News Column

Researchers Submit Patent Application, "Fibrous Scaffold for Use in Soft Tissue Engineering", for Approval

July 24, 2014



By a News Reporter-Staff News Editor at Gene Therapy Weekly -- From Washington, D.C., NewsRx journalists report that a patent application by the inventors Santerre, J. Paul (Whitby, CA); Kandel, Rita (Toronto, CA), filed on February 20, 2014, was made available online on July 10, 2014 (see also Mount Sinai Hospital).

The patent's assignee is Mount Sinai Hospital.

News editors obtained the following quote from the background information supplied by the inventors: "In an autopsy study, 97% of individuals 50 years or older had intervertebral disc degeneration, a disease process that involves both the annulus fibrosus and nucleus pulposus [1]. The etiology of this process is unknown but may be due to the relative avascularity of the tissue [2], calcification of the cartilage endplate [3], mechanical factors [4], vertebral body microfractures [5], loss of notochordal cells and/or genetic factors [6]. The low back pain that can develop in association with this disease is one of the most common afflictions in today's society and approximately eighty percent of people will experience at least one episode of low back pain at some time in their lives [7]. The direct costs of diagnosing and treating low back pain in the United States, as estimated by the American Chiropractic Association, is approximately $25 billion annually [8]. There is no optimal treatment for chronic back pain currently. Although there are several surgical options these all have limitations. Spinal fusion of diseased disc tissue may relieve pain faster, but it can result in reduced flexibility and the potential to develop degenerative changes in adjacent segments [9]. The intervertebral disc can be replaced with a synthetic prosthesis but this treatment is only appropriate for selected individuals [10, 11] and they can loosen over time [1,2]. Discectomy does not restore disc height and thus does not treat the underlying disease process. Therefore, there is a great interest in developing alternative biological treatments for this disease. One of the options is to tissue engineer a functional intervertebral disc that could be used to replace the degenerated disc [13].

"The human spine consists of 33 vertebral bodies each separated, with the exception of C1 and C2 and the coccyx, by an intervertebral disc (IVD). The IVD anchors adjacent vertebral bodies and by doing so allows for spinal stabilization, load bearing, and movement. The intervertebral disc is a specialized structure consisting of three components, a gel-like nucleus pulposus (NP) which is surrounded by annulus fibrosus (AF), which are sandwiched between cartilage end plates (CEP) and vertebral bodies [14]. The normal function of the disc is dependent on maintenance of the composition, organization, and integrity of the different components.

"The annulus fibrosus (FIG. 3) is the most complex of these 3 tissues present in the disc. It consists of approximately 10-20 lamellar sheets each composed of collagen fibres oriented parallel to each other and about 65.degree. from the vertical. Although the angle is the same, the direction of the inclination alternates with each sheet such that the fibres in one lamella are 65.degree. to the right, while in the next lamella they are 65.degree. to the left. Every second lamella has the same orientation. This very specific collagen organization allows the disc to rotate and flex. Collagen makes up about 70% of the dry weight of the annulus. Type I collagen is the predominant collagen but types II, III, V, VI and type IX collagen are also present in lesser amounts.

"To date, many studies have focused on the regeneration of NP [15-17] rather than AF tissue, probably because of the structural complexity of the AF tissue [18]. Even though AF tissue engineering has been attempted using various polymeric scaffolds including PDLLA/45S5 Bioglass.RTM. composite films [19], atelocollagen honeycomb [20], collagen-GAG [21], collagen-hyaluronan [22], polyglycolic acid/polylactic acid [23], and alginate [24] materials, in all of these scaffolds AF tissue formation has been limited and none has recapitulated the complex structure of the AF. Furthermore some scaffolds may not be optimal for this use. For example when polylactides, polyglycolides, and their copolymers degrade, they form acidic degradation products that can decrease the local pH, and overwhelm the tissue buffering and cell regulating capacities, which adversely affect biocompatibility [25]. Furthermore an acidic environment in the disc has been shown to greatly inhibit the rates of extracellular matrix synthesis [26], which may actually affect tissue formation. For these reasons there has been an interest in developing new polymers."

As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventors' summary information for this patent application: "The present invention relates to a fibrous scaffold for use as a substrate in soft tissue applications or for culturing soft tissues, in particular for preparing AF tissue. In aspects of the invention the fibrous scaffold is a nanofiber porous scaffold comprising polyurethane polymers optionally with components that increase surface energy of the scaffold. In particular aspects the fibrous scaffold is a nanofiber porous scaffold comprising a polyurethane formulation comprising a polyurethane base polymer and novel anionic dihydroxyl oligomers (ADO).

"In aspects of the invention, a polyurethane formulation is provided comprising a blend of polyurethane polymers and selected oligomers that increase surface energy in a scaffold or substrate formed from the formulation. In aspects of the invention, a polyurethane formulation is provided comprising fibres comprising a blend of polycarbonate urethane polymers and selected oligomers that increase surface energy in a scaffold or substrate formed from the formulation. In particular aspects of the invention, the fibres are random. In other particular aspects of the invention, the fibres are aligned.

"In aspects of the invention, the selected oligomers are novel anionic dihydroxyl oligomers (ADO). Thus, the invention provides novel anionic dihydroxyl oligomers having one or more of the following properties: a) about 50% to about 70%, about 50% to 60% or about 55% to 65% of its side chains comprise carboxylic acid groups; b) absorption bands in the about 600 cm.sup.-1 to about 4000 cm.sup.-1 region by Fourier transform infrared spectroscopy (FTIR); and c) a peak corresponding to a urethane group at about 1680-1750 cm.sup.-1, in particular 1720 to 1740 cm.sup.-1, by FTIR.

"The invention also relates to a process for producing the novel anionic dihydroxyl oligomer comprising linking a polyether diol with a carboxylic ester in the presence of a polyisocyanate to produce an oligomeric product, and hydrolzying the oligomeric product to produce the anionic dihydroxyl oligomer. The invention also contemplates an anionic dihydroxyl oligomer produced by a method of the invention.

"The invention further relates to a fibrous scaffold or substrate produced or fabricated from a polyurethane formulation described herein, and a process for producing a fibrous scaffold of the invention.

"In an aspect, the invention provides a fibrous scaffold for culturing soft tissues on its surface said scaffold comprising fibres comprising a blend of polyurethane polymers and oligomers wherein the oligomers increase surface energy of the scaffold and comprise polar groups that are exposed on the surface of the fibrous scaffold. In a particular aspect, the invention provides a fibrous scaffold for culturing soft tissues on its surface comprising fibres comprising a blend of polycarbonate urethane polymers and anionic dihydroxyl oligomers, wherein the fibres are aligned or random.

"The invention provides an engineered biological material comprising in combination a fibrous scaffold of the invention and a soft tissue, in particular intervertebral disc tissue or a portion thereof, more particularly annulus fibrosus (AF) tissue. Further, the invention provides tissues derived from the biological material, and a process for producing the engineered biological material. Still further, the invention provides a construct comprising an engineered biological material of the invention or tissue therefrom.

"In an aspect the invention provides an engineered biological material comprising or enriched for annulus fibrosus (AF) tissue. In particular, the invention relates to an engineered biological material comprising a continuous layer of annulus fibrosus (AF) tissue. The tissue formed in vitro mimics the organization of AF tissue in vivo. In particular, the collagen content of the AF tissue is or will be substantially the same as native AF tissue following implantation. The collagen content of the in vitro-formed AF tissue will be sufficient to support function following implantation and amenable to remodeling to reach a collagen content that approached that of native AF. More particularly the engineered biological material is characterized by lamellar sheets each composed of collagen fibres oriented parallel to each other and about 50-70.degree., more particularly 60-65.degree., most particularly 65.degree. from the vertical. The engineered biological material may also comprise collagen, predominantly Type I collagen and types II, III, V, VI and type IX collagen are generally present in lesser amounts. In an embodiment an engineered biological material of the invention comprises in combination a fibrous scaffold of the invention and a continuous layer of annulus fibrosus tissue, preferably on the scaffold.

"In an embodiment, the invention provides an engineered biological material comprising in combination annulus fibrosus tissue and a fibrous scaffold for the annulus fibrosus tissue, the annulus fibrosus tissue being reconstituted on the fibrous scaffold in vitro from isolated annulus fibrosus cells and being a continuous layer comprising annulus fibrosus cells and an extracellular matrix.

"In an aspect the invention provides a process for producing an engineered biological material comprising: forming a layer of isolated annulus fibrosus cells on a fibrous scaffold of the invention, and; culturing the annulus fibrosus cells in culture media so that the annulus fibrosus cells accumulate extracellular matrix and form a continuous layer of annulus fibrosus tissue.

"In another aspect, the invention provides a process for producing an engineered biological material of the invention comprising isolating annulus fibrosus cells from intervertebral disc; forming a layer of the annulus fibrosus cells on a fibrous scaffold, and; culturing the annulus fibrosus cells in culture media under suitable conditions so that the annulus fibrosus cells accumulate extracellular matrix and form annulus fibrosus tissue, in particular a continuous layer of annulus fibrosus tissue. In an embodiment the fibrous scaffold is a nanofiber porous scaffold comprising polyurethane and optionally ADO, in particular a polycarbonate urethane polymer and ADO.

"The invention also relates to annulus fibrosus tissue derived from the engineered biological materials of the invention. Still further the invention contemplates an intervertebral disc construct comprising annulus fibrosus tissue derived from an engineered biological material of the invention.

"The cells (e.g. annulus fibrosus cells) in engineered biological materials or constructs of the invention may be transformed with recombinant vectors containing an exogenous gene encoding a biologically active protein that corrects or compensates for a genetic deficiency, or stimulates cell growth or stimulates extracellular matrix production by cells, or alternatively, encoding a drug. Therefore, the invention also contemplates an engineered biological material or construct of the invention wherein cells (e.g. annulus fibrosus cells) in the engineered biological material or construct are transformed with recombinant vectors containing an exogenous gene encoding a biologically active protein which can correct or compensate for a genetic deficiency or have a stimulatory effect, or encoding a drug.

"The invention still further relates to a system for testing a substance or agent that affects a soft tissue (e.g. annulus fibrosus tissue) comprising: generating and/or culturing an engineered biological material or construct of the invention comprising the soft tissue in the presence of a substance or agent which is suspected of affecting the soft tissue (e.g. annulus fibrosus tissue), and comparing the biochemical composition and/or physiological organization of the soft tissue with the biochemical composition and/or physiological organization of the soft tissue of the engineered biological material or construct generated and/or cultured in the absence of the substance or agent to determine its effect on the tissue.

"The invention still further relates to a method of using the biological materials, tissues therefrom or constructs of the invention to test pharmaceutical preparations for efficacy in the treatment of diseases of intervertebral disc.

"Still another aspect of the present invention provides a method of conducting a drug discovery business comprising: (a) identifying agents that affect the biochemical composition and/or physiological organization of an engineered biological material or tissues thereof, or a construct of the invention; (b) conducting therapeutic profiling of agents identified in step (a), or further analogs thereof, for efficacy and toxicity in animals; and formulating a pharmaceutical preparation including one or more agents identified in step (b) as having an acceptable therapeutic profile.

"In certain embodiments, the subject method can also include a step of establishing a distribution system for distributing the pharmaceutical preparation for sale, and may optionally include establishing a sales group for marketing the pharmaceutical preparation.

"Yet another aspect of the invention provides a method of conducting a target discovery business comprising: (a) providing one or more engineered biological material, tissues therefrom or a construct of the invention for identifying agents by their ability to affect the biochemical composition and/or physiological organization of the engineered biological material, tissues therefrom or construct; (b) (optionally) conducting therapeutic profiling of agents identified in step (a) for efficacy and toxicity in animals; and licensing, to a third party, the rights for further drug development and/or sales for agents identified in step (a), or analogs thereof.

"The invention provides methods of using an engineered biological material or tissues obtained therefrom or construct of the present invention as an implant to replace or repair damaged, degenerated or deficient soft tissues, in particular AF tissue or intervertebral discs or portions thereof, and methods for repairing damaged or degenerated soft tissues, in particular AF tissue or intervertebral discs or portions thereof. Methods of the invention may be used to treat vertebrates suffering from degenerated intervertebral disc conditions and in particular to treat humans with such conditions.

"Therefore, the invention contemplates a method of replacing or repairing damaged, degenerated or deficient AF tissue or intervertebral discs or portions thereof (preferably AF) of a patient comprising implanting an engineered biological material (or tissue therefrom) or construct of the invention into the site of the damaged, degenerated or deficient AF tissue or intervertebral disc of the patient. Methods for enhancing healing of an intervertebral disc in a patient are contemplated which comprise inserting an engineered biological material (or tissue therefrom) or construct of the invention into the site of a damaged intervertebral disc.

"In an embodiment, the invention provides a method for replacing or repairing a degenerated or damaged annulus fibrosus tissue of an intervertebral disc comprising implanting in the disc space, after the removal of the degenerated or damaged annulus fibrosus tissue, an engineered biological material of the invention comprising a continuous layer of annulus fibrosus tissue, or annulus fibrosus tissue obtained therefrom.

"In another aspect of the invention, a method for repairing damaged or degenerated intervertebral discs is provided comprising evacuating tissue from the annulus fibrosus portion of a degenerated intervertebral disc space, preparing an engineered biological material of the invention using annulus fibrosus cells from the evacuated tissue, and implanting the biological material or tissue therefrom in the evacuated annulus fibrosus space.

"The invention also contemplates methods for using the engineered biological materials and tissues and cells therefrom, and constructs of the invention in gene therapy.

"A biological material or construct of the invention can be used as an in vitro model for investigating the metabolism and degeneration of soft tissue and cells, in particular annulus fibrosus cells and tissues.

"These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings.

DESCRIPTION OF THE DRAWINGS

"The invention will be better understood with reference to the drawings in which:

"FIG. 1 shows SEM images of polycarbonate urethane fibrous scaffolds in the absence (0% ADO) or presence of increasing amounts of ADO (.times.5000 magnification).

"FIG. 2 is a graph showing AF cell attachment to scaffolds in the presence of serum (5% FBS), serum free, or serum-free media with cycloheximide. * indicates significant difference.

"FIG. 3 is a diagram showing annulus fibrosus tissue structure.

"FIGS. 4A and B show SEM images of random polycarbonate urethane fibrous scaffold with 5% ADO and 0.05% ADO.

"FIG. 5 is a graph showing AF cell attachment to polycarbonate urethane fibrous scaffolds with 0.05% ADO, 0.5% ADO, 5% ADO and without ADO. * indicates significant difference.

"FIG. 6 shows the following: (A) graph showing collagen content of tissue formed by AF cells cultured on fibrous scaffolds comprising polyurethane (PU), 0.05% ADO-PU, 0.5% ADO-PU, and 5% ADO-PU. (B) A graph showing retained and newly synthesized collagen of AF cells cultured on fibrous scaffolds comprising polyurethane (PU), 0.05% ADO-PU, 0.5% ADO-PU, and 5% ADO-PU. (C) SEM image taken on a cross-section showing layers of tissue formed on the scaffold containing 0.5 wt % oligomer.

"FIG. 7 shows SEM images of (A) a random fibrous porous scaffold made of polyurethane with 0.5% wt oligomer and (B) aligned fibrous porous scaffold made of polyurethane with 0.5% wt oligomer.

"FIG. 8 shows SEM images of cells grown on (A) a random scaffold for 5 days and (B) cells grown on an aligned scaffold for 5 days.

"FIG. 9 is a FTIR spectrum of the anionic dihydroxyl oligomer (ADO) (A), and the oligomer precursor (B).

"FIG. 10 is a graph showing water contact angle measurements of PU materials containing 0%, 0.05%, 0.5% and 5% ADO content (wt %). The results are reported as mean.+-.standard error of the mean. * indicates significant difference from all other scaffolds (n=10).

"FIG. 11 shows SEM images of as-made PU scaffolds containing various amounts of ADO content (at 0%, 0.05%, 0.5% and 5% wt %).

"FIG. 12 are graphs showing: (A) Percent AF cell attachment 24 hours after seeding PU scaffolds formed in the presence of ADO (0.05%, 0.5% and 5% (wt %)) or absence of ADO in the presence of 5% fetal bovine serum. (B) AF cell attachment at 24 hours after seeding in serum-free DMEM in the absence or presence of cyclohexamide (10 .mu.g/ml). The data are presented as a percent decrease in attachment and was calculated by dividing the % cell attachment for the test condition by the percent attachment in the presence of serum. The results from 3 different experiments were pooled and expressed as mean.+-.standard error of the mean (n=9). * indicates significant difference, p

"FIG. 13 shows SEM images of AF cells attached onto PU scaffolds containing (A) 0%, (B) 0.05%, (C) 0.5%, and (D) 5% ADO 24 hours after cell seeding.

"FIG. 14 are graphs showing extracellular matrix accumulation on PU scaffolds containing 0%, 0.05%, 0.5% and 5% ADO (wt %) after 7 days of culture. The glycosaminoglycan (GAG) content (A) and collagen content (B) were determined as described in the Examples. The data was pooled and expressed as mean.+-.standard error of the mean. * indicates significant difference from scaffolds containing 0.05% or no ADO (n=9)."

For additional information on this patent application, see: Santerre, J. Paul; Kandel, Rita. Fibrous Scaffold for Use in Soft Tissue Engineering. Filed February 20, 2014 and posted July 10, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=1695&p=34&f=G&l=50&d=PG01&S1=20140703.PD.&OS=PD/20140703&RS=PD/20140703

Keywords for this news article include: Tissue Engineering, Biomedical Engineering, Biomedicine, Genetics, Urethane, Carbamates, Biochemical, Biochemistry, Therapeutics, Bioengineering, Back Pain Relief, Extracellular Space, Mount Sinai Hospital, Neurologic Manifestations, Extracellular Matrix Proteins.

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Source: Gene Therapy Weekly


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