualitative methods. Also, in others experiments, viable and non-viable seeds with different specific humidity levels could be classified using the same technique