WO2010060090A1 - Systems and methods to affect anatomical structures - Google Patents
Systems and methods to affect anatomical structures Download PDFInfo
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- WO2010060090A1 WO2010060090A1 PCT/US2009/065754 US2009065754W WO2010060090A1 WO 2010060090 A1 WO2010060090 A1 WO 2010060090A1 US 2009065754 W US2009065754 W US 2009065754W WO 2010060090 A1 WO2010060090 A1 WO 2010060090A1
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- WIPO (PCT)
- Prior art keywords
- bone
- anatomical structure
- nanofiber
- affecting
- mesh
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Definitions
- the various embodiments of the present disclosure relate generally to systems and methods to affect anatomical structures. More particularly, the various embodiments of the present disclosure are related to systems and methods to regenerate bone.
- Bone is one of the few adult tissues with the capacity for true self-healing. Unlike soft tissue injuries, which generally result in the formation of scar tissue, bone healing concludes with the actual regeneration and reconstitution of the injured tissue, including the biochemical and biomechanical properties.
- the need for bone regeneration arises in cases of bone loss due to trauma, tumor resection, or disease.
- Traditional orthopedic practice relies on the ability of a surgeon to drill, cut, ream, and realign bone.
- the success of these procedures requires technical skill and well- designed hardware. In a majority of cases, this is sufficient for healing, due largely to the remarkable capacity of bone for self-regeneration.
- Certain fractures and bone defects require additional bone augmentation.
- the current procedures for bone augmentation include autologous and allogenic bone grafting, and more recently, ceramic and composite substitutes for bone grafts.
- the clinical gold standard for bone regeneration has been autologous bone grafting, as it provides osteogenic cells and osteoinductive factors for bone healing.
- autologous bone grafting has been successful in many cases, it has significant disadvantages including limited graft material and morbidity of the donor site. These limitations have led to increased use of allograft bone as a substitute for autologous bone. Allograft bone, however, is inferior to autologous bone due to its reduced biological activity after processing.
- allograft bone grafting is associated with a high rate of complications and late fractures as well as a risk of disease transmission. The high failure rate of allografts is largely attributable to the inability of the graft to revascularize and remodel.
- Ceramic and polymer based bone graft substitutes have recently been introduced and are being used frequently. But, ceramics tend to be brittle, and polymers suffer from limited bioactivity and strength and may need to be supplemented with osteogenic cells and growth factors.
- Tissue engineering research has focused on therapeutic strategies for repairing bone defects by the delivery of biological agents along with biodegradable scaffolds.
- Both two- and three-dimensional scaffolds have been designed to provide a template for bone repair.
- the focus has been to create three-dimensional scaffolds having adequate strength to support in vivo loading, most scaffolds do not provide an optimal environment for cellular function, and suffer from slow resorption kinetics.
- exogenous cells delivered at the center of these scaffolds in vivo may not survive due to the initial lack of vascularity at the defect.
- two- dimensional membranes have been used for bone repair by placing them along the periosteal surface to demarcate the osseous from the non-osseous region. Though this technique (called guided bone/tissue regeneration) has been highly successful in the dental field for bone regeneration, it has not been quantitatively evaluated for the load bearing case of long bone defects.
- the various embodiments of the present invention are directed to systems and methods to affect anatomical structures. More particularly, the various embodiments of the present invention are directed to systems and methods to regenerate bone.
- One aspect of the present invention includes a system for affecting an anatomical structure, comprising: a nanofiber mesh configured to substantially conform to an anatomical structure; and an active agent.
- the system can further comprise a carrier substance, such as a hydrogel.
- the nanofiber mesh defines a fillable space, and the carrier substance can be disposed within the fillable space, to substantially fill the space.
- the nanofiber mesh can have an average pore size of less than about 100 micrometers, and the nanofiber mesh can further comprise a plurality of macropores.
- the active agent can comprise at least one member of the transforming growth factor-beta (TGF- ⁇ ) superfamily, such as bone morphogenetic protein (BMP).
- TGF- ⁇ transforming growth factor-beta
- BMP bone morphogenetic protein
- the system for affecting an anatomical structure is used on a bone, and the carrier substance comprises alginate or a derivative thereof, the at least one member of the transforming growth factor-beta (TGF- ⁇ ) superfamily comprises a bone morphogenetic protein (BMP).
- the active agent can comprise a plurality of cells, such as mesenchymal progenitor cells.
- Another aspect of the present invention includes a method for affecting an anatomical structure, comprising: adapting a nanofiber mesh to an anatomical structure of a subject so that the nanofiber mesh substantially conforms to the anatomical structure, wherein at least a portion of the nanofiber mesh defines a fillable space; and flowing a carrier substance into the fillable space, wherein the carrier substance comprises an active agent, and wherein at least a portion of the carrier substance is disposed within the fillable space.
- the nanofiber mesh can have an average pore size of less than about 100 micrometers.
- the carrier substance can comprise a hydrogel, and the carrier substance can further comprise the active agent.
- the active agent can comprise at least one member of the transforming growth factor-beta (TGF- ⁇ ) superfamily.
- the anatomical structure comprises a bone
- the carrier substance comprises alginate or a derivative thereof
- the at least one member of the transforming growth factor-beta (TGF- ⁇ ) superfamily comprises a bone morphogenetic protein (BMP).
- a method for affecting an anatomical structure can further comprise regenerating at least a portion of the bone of a subject, wherein the bone morphogenetic protein is present in an initial concentration of less than about 100 micrograms per kilogram of body weight of the subject.
- Methods for affecting an anatomical structure can also comprise using an active agent that comprises a plurality of mesenchymal progenitor cells.
- a kit for affecting an anatomical structure comprising: a membrane configured to substantially conform to an anatomical structure; and a flowable hydrogel system comprising at least one active agent.
- the membrane can be a nanofiber mesh having an average pore size of less than about 100 micrometers, and the nanofiber mesh can further comprise a plurality of macropores.
- the kit is used to affect a bone
- the flowable hydrogel comprises alginate or a derivative thereof
- the active agent comprises a bone morphogenetic protein, a plurality of mesenchymal progenitor cells, or combinations thereof.
- Figure IA is a representative scanning electron microscope (SEM) image of a nanofiber mesh at 10,000X magnification.
- Figure IB is a histogram of nanofiber diameter of a representative nanofiber mesh.
- Figure 2A provides SEM images of random and aligned nanofiber meshes at 2000X magnification.
- Figure 2B graphically depicts fiber angle measurements of nanofiber meshes.
- Figure 3 is a perspective view of one embodiment of a system for affecting an anatomical structure, such as a long bone.
- Figure 4 is a cross section of one embodiment of a system for affecting an anatomical structure, such as a long bone.
- Figure 5 is a perspective view of one embodiment of a system for affecting an anatomical structure having macropores.
- Figure 6 illustrates the infiltration of cells and angiogenesis promoted by one embodiment of a system for affecting an anatomical structure having macropores.
- Figures 7A-C provide (A) a SEM image at low (1000X) magnification; (B) a SEM image at high (7000X) magnification; and (C) a nanofiber diameter histogram.
- Figures 8A-D demonstrate sample cellularity through DNA quantity for cells cultured on (A) tissue culture plates and (B) nanofiber meshes. To compare the cellularity on nanofiber meshes with that on tissue culture plates, the data was plotted again for (C) hMSCs and (D) hAFS cells. (* - significantly different than the other cell type at same time point; # - significantly different than the previous time point for same cell type).
- Figures 9A-B graphically depict the alkaline phosphatase activity of cell lysates on (A) tissues culture plates and (B) nanofiber meshes. (* - significantly different than the other cell type at same time point; # - significantly different than a previous time point for same cell type; $ - significantly different than hMSC peak on plate at day 7).
- Figure 10 graphically depicts calcium quantification deposited by cells. (# - significantly different than growth media; * - significantly different than other cell type on same surface; $ - significantly different than plate with same cell type).
- Figure 11 graphically depicts calcein staining quantified by fluorescence (* - significantly greater than MSC osteogenic media; # - significantly greater than growth media with same cell type).
- Figure 12 is a schematic of the cell infiltration model.
- Figures 13A-B illustrate the kinetics of cell infiltration on uncoated random nanofiber meshes. (* indicates significantly greater than cell number on day 0. # indicates significantly greater than cell number on days 3 and 4. Significance was set at p ⁇ 0.05).
- Figure 14 graphically depicts the effect of coating and fiber alignment on cell infiltration at 48 hours after strip removal. (** indicates significantly greater than collagen with same fiber orientation. $ indicates greater than random orientation with same coating. Significance was set at p ⁇ 0.05).
- Figures 15A-B demonstrate the role of cell proliferation in the cell infiltration model. (* indicates significantly greater than random uncoated with same MMC condition (p ⁇ 0.05). $ indicates significantly greater than random GFOGER with same MMC condition (p ⁇ 0.05)).
- Figure 16 graphically depicts the early effect of GFOGER coating on hMSC number. (* indicates significantly greater than uncoated in the same group).
- Figures 17A-C demonstrate the effect of coating and fiber alignment on cell number and osteogenic differentiation of hMSCs in osteogenic media through (A) DNA amount, (B) alkaline phosphatase (ALP) activity, and (C) calcium deposition.
- Figure 18 is radiographs of femurs implanted with nanofiber mesh tubes at 4 and 12 weeks.
- Figure 19A illustrates regenerated bone volume from ⁇ CT analysis
- Figure 19B demonstrates failure strength derived from the maximum torque in torsional testing.
- Figure 19C illustrates cross sections of ⁇ CT images of regenerated bone.
- Figure 20 shows histological analysis of newly formed bone with hematoxylin and eosin staining at 12 weeks.
- the defect in both groups contains a combination of newly formed bone (nb), fibrous tissue (ft) and marrow (m). Scale bars are 200 ⁇ m.
- Figure 21 A is a SEM image of an electrospun nanofiber mesh.
- Figure 2 IB illustrates a hollow tubular implant without perforations made from nanofiber meshes.
- Figure 21C illustrates hollow tubular implant with perforations made from nanofiber meshes.
- Figure 21D illustrates implants in segmental bone defect.
- Figure 21E displays a photograph of the defect in a subject following placement of a perforated mesh tube.
- Figure 2 IF displays a photograph of the defect in a subject after a one week incubation with the nanofiber mesh tube.
- Figure 21 G graphically depicts alginate release kinetics over 21 days in vitro.
- Figure 22 provides representative radiographs at 4 and 12 weeks.
- Figure 23A illustrates ⁇ CT images of bone regeneration at 4 and 12 weeks.
- Figure 23B graphically depicts quantification of regenerated bone volume.
- Figure 23 C graphically depicts mean density of regenerated bone.
- Figures 24A-B demonstrate the mechanical properties, (A) maximum torque and (B) torsional stiffness, of femora at 12 weeks.
- Figures 25A-B provide a histological analysis at 12 weeks (A) 4X magnification, (B) 1OX magnification.
- White arrows point to osteocytes embedded in lacunae.
- Black arrows point to osteoblasts lining the bone surface.
- Scale bar is 100 ⁇ m.
- Figure 26 illustrates vascular ingrowth at a defect site at 3 weeks. Scale bar is 1 mm and applies to all images. "Peri" refers to periphery of defect.
- Figure 27 displays representative radiographs at 2, 4 and 12 weeks.
- Figure 28 illustrates three-dimensional in vivo ⁇ CT images at 4 and 12 weeks.
- Figures 29A-B demonstrate quantitative ⁇ CT analysis of new bone volume. (* indicates significantly different (p ⁇ 0.05)).
- Figures 30A-B demonstrate mean density (A) and connectivity density (B) of the newly formed bone, obtained from ⁇ CT analysis. (* indicates significantly different (p ⁇ 0.05)).
- Figure 31 illustrates the biomechanical properties of regenerated femurs at 12 weeks.
- the various embodiments of the present invention provide systems and methods to affect anatomical structures of a subject.
- the term "subject” refers to animals and plants, as well as cells and tissues derived therefrom.
- the systems and methods of the present invention are applicable to a broad range of vertebrate animals, preferably mammals, and more preferably humans. Mammals include, but are not limited to, humans, primates, horses, cows, sheep, pigs, goats, dogs, cats, rabbits, guinea pigs, rats, and mice.
- the term “anatomical structure” can refer to many anatomical structures of animals or plants.
- an anatomical structure of an animal can include, but is not limited to, bone, cartilage, tendons, ligaments, muscle, tumors, nerve, skin, organs, and tissues.
- an anatomical structure is bone, such as short bones, flat bones, and long bones.
- the term "anatomical structure” also encompasses defects, injuries, trauma, lesions, and the like that are associated with an anatomical structure.
- the phrase "to affect an anatomical structure” refers to acting on or producing an effect or a change in the structure or function of an anatomical structure. Therefore, “to affect an anatomical structure” refers to a difference in degree from a first state, such as an untreated state in a subject, to a second state, such as a treated state in a subject. Consequently, the various embodiments of the systems and methods of the present invention can function to alter one or more various anatomical, physiological, and pathological properties of an anatomical structure.
- the alteration of one or more various anatomical, physiological, and pathological properties of an anatomical structure can include, but are not limited to, promoting or inhibiting cellular infiltration, promoting or inhibiting cellular proliferation, promoting or inhibiting cellular differentiation, promoting or inhibiting cellular survival, promoting or inhibiting angiogenesis, promoting or inhibiting an immune response, or promoting or inhibiting matrix synthesis or organization, among others.
- the alteration of one or more anatomical property, physiological property, pathological property, or various combinations thereof will depend upon the desired therapeutic effect.
- alteration of one or more anatomical, physiological, or pathological properties can include promotion of cellular proliferation, infiltration, and differentiation as well as promotion of angiogenesis in the defect site.
- alteration of one or more anatomical, physiological, or pathological properties can include inhibition of cellular proliferation, infiltration, and differentiation or inhibition of angiogenesis into the defect site.
- alteration of one or more anatomical, physiological, or pathological properties can include inhibition of cellular proliferation or survival, inhibition of angiogenesis, or stimulation of an immune response.
- a system for affecting an anatomical structure comprises: a nanofiber mesh configured to substantially conform to an anatomical structure or defect region; and an active agent.
- a nanofiber mesh 100 can comprise a plurality of nanofibers 105, as shown in Figure 1.
- the term "plurality" means more than one.
- the nanofiber mesh 100 is a porous, substantially flattened structure composed of a plurality of interconnected nanofibers 105, as shown in Figure IA.
- the nanofiber mesh 100 is plurality of interconnected microfibers (i.e., fibers having an average longest cross-sectional dimension greater than about 1000 nm).
- the nanofiber mesh 100 can comprise a one or more of nanofibers and one or more macrofibers.
- a nanofiber 105 comprises many classes of materials that are continuous, flexible, filaments or are in discrete elongated pieces, similar to lengths of thread.
- a "nanofiber” means a continuous, flexible, filamentous material having a relatively high aspect ratio (i.e., ratio of length to average longest cross-section).
- an aspect ratio can be at least about 5:1.
- an aspect ratio can be at least about 10: 1, at least about 100:1, or at least about 1000:1.
- a nanofiber 105 can have many cross-sectional shapes, including, but not limited to, a rectangular shape, a circular shape, a semicircular shape, a square shape, a pentagonal shape, a triangular shape, a hexagonal shape, an octagonal shape, a star-shape, a starburst shape, a "U" shape, a lobed shape, a multi-lobed shape, an arbitrary shape, or combinations thereof or therebetween.
- a nanofiber 105 has a circular cross-sectional shape; however, one of ordinary skill in the art would realize that the cross-sectional shape of the nanofiber 105 can be varied to impart additional properties to the nanofiber 105 or nanofiber mesh 100, such as increased surface area.
- the cross-sectional shape of the nanofiber 105 will determine the average longest cross-sectional dimension of the nanofiber 105.
- the average longest cross-sectional dimension of a nanofiber 105 having a circular cross-sectional shape will be the diameter of the nanofiber 105.
- the average longest cross-sectional dimension of a nanofiber 105 having a rectangular cross-sectional shape will be the length (with the proviso that the so-called length is greater than the so-called width) of the rectangular cross-section of the nanofiber 105.
- the average longest cross-sectional dimension of a nanofiber 105 having a starburst cross-sectional shape will be the distance between the two furthest points of the starburst cross- section of the nanofiber 105.
- a nanofiber 105 has an average longest cross-sectional dimension ranging from about 10 nm to about 1000 nm. In another embodiment of the present invention, a nanofiber 105 has an average longest cross-sectional dimension ranging from about 50 nm to about 600 nm. In yet another embodiment of the present invention, a nanofiber 105 has an average longest cross-sectional dimension ranging from about 50 nm to about 300 nm. In yet another embodiment of the present invention, a nanofiber 105 has an average longest cross-sectional dimension ranging from about 50 nm to about 300 nm ( Figure IB).
- a nanofiber 105 has an average longest cross-sectional dimension ranging from about 50 nm to about 150 nm. In an exemplary embodiment of the present invention, a nanofiber 105 has an average longest cross- sectional dimension of about 150 nm.
- the nanofibers 105 of the present invention can be made of many biocompatible materials, including degradable materials, non-degradable materials, or combinations thereof.
- the nanofibers 105 are made of poly ( ⁇ -caprolactone). In an exemplary embodiment of the present invention, the nanofibers 105 are made of
- the nanofiber mesh 100 is produced by electrospinning. Electrospinning has recently emerged as a technique to produce polymeric scaffolds for tissue engineering, with fiber diameters ranging from tens of nanometers to as large as 10 micrometers.
- the nanofiber mesh obtained by this process is a unique scaffold membrane that possesses structural features with a size scale similar to extracellular matrix (ECM) components, high porosity and large surface area to volume ratios. These properties allow for enhanced cellular attachment and spreading. Therefore, nanofiber meshes serve as an effective delivery vehicle for cells to a defect site in vivo.
- ECM extracellular matrix
- the nanofiber mesh 100 may have a network of randomly-oriented nanofibers 105 or may have a network of substantially aligned nanofibers 105, as shown in Figures 2A-B.
- a substantially aligned nanofiber mesh 100 has a majority of nanofibers 105 oriented in the same direction. Stated another way, a substantially aligned nanofiber mesh 100 comprises a plurality of nanofibers 105, where more than about 85% of the nanofibers are aligned substantially parallel to an arbitrary line (i.e., within -45° and +45° of the arbitrary line). Conversely, a randomly-oriented nanofiber mesh 100 comprises a plurality of nanofibers 105, where less than about 85% of the nanofibers are aligned substantially parallel to an arbitrary line. In an exemplary embodiment of the present invention, a randomly-oriented nanofiber mesh 100 comprises a plurality of nanofibers 105, where less than about 55% of the nanofibers are aligned substantially parallel to an arbitrary line.
- the nanofiber mesh 100 is a porous, substantially flattened structure composed of a plurality of interconnected nanofibers 105, as shown in Figure IA.
- the nanofiber mesh 100 can have an average thickness ranging from about 50 nm to about 10 mm. In an exemplary embodiment of the present invention, the nanofiber mesh 100 has an average thickness ranging from about 100 nm to about 1 mm. In another exemplary embodiment, the nanofiber mesh 100 has an average thickness ranging from about 300 ⁇ m to about 400 ⁇ m.
- the plurality of interconnected nanofibers 105 creates a network or mesh of fibers, which confers the nanofiber mesh 100 with plurality of pores 110 (Figure IA). These pores 110 may have varying characteristics, such as size, shape, and tortuosity.
- the pores 110 are constructed such that they pass directly through the nanofiber mesh 100.
- the pores 110 have an average longest cross-sectional dimension of less than about 100 ⁇ m.
- the pores 110 have an average longest cross-sectional dimension of less than about 10 ⁇ m.
- the pores 110 have an average longest cross- sectional dimension of less than about 5 ⁇ m.
- the pores 110 have an average longest cross-sectional dimension of less than about 2 ⁇ m.
- the pores 110 have an average longest cross-sectional dimension of about 1 ⁇ m.
- the nanofiber mesh 100 is a substantially flattened structure, composed of a network of biocompatible nanofibers 105, that can be configured to substantially conform to an anatomical structure. Configuring the nanofiber mesh 100 to substantially conform to an anatomical structure may take place during construction of the mesh. Alternatively, the nanofiber mesh 100 may be configured after construction, for example during implantation. In yet another alternative, the nanofiber mesh 100 may be configured during construction and further configured after construction.
- the nanofiber mesh 100 When implanted, the nanofiber mesh 100 can be configured to define a fillable space. Therefore, the implanted nanofiber mesh 100 provides a porous permeable membrane between the established fillable space of the anatomical structure and the milieu surrounding the anatomical structure.
- the phrase "substantially conform to an anatomical structure" refers to situations where the nanofiber mesh 100 may delineate the entire boundary of the space, or alternatively, the nanofiber mesh 100 may delineate only a portion of the space and the remainder of the boundary being delineated by the anatomical structure or adjacent structures or tissues of the subject.
- the parameters and configuration of the fillable space established by implantation of the nanofiber mesh 100 is approximately equivalent to the anatomical structure or defect associated therewith that is desired for treatment with the systems and methods of the present invention.
- Figure 3 illustrates one example of a system 300 for affecting an anatomical structure, such as bone 305.
- the system comprises a nanofiber mesh 100 is configured to substantially conform to an anatomical structure, in this case a long bone 305.
- the nanofiber mesh 100 is configured to define a fillable space 310.
- the nanofiber mesh 100 is wrapped around the bone defect to establish a fillable space 310 for bone growth.
- an active agent 320 can include, without limitation, agents for gene therapy, analgesics, anti-arthritics, anti-asthmatic agents, anticholinergics, anti-convulsants, anti-depressants, anti-diabetic agents, anti-diarrheals, anesthetics, antibiotics, antigens, anti-histamines, anti-infectives, anti-inflammatory agents, antimicrobial agents, anti-migraine preparations, anti-nauseants, anti-neoplasties, anti-parkinsonism drugs, anti-pruritics, anti-psychotics, anti-pyretics, anti-spasmodics, anorexics, anti-helminthics, antiviral agents, nucleic acids, DNA, RNA, polynucleotides, nucleosides, nucleotides, amino acids, peptides,
- the active agent 320 comprises at least one member of the transforming growth factor-beta (TGF- ⁇ ) superfamily.
- TGF- ⁇ transforming growth factor-beta
- TGF- ⁇ superfamily include but are not limited to, TGF- ⁇ (e.g., TGF- ⁇ l, TGF- ⁇ 2, TGF- ⁇ 3) bone morphogenetic protein (BMP) (e.g., BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMPlO, BMPI l, BMP12, BMP13, BMP14, BMP15), inhibin (e.g., Inhibin A, Inhibin B), activin (e.g., Activin A, Activin B, Activin AB), growth differentiation factor (GDFl, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDFlO, GDFI l, GDF15), Nodal,
- the active agent can be recombinant human BMP2 (rhBMP2) or recombinant human BMP7 (rhBMP7), or combinations thereof.
- the active agent 320 may comprise a growth factor, such as insulin-like growth factor 1 (IGF-I), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), or fibroblast growth factor (FGF).
- IGF-I insulin-like growth factor 1
- VEGF vascular endothelial growth factor
- PDGF platelet-derived growth factor
- FGF fibroblast growth factor
- the active agent 320 can comprise a cell or a plurality of cells, such as a mesenchymal progenitor cell, which can be derived from amniotic fluid, bone marrow, adipose tissue, and the like.
- a mesenchymal progenitor cell which can be derived from amniotic fluid, bone marrow, adipose tissue, and the like.
- MSCs bone marrow derived mesenchymal stem cells
- Other cells that may be suitable for applications related to regeneration and remodeling of bone include, but are not limited to, osteoblasts, osteoclasts, osteocytes, and bone-lining cells
- the nanofiber mesh 100 can be functionalized or surface-modified with one or more active agents.
- the nanofiber mesh 100 can be functionalized or surface-modified with an osteogenic biomolecule.
- biomolecule refers many organic molecules that are produced by a living organism, including large polymeric molecules such as proteins, polysaccharides, and polynucleotides, including smaller peptides, oligosaccharides, and nucleic acids, as well as small molecules such as primary metabolites, secondary metabolites, and other biological products.
- the nanofiber mesh can be functionalized or surface- modified to mimic the extracellular matrix.
- the osteogenic biomolecule can include proteoglycans, proteins, peptides, polysaccharides, and components thereof, among others.
- “mimicking the extracellular matrix” refers to biomolecules (e.g., proteoglycans, proteins, peptides, polysaccharides, and components thereof) having structural identity to a component of the extracellular matrix or sufficient structural homology to a component of the extracellular matrix, thereby effectively replicating the same interactions associated with that component of the extracellular matrix.
- biomolecules can include, but are not limited to, glycosaminoglycans (e.g., chondroitin sulfate, heparin sulfate, keratin sulfate, hyaluronic acid), or proteins (e.g., collagen, elastin, fibronectin, laminin) or peptides derived from these proteins (e.g., glycine-phenylalanine-hydroxyproline-glycine-glutamate- arginine (GFOGER) domain from residues 502-507 of the ⁇ i(I) chain of type I collagen), among others.
- the system for affecting an anatomical structure can further comprise a carrier substance 315.
- the term "carrier substance" can comprise many media, including but not limited to, a fluid, liquid, solid, solution, suspension, emulsion, gas, vapor, gel, dispersion, a flowable material, a multiphase material, or combination thereof.
- the carrier substance comprises a flowable polymer.
- the flowable polymer can comprise many suitable hydrophilic, hydrophobic, and amphiphilic polymers known in the art.
- the flowable polymer can comprise a hydrophilic polymer, including, but not limited to, acrylates, acrylamides, acetates, acrylic acids, vinyl alcohols, glycols, polysaccharides, co-polymers thereof, or combinations thereof.
- the polymer can have many topologies including, but not limited to, a branched topology, a graft topology, a comb topology, a star topology, a cyclic topology, a network topology, or combinations thereof, among others.
- the crosslinker of the polymer particles can be many suitable crosslinkers known in the art including, but not limited to, N 1 N', methylenebis(acrylamide), poly(ethylene glycol) (PEG) diacrylate, NN'-dihydroxyethylene-bisacrylamide, NO- (dimethacryloyl)hydroxylamine, ethylene glycol dimethacrylate, divinylbenzene, divalent cations (e.g., Ca 2+ and Mg 2+ ), or combinations thereof.
- the carrier substance comprises an alginate or a derivative thereof. Such derivatives are described in U.S. Patent No. 6,642,363, which is incorporated by reference in its entirety.
- An example of an alginate derivative is alginate modified with a G 4 RGDASSP peptide sequence.
- an embodiment of the present invention comprises a method for affecting an anatomical structure, which involves adapting a nanofiber mesh 100 to an anatomical structure of a subject so that the nanofiber mesh substantially conforms to the anatomical structure, and at least a portion of the nanofiber mesh 100 defines a fillable space 310 followed by flowing a carrier substance 315 into the fillable space 310.
- the flowable carrier substance 315 is capable of substantially filling the fillable space 310.
- the flowable aspect of the carrier substance provides a significant advancement over prior art methods, which utilize pre-formed gels.
- a flowable carrier substance 315 permits complete or near complete filling of the space 310 created by the nanofiber mesh 100.
- the fillable space 310 can be filled with a carrier substance 315, such as a hydrogel, comprising an active agent 320.
- a carrier substance 315 such as a hydrogel, comprising an active agent 320.
- membranes i.e., polymer films, which can be made of many the degradable materials, non-degradable materials, or combinations thereof, described above).
- the carrier substance 315 can contain the active agent 320.
- the carrier substance comprises alginate or a derivative thereof, and the active agent comprises a member of the TGF- ⁇ superfamily, such as a bone morphogenetic protein (BMP).
- BMP bone morphogenetic protein
- the nanofiber mesh and flowable hydrogel carrier operate synergistically to promote new bone growth.
- the nanofiber mesh 100 is positioned on the periosteal surface to define a space 310, as shown in Figure 4.
- the flowable hydrogel carrier 315 which contains the active agent 320, operates to effectively fill the defect space 310 defined by the at least a portion of the nanofiber mesh 100 and the bone 305.
- the polymerized hydrogel 315 retains the active agent 320 at the defect site, which permits sustained delivery of the active agent 320, thereby promoting the infiltration and proliferation of osteoprogenitor cells.
- the nanofiber mesh 100 provides a substrate to facilitate the migration and proliferation of progenitor cells on the periosteal surface.
- the porosity of the nanofiber mesh 100 permits sufficient retention of the active agent 320 at the defect site, while allowing for the release of the active agent 320 (through diffusion or hydrogel degradation), which establishes a gradient to attract osteoprogenitor cells to the defect site.
- the porosity of the nanofiber mesh 100 permits the infiltration of nutrients to the defect site.
- the nanofiber mesh can be attached to the anatomical structure with the use of various biocompatible fastening elements, such as sutures, staples, tacks, screws, adhesives, or other fastening elements known in the art of fixing articles to anatomical structures.
- the nanofiber mesh may simply be surgically placed on the anatomical structure without using an attachment element.
- the nanofiber mesh 100 can be modified to comprise a plurality of macropores 505, as shown in Figure 5. These macropores 505 can have an average longest cross-section dimension ranging from about 100 ⁇ m to about 2000 ⁇ m.
- the macropores 505 can have an average longest cross-section dimension of about 1 mm, and these macropores can be spaced approximately 1.5 mm apart. Although not wishing to be bound by any particular theory, it is believed that the macropores 505 facilitate the invasion of vasculature 605 and the migration of cells 610 into the defect site, such as osteoprogenitor cells. (Figure 6).
- design considerations include, but are not limited to, the number of macropores 505, the average longest cross- sectional dimension of the macropore 505, the distribution of macropores 505 on the nanofiber mesh 100, and the shape of the macropores 505.
- the composition and viscosity of the carrier substance 315 may be varied depending upon the composition and design of the nanofiber mesh 100 as well as the composition of the active agent 320.
- the carrier substance 315 can further comprise various thickening agents, diluents, and suspending agents to facilitate suspension, retention, and controlled diffusion of the active agent in the carrier substance.
- active agents may be suspended in a carrier substance that demonstrates reduced viscosity through the use of nanoparticles or microtubes.
- the systems and methods of the present invention contemplate a method for treating a bone defect, comprising adapting a nanofiber mesh to a bone defect of a subject so that the nanofiber mesh substantially conforms to the bone defect, and at least a portion of the nanofiber mesh defines a fillable space; and flowing a carrier substance into the fillable space, the carrier substance comprising a therapeutically effective amount of an active agent.
- therapeutically effective amount or "effective amount” as used herein is an amount of a compound that produces a desired therapeutic effect in a subject, such as inducing bone regeneration.
- the precise therapeutically effective amount is an amount of the composition that will yield effective results in terms of efficacy of treatment in a given subject.
- This amount (i.e., dosage) may vary depending upon a number of factors, including, but not limited to, the characteristics of the active agent (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease stage, general physical condition, and responsiveness to a given dosage), the nature of the carrier substance, and the composition of the nanofiber mesh.
- One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly.
- a method of treating a bone defect comprises administering a therapeutically effective dose of at least one TGF- ⁇ member of the TGF- ⁇ superfamily, such as a bone morphogenetic protein (e.g., rhBMP-2, rhBMP-7).
- a therapeutically effective amount of bone morphogenetic protein is less than about 100 micrograms per kilogram of body weight of the subject.
- a therapeutically effective amount of bone morphogenetic protein is less than about 50 micrograms per kilogram of body weight of the subject.
- a therapeutically effective amount of bone morphogenetic protein is less than about 25 micrograms per kilogram of body weight of the subject.
- a therapeutically effective amount of bone morphogenetic protein is about 20 micrograms per kilogram of body weight of the subject.
- Nanofiber meshes were utilized in a three -pronged approach.
- nanofiber meshes were validated their ability to robustly support osteogenic cell functions, including proliferation and matrix mineralization. We also demonstrated their efficacy as a cell delivery vehicle.
- a nanofiber mesh based hybrid system for growth factor delivery was developed for bone repair and tested in a challenging animal model. The delivery of bone morphogenetic protein (BMP) via this system resulted in the functional restoration of limb function, and in fact proved more efficacious than the current clinical standard for BMP delivery.
- BMP bone morphogenetic protein
- EXAMPLE 1 COLONIZATION AND OSTEOGENIC DIFFERENTIATION OF DIFFERENT STEM CELL SOURCES ON ELECTROSPUN NANOFIBER MESHES
- hMSCs human MSCs
- hAFS human AFS
- a polymer solution was made by dissolving 13% (w/v) poly ( ⁇ -caprolactone) (PCL) (Sigma-Aldrich, St. Louis, MO) in a 40:60 volume ratio of dichloromethane (DCM) :dimethy If ormamide (DMF) (Sigma-Aldrich). PCL pellets were added to the solvent mixture, and gently stirred for 16-24 hours.
- the polymer solution was loaded in a 3 mL syringe (Becton-Dickinson, Franklin Lakes, NJ), and a 22 gauge blunt stainless steel needle (Jensen Global Inc., Santa Barbara, CA) was attached to the syringe end.
- the syringe was mounted on a syringe pump (Harvard Apparatus, Holliston, MA), and the pump was set to infuse at a rate of 0.75 mL/hr.
- a flat, 6 x 6 inch copper plate (McMaster-Carr, Atlanta, GA) covered with aluminum foil was used to collect the fibers, and placed at a distance of 20 cm from the needle end. Fibers were electrospun for 50 minutes at a voltage of 14 kV, supplied by a high voltage power supply (Gamma High Voltage Research, Ormond Beach, FL), to obtain a thin sheet of nanofiber mesh. To remove any residual solvent, the meshes were placed in a dessicator for at least one day before further use.
- Nanofiber mesh morphology The morphology of the nanofiber meshes was examined using a Scanning Electron Microscope (SEM). A small piece of the dry nanofiber mesh was cut and mounted on a metal stub using double-sided adhesive tape. A thin layer of gold was then deposited on the mesh sample for 80 seconds using a sputter coater (Quorum Technologies, East Granby, CT). The gold-coated sample was then viewed under a Hitachi S-800 Field Emission SEM (Hitachi HTA, Pleasanton, CA) with 1OkV accelerating voltage.
- SEM Scanning Electron Microscope
- the diameters of the fibers were quantified by analyzing the SEM images (at 700Ox magnification) using a custom MATLAB ® (MATLAB ® 7.0 R14, The MathWorks Inc., Natick, MA) program. A total of at least 75 distinct fibers were measured from four randomly chosen locations.
- hAFS cells were subcultured routinely at a dilution of 1:4 to 1:8, and not permitted to expand beyond 70% confluence.
- the hAFS cells were passaged in ⁇ MEM (Minimum Essential Medium Alpha) supplemented with 16% fetal bovine serum (ES Cell-qualified FBS), 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 2 mM L-glutamine (Invitrogen, Carlsbad, CA), 18% Chang B and 2% Chang C (Irvine Scientific, Santa Ana, CA).
- ⁇ MEM Minimum Essential Medium Alpha
- ES Cell-qualified FBS 16% fetal bovine serum
- penicillin 100 ⁇ g/ml streptomycin
- 2 mM L-glutamine Invitrogen, Carlsbad, CA
- 18% Chang B and 2% Chang C Irvine Scientific, Santa Ana, CA.
- the control groups received hMSC growth media, whereas the osteogenic groups were further supplemented with 10 nM dexamethasone, 6 mM ⁇ -glycerol phosphate, 50 ⁇ g/ml ascorbic acid 2-phosphate and 50 ng/ml L-thyroxine (Sigma- Aldrich).
- hMSCs and hAFS cells were then seeded onto nanofiber meshes in approximately 200 ⁇ L of hMSC media such that the density of cells was 20,000 cells/cm 2 .
- Cells were also cultured in tissue culture treated 24-well plates at the same density for comparison. Media was changed every 3-4 days, and the constructs were cultured for up to 4 weeks.
- DNA content was assessed by determining the DNA content.
- Samples were harvested after 1, 7, 14 and 28 days to evaluate the construct cellularity, which was assessed by determining the DNA content.
- the cells were first lysed by freeze-thawing the constructs three times in 0.05% Triton X-100 (Sigma- Aldrich) with vigorous vortexing. To freeze the samples, they were placed in dry ice cooled methanol (Sigma-Aldrich) for 5 minutes. Samples were then thawed in a room temperature water bath.
- the DNA amount in the lysate was quantified using the PicoGreen dsDNA Quantitation Kit (Molecular Probes, Eugene, OR), and standardized using Lambda DNA solutions of known concentrations.
- a working solution of the PicoGreen ® reagent was made following the manufacturer's protocol, and incubated with experimental samples in the dark for 5 minutes at room temperature. The fluorescence was measured on a plate reader (HTS 7000, Perkins- Elmer, Waltham, MA) at an excitation of 485-nm and emission of 535-nm. All samples were run in triplicate, and the DNA content was normalized to the culture surface area of the samples.
- Alkaline Phosphatase (ALP) activity was performed. In this assay, the release of /?-nitrophenol from /?-nitrophenyl phosphate by the ALP enzyme is measured. The same lysate solution that was used to determine DNA content was used for this purpose.
- the ALP substrate working solution was made by mixing equal parts of 20 mM p-nitrophenyl phosphate, 1.5 M 2- Amino-2-Methyl-l-Propanol (pH 10.25) and 10 mM MgCl 2 . The experimental samples were mixed with the freshly made substrate working solution, and incubated for 1 hour at 37° C.
- the reaction was stopped by adding IN NaOH, and the absorbance was measured at 405 nm on a plate reader (PowerWave XS, Biotek, VT). All samples were run in triplicate and compared to p-nitrophenol standards. The ALP activity was normalized by the incubation time and the amount of DNA obtained from the PicoGreen ® assay.
- Calcein staining and quantification For visualization of the mineral deposited by the cells, the samples were stained using calcein on day 28. Briefly, a stock solution of 100 ⁇ g/ml calcein (Sigma-Aldrich) in PBS (pH 7.4) was added to the media on top of the samples, such that the final concentration of calcein was 10 ⁇ g/ml. The samples were incubated in the calcein solution for 4 hours in the incubator. After rinsing twice with PBS and fixing with 10% neutral buffered formalin (EMD Chemicals, Gibbstown, NJ), samples were rinsed with excess of DI water.
- the fluorescence of the samples was read on a fluorescence plate reader (HTS 7000, Perkins-Elmer) at an excitation of 485-nm and emission of 535-nm. Following this, the same samples were imaged using an inverted microscope (Axio Observer.Zl, Carl Zeiss) and a FITC filter.
- FTIR Fourier Transform Infrared
- AFS cells were seeded on to nanofiber mesh samples (15 x 10 mm) at a density of 200,000 cells/cm 2 . The cells were allowed to attach to the mesh overnight. On the following day, each cell seeded mesh was wrapped around a cylindrical collagen scaffold (dry dimensions: 4 mm diameter and 9 mm length) aseptically, such that the cells were facing the scaffold. The scaffolds were punched from a fibrous collagen sheet (average pore size 61.7 ⁇ m, 93.7% pore volume, Kensey Nash, Exton, PA).
- the mesh was held in position by placing two interrupted silk sutures through the mesh and scaffold at the two ends of the scaffold. For comparison, we also seeded 300,000 cells throughout collagen scaffolds by pipetting the cell suspension directly in the scaffolds. There was no nanofiber mesh in this control group.
- the constructs were statically cultured in hAFS cell growth media. After two weeks, the mesh was taken off, following which the mesh and scaffold were stained with the Live/Dead ® staining kit (Molecular Probes, Invitrogen) to visualize the cell migration into the scaffold.
- Live/Dead ® staining kit Molecular Probes, Invitrogen
- Results are presented as mean + standard error of the mean (SEM). Analysis of variance (ANOVA) was performed on data, with pairwise comparisons done using the Tukey multiple comparison procedure. A /?-value ⁇ 0.05 was considered significant. Residuals were used for diagnosing the appropriateness of the model by analyzing the constancy of error variance and normality of error terms. Wherever required, remedial measures were taken by transforming the data according to the Box-Cox procedure, or by using weighted least squares to make the error variance constant and the error distribution normal. Minitab ® 15 (Minitab Inc., State College, PA) was used to perform the statistical analysis.
- hMSCs and hAFS cells were seeded on electrospun nanofiber meshes and tissue culture plates, and cultured in osteogenic media for up to 28 days.
- the viability of the cells on the meshes was assessed on days 1, 7, 14 and 28 by the Live/Dead ® staining kit.
- the live cells are stained green, whereas the dead cells appear red.
- DNA from the samples was extracted and quantified to estimate the number of cells on the meshes, as well as in the culture wells.
- the Live/Dead images illustrate that both cell types attached to the nanofiber meshes by day 1 and were able to spread out by day 7.
- the pelleting phenomenon does not occur on the nanofiber meshes, even at a later time points.
- the number of cells did not change significantly after that, suggesting confluency of the cells on the nanofiber meshes.
- ALP activity Osteogenic differentiation of hMSCs and hAFS cells.
- the osteogenic differentiation of the cells was first investigated by analyzing the ALP activity of the cells ( Figure 9).
- ALP is a membrane-bound enzyme that hydrolyzes phosphate esters, which results in inorganic phosphate being available for incorporation into mineral deposits.
- ALP activity peaked at day 7 for MSCs, whereas for AFS cells it increased slowly but continuously up to day 28 (Figure 9A).
- the maximum ALP activity of the hAFS cells was greater than the hMSC peak.
- hMSCs demonstrated a similar earlier rise in ALP activity, on day 14, compared to day 28 for hAFS cells (Figure 9B).
- the ALP activity of the hMSCs was significantly greater than that of hAFS cells on all time points other than day 1.
- the ALP response on meshes is delayed compared to that on the plate, as seen by the later increase in ALP activity.
- the ALP activity of hMSCs on meshes was sustained longer than plates, with the maximum value on meshes at day 28 greater than that observed on plates at day 7 (p ⁇ 0.05).
- Osteogenic differentiation of hMSCs and hAFS cells matrix mineralization.
- the osteogenic differentiation of the cells was further investigated by quantifying and staining the calcium deposits and by analyzing the chemical nature of the deposited mineral by FTIR spectroscopy.
- An ANOVA on the calcium deposited by the cells revealed that both cell type and the culture surface had a significant effect on the calcium levels ( Figure 10).
- Under osteogenic stimulation all groups demonstrated increased calcium deposition, compared to growth media, indicating that cells are able to differentiate to an osteoblastic phenotype on the surfaces.
- Calcium levels in the hMSC growth media groups were negligible.
- hAFS cells deposited a higher amount of calcium than hMSCs on both plates and meshes. Also, both cell types deposited more calcium on meshes compared to plates.
- FTIR spectroscopy was used to characterize the composition of the mineral that was deposited by the cells on the nanofiber meshes under osteogenic stimulation.
- an acellular piece of PCL mesh was also scanned.
- the cellular samples displayed amide I/II peaks at 1655 and 1550 cm “1 , a carbonate peak at 870 cm “1 , and a doublet split phosphate peak at 560 and 605 cm “1 , which were not seen in the acellular mesh. There was also a peak at 1050 cm "1 in the cellular samples, but it overlapped with a PCL mesh peak.
- Nanofiber mesh as a cell delivery vehicle.
- AFS cells were seeded on nanofiber meshes and wrapped around cylindrical collagen scaffolds in vitro. The constructs were cultured for two weeks and then stained with the Live/Dead ® staining kit. In the cell-seeded scaffold, more cells were present at the periphery of the scaffold, even though cells were seeded throughout. A large number of dead cells were also detected in the interior of the scaffold.
- the cell-seeded mesh was wrapped around a collagen scaffold, we found that numerous cells had migrated off the mesh onto the collagen scaffold and had high viability. The majority of cells were located close to the peripheral surface. Interestingly, we noted that the top surface was completely covered with cells.
- Electrospun nanofiber meshes are a unique type of scaffold with structural features that, at least by scale, resemble the extracellular matrix (ECM). In addition, they exhibit large surface area and high porosity, making them suitable as a scaffold for guiding tissue regeneration by host cells. For example, in the case of a large diaphyseal bone defect, the nanofiber mesh may be able to provide cues for progenitor cells from the periosteum and marrow space to migrate into the defect region and deposit mineralized matrix.
- Example 1 demonstrated that nanofiber meshes made from a synthetic polymer are able to support the attachment, colonization and osteogenic differentiation of progenitor cells. Synthetic polymers, however, lack biological ligands, and are not capable of directing intracellular signaling and response.
- Nanofiber meshes have also been fabricated from natural materials like collagen and fibrinogen, but these are limited by poor mechanical strength and handling characteristics for in vivo applications.
- Approaches that incorporate bioactive molecules within a synthetic polymer backbone may provide an optimal combination of biological activity and mechanical integrity.
- Tremendous advances have been made in imparting biofunctionality to synthetic materials by coating them with ECM components.
- These biomimetic material surfaces present adhesion motifs to engage the cell signal transduction machinery for directing cellular response and tissue repair.
- adhesive proteins such as type I collagen, fibronectin and laminin have been immobilized on material surfaces, these approaches are limited by protein purification and processing issues, and a potential host immunogenic response.
- ECM-mimetic peptides can be synthesized and purified with relative ease, and furthermore, can be designed to trigger a specific cellular response.
- One such peptide that has been recently investigated is a triple-helical, collagen- mimetic oligopeptide containing the glycine -phenylalanine-hydroxyproline-glycine-glutamate- arginine (GFOGER) domain from residues 502-507 of the ⁇ i(I) chain of type I collagen.
- Another set of guidance strategies consists of topographical cues to influence cellular responses. It is now accepted that surface morphology, including roughness and texture, modulates cellular response. For instance, titanium implants with rough microtopographies reduced cell number, increased differentiation and enhanced implant integration. The electrospinning process can be easily adapted to obtain fibrous matrices with varying structure. Fiber alignment, especially, has generated significant interest due to the fact that a number of native and regenerating tissues display an ordered architecture. Studies have shown that alignment of fibers along a particular direction affects cellular attachment and morphology as well as matrix deposition. Neurites grown on aligned nanofibers display contact guidance and improved migration. However, the effect of fiber alignment on human MSCs (hMSCs) function needs to be investigated. In addition, the combined effect of nanofiber mesh orientation and surface composition has not been studied extensively.
- Nanofiber meshes were functionalized with the GFOGER peptide to improve cell adhesion and osteogenic differentiation.
- An oriented topography was obtained by electrospinning aligned nanofibers in order to enhance cellular migration.
- An in vitro model was developed to examine hMSC infiltration on top of nanofiber meshes, and isolated the contribution of cell proliferation versus migration. The individual and combined effects of nanofiber functionalization and orientation on hMSC function were investigated.
- the efficacy of GFOGER coating to improve nanofiber mesh based repair of segmental bone defects in vivo was assessed MATERIALS AND METHODS
- Nanofiber meshes were made by electrospinning, and characterized as described in Example 1. Briefly, a 12% (w/v) solution of poly ( ⁇ -caprolactone) (PCL) was made in a 90:10 volume ratio of hexafluoro-2-propanol (HFP) :dimethy If ormamide (DMF) (Sigma-Aldrich, St. Louis, MO). A 3 mL syringe (Becton- Dickinson, Franklin Lakes, NJ) was filled with the PCL solution and fitted with a 22 gauge blunt-end needle (Jensen Global Inc., Santa Barbara, CA).
- PCL poly ( ⁇ -caprolactone)
- HFP hexafluoro-2-propanol
- DMF diimethy If ormamide
- the syringe was placed on a syringe pump (Harvard Apparatus, Holliston, MA), which was adjusted for a flow rate of 0.75 mlVhr.
- a syringe pump Hard Apparatus, Holliston, MA
- a flat copper plate was placed at a distance of 20-23 cm from the needle tip.
- meshes with fibers aligned along the same direction they were collected on a mandrel rotating at -2500 rpm and placed 8-10 cm from the need tip.
- the syringe needle was attached to the positive end of a high voltage power supply (Gamma High Voltage Research, Ormond Beach, FL), and the collector was grounded to create the electrostatic field required for electrospinning. After applying a voltage of 13-20 kV, the polymer solution was ejected from the needle towards the collector and deposited as nano-scaled fibers. The fibers were collected for 45-60 minutes to obtain meshes with sufficient thickness for cell culture experiments.
- a high voltage power supply Gamma High Voltage Research, Ormond Beach, FL
- the nanofiber meshes were sputter coated with gold (Quorum Technologies, East Granby, CT) and their morphology visualized by Scanning Electron Microscopy (SEM; Hitachi HTA, Pleasanton, CA). A custom MATLAB ® (The MathWorks Inc., Natick, MA) program was used to calculate the individual fiber diameter from the SEM images.
- the alignment of the fibers in aligned nanofiber meshes was quantified by measuring fiber angle relative to the direction of rotation, using the Image-Pro software (Media Cybernetics, Inc., Bethesda, MD). In the case of random nanofiber meshes, the angles were measured with respect to an arbitrarily set line.
- GFOGER peptide preparation and nanofiber surface coating The peptide, GGYGGGPC(GPP) 5 GFOGER(GPP) 5 GPC, was synthesized by the Emory University Microchemical Facility (Atlanta, GA). This peptide contains the GFOGER motif, where O refers to hydroxyproline.
- the purified peptide was lyophilized as a trifluoroacetic acid (TFA) salt. The peptide was reconstituted at a concentration of 10 mg/mL in a 0.1% TFA solution containing 0.01% sodium azide (NaN 3 ).
- Nanofiber mesh samples were sterilized by ethanol evaporation, wetted with 70% ethanol and rinsed with excess PBS. The samples were then passively adsorbed with GFOGER by submerging them in the dilute GFOGER solution for 2 hours at room temperature or overnight at 4°C. For comparison, samples were also coated with a 50 ⁇ g/mL purified rat type I collagen (Trevigen Inc., Gaithersburg, MD) solution or left uncoated in PBS. After rinsing again with PBS to remove any unbound peptide, the samples were ready for analysis or cell seeding.
- PBS phosphate-buffered saline
- GFOGER surface coating Analysis of GFOGER surface coating.
- the GFOGER adsorbed on the surface of the nanofibers was visualized and quantified using a biotinylated version of the GFOGER peptide.
- Biotin was conjugated to the carboxyl end of the peptide using the EZ-Link Amine-PEG 3 - Biotin kit (Pierce Biotechnology), and the unreacted biotin was removed by dialysis.
- the GFOGER coated nanofiber mesh samples were incubated with 10 ⁇ g/mL fluorescein conjugated NeutrAvidin ® (Molecular Probes, Eugene, OR) for 30 minutes at room temperature in the dark. After rinsing with excess PBS, images were taken on an inverted fluorescence microscope (Axio Observer.Zl, Carl Zeiss, Thorn wood, NY) using a FITC filter.
- the saturation of the surface by GFOGER was investigated by incubating meshes with varying GFOGER concentrations of 0-50 ⁇ g/mL.
- GFOGER concentrations 0-50 ⁇ g/mL.
- an ELISA was performed using an anti-biotin antibody. Non-specific adsorption of the antibody was first blocked by immersing the nanofiber meshes in 0.25% heat denatured serum albumin with 0.0005% Tween-20, 1 mM EDTA, and 0.025% NaN 3 in PBS for 1 hour at 37°C.
- the meshes were then incubated with an anti-biotin antibody (diluted 1:2000) conjugated to alkaline phosphatase (clone BN-34, Sigma-Aldrich) for 1 hour at 37°C.
- An alkaline phosphatase substrate, 4-methylumbelliferyl phosphate was used at a concentration of 60 ⁇ g/mL in diethanolamine buffer (pH 9.5) to measure the amount of bound antibody.
- the fluorescence was read on a plate reader (HTS 7000, Perkins -Elmer, Waltham, MA) at an excitation of 360 nm and emission of 465 nm.
- hMSC Human mesenchymal stem cell
- hMSC growth media This is termed the hMSC growth media. After the cells reached a confluency of -70%, they were harvested with 0.25% trypsin-EDTA (Invitrogen), counted, and either expanded again or seeded on nanofiber meshes. Passage 2-3 hMSCs were used in all experiments.
- hMSCs were then seeded on the nanofiber mesh samples at a density of 40,000 cells/cm in 200 ⁇ L of hMSC growth media. After 24 hours, the strip was removed, and the cell infiltration into the gap was observed at various time points.
- DAPI 4',6-diamidino-2-phenylindole
- Some samples were, in addition, stained with rhodamine phalloidin (Molecular Probes) to visualize the cell alignment. After samples were taken down, they were rinsed with PBS and fixed with 4% formaldehyde for 10 minutes. The samples were then incubated in 0.05% Triton-X (Sigma-Aldrich) for 5 minutes, rinsed in PBS and incubated in 5 units/mL rhodamine phalloidin for 20 minutes. After a PBS rinse step, the samples were incubated in 5 ⁇ g/mL DAPI for 5 minutes.
- Mitomycin C which is a known inhibitor of cell proliferation, cross links the strands of DNA, thereby inhibiting DNA replication.
- the effect of mitomycin C on hMSC proliferation was first studied on tissue culture plates. hMSCs were plated at a density of 20,000 cells/cm 2 , and after 24 hours, they were incubated with 10 ⁇ g/mL mitomycin C for 60 minutes. Control samples remained in culture media.
- Cell proliferation was assessed using 5- bromo-2'-deoxyuridine (BrdU) labeling, according to the manufacturer's instructions (FLUOS, Roche Diagnostics, Mannheim, Germany). Forty eight hours after cell seeding, the cells were incubated in hMSC growth media containing 10 ⁇ M BrdU for 24 hours in the incubator. Cells were fixed with an ethanol based fixative for 45 minutes, and denatured with 4 M HCl for 20 minutes to allow for antibody access. After neutralizing the pH with PBS, the cells were incubated with a monoclonal anti-BrdU-antibody conjugated with fluorescein for 45 minutes at 37°C.
- PrdU 5- bromo-2'-deoxyuridine
- the cells were finally stained with DAPI for the total number of cells and analyzed using fluorescence microscopy, as mentioned above.
- DAPI fluorescence microscopy
- the effect of mitomycin C was next studied in the cell infiltration model on nanofiber meshes.
- Cells were seeded on rectangular nanofiber mesh samples at a density of 40,000 cells/cm 2 , as above. Twenty four hours post seeding, mitomycin C was added to the media at a final concentration of 10 ⁇ g/mL, and incubated for 60 minutes. The media was slowly aspirated, fresh media was added and the stainless strip was removed. After a further 48 hours, the samples were taken down, stained with DAPI and analyzed for cell infiltration into the gap.
- hMSCs Osteogenic differentiation and total DNA content on nanofiber meshes. Random and aligned nanofiber mesh samples were coated with GFOGER, collagen, or left uncoated. hMSCs were seeded on circular mesh samples (diameter: 12 mm), cut using biopsy punches (Acuderm, Ft. Lauderdale, FL), at a density of 20,000 cells/cm 2 . Four days after seeding, the hMSC growth media was replaced with osteogenic media, which consists of the hMSC growth media supplemented with 10 nM dexamethasone, 6 mM ⁇ -glycerol phosphate, 50 ⁇ g/ml ascorbic acid 2-phosphate and 50 ng/ml L-thyroxine (Sigma- Aldrich).
- Nanofiber meshes were used to create tubular implants, having a diameter of approximately 5 mm and a length of 13 mm, without any perforations.
- the mesh tubes were coated with 50 ⁇ g/mL GFOGER overnight at 4°C, while control samples were left in PBS.
- Female Sprague-Dawley rats aged 13 weeks were used for this study. Both femurs were stabilized by fixation plates, and 8 mm segmental defects were created in the mid-diaphyses.
- Nanofiber mesh tubes were rinsed with PBS, and implanted in the defect, such that the periphery of the defect was covered with nanofiber mesh with the center empty.
- Radiographs were obtained at 4 and 12 weeks, and ex vivo ⁇ CT analysis was performed at 12 weeks to detect bone formation. Torsional testing was performed on extracted femurs at 12 weeks to test their biomechanical properties. Histological analysis consisted of embedding in glycol methacrylate (GMA), obtaining 5 ⁇ m sections, and staining with H&E.
- GMA glycol methacrylate
- the resulting nanofiber mesh is a porous structure, but due to the multiple fiber layers, the effective pore size appeared to be less than 2 ⁇ m, much less than hMSC dimensions.
- a flat stationary collector was used to obtain random nanofiber meshes with no dominant fiber orientation.
- the mean and median fiber diameters for random meshes were found to be 168.0 nm and 122.8 nm respectively.
- the distribution of fiber diameters indicated that the highest frequency occurred for fibers between 75 nm to 125 nm, with 90% of the fibers between 50-300 nm.
- a rotating mandrel was used to orient the fibers along the direction of rotation of the mandrel surface (Figure 2A).
- the mean and median fiber diameters were observed to be 256.4 nm and 227.4 nm.
- the fibers of the aligned meshes were found to be significantly larger in diameter than those in the random meshes (Mann- Whitney test; p ⁇ 0.001).
- the angle of the fibers with respect to an arbitrary line was measured to quantify fiber alignment (Figure 2B). Though there is a moderate spread in the orientation of the fibers, a preferred fiber direction was observed, with 89% of the fibers between -45 to 45°. For random meshes, this metric was calculated to be only 52%. Using a Z test for proportions, the alignment was found to be significantly higher in the case of aligned meshes (p ⁇ 0.0001). hMSCs seeded on aligned nanofiber meshes exhibited a polarized morphology along the preferred fiber direction, while in the case of random nanofiber meshes, the cells did not display any regular orientation (Figure 3C).
- the collagen-mimetic peptide GGYGGGPC(GPP) 5 GFOGER(GPP) 5 GPC, containing the GFOGER motif, was synthesized by stepwise solid-phase procedures.
- the amino acid sequences adjacent to GFOGER enable formation of a stable right-handed triple helical configuration, which is required for cell adhesion.
- the GFOGER peptide was passively adsorbed on the surface of nanofiber meshes at a concentration of 50 ⁇ g/mL.
- the adsorbed peptide was visualized by coating the meshes with a biotinylated GFOGER peptide and incubating in a fluorescein conjugated NeutrAvidin ® .
- An ELISA was performed to quantify the amount of the GFOGER peptide adsorbed on the nanofibers with varying peptide concentration.
- the saturation curve indicated that the relative surface density increased with increasing peptide concentration, with the surface being saturated at a concentration of approximately 20 ⁇ g/mL.
- the peptide concentration at which the surface is 50% saturated, was calculated to be 3.45 ⁇ g/mL .
- a peptide concentration of 50 ⁇ g/mL was used to ensure saturation of the surface for maximal biologic response.
- GFOGER coated samples displayed higher cell numbers, compared to both uncoated and collagen samples.
- both GFOGER and collagen coated samples had higher cell counts than the uncoated samples.
- Fiber alignment enhanced cell infiltration on uncoated and collagen coated samples. However, this effect was not seen on the GFOGER coated meshes, probably due to the fact that the gap was saturated with cells, even on the GFOGER coated, random meshes.
- Analysis of the middle- third region of the gap revealed that the cell counts displayed the following order for both random and aligned meshes: GFOGER > Collagen > Uncoated. Fiber alignment enhanced the cell infiltration on both uncoated and collagen coated samples, similar to the observation in the entire gap.
- hMSCs were seeded on nanofiber meshes, with a stainless steel strip placed on top, and allowed to attach for 24 hours (Figure 15A).
- the cells were incubated with mitomycin C for 60 minutes, and the strip was removed. After 48 hours of strip removal, the samples were stained with DAPI and analyzed for cell infiltration ( Figure 15B).
- the analysis of both the entire gap and the middle-third gap regions revealed that in the absence of mitomycin C, both GFOGER coating and fiber alignment enhanced cell infiltration, as observed before. With mitomycin C incubation, the overall cell infiltration decreased, indicating that cell proliferation contributed significantly to the infiltration.
- EXAMPLE 3 A NOVEL HYBRID SYSTEM FOR GROWTH FACTOR DELIVERY PROMOTES FUNCTIONAL REPAIR OF LARGE BONE DEFECTS
- the primary objective of this Example was to develop and test a hybrid growth factor delivery system for bone repair that utilizes an electrospun nanofiber mesh and injectable alginate hydrogel.
- the effect of a perforated nanofiber mesh design on bone repair was investigated.
- PCL Poly ( ⁇ -caprolactone) pellets (Sigma- Aldrich, St. Louis, MO) were dissolved in a 90:10 volume ratio of hexafluoro-2-propanol (HFP) :dimethy If ormamide (DMF) (Sigma-Aldrich) to obtain a 12% (w/v) polymer solution. DMF was first slowly added to HFP to prevent excessive heat generation, and mixed well on a stir plate for 5 minutes. The PCL pellets were then added to the solvent solution, and gently stirred for 16-24 hours. The solution was visually inspected to ensure a homogeneous and clear solution.
- HFP hexafluoro-2-propanol
- DMF diimethy If ormamide
- the polymer solution was loaded in a 3 mL syringe (Becton-Dickinson, Franklin Lakes, NJ), and a 22 gauge blunt stainless steel needle (Jensen Global Inc., Santa Barbara, CA) was attached to the syringe end.
- the syringe was mounted on a syringe pump (Harvard Apparatus, Holliston, MA) set at a rate of 0.75 mL/hr.
- the fibers were collected on a flat copper plate (McMaster-Carr, Atlanta, GA) which was placed at a distance of 20-23 cm from the needle end.
- Fibers were electrospun for 5 hours at a voltage of 13-20 kV, supplied by a high voltage power supply (Gamma High Voltage Research, Ormond Beach, FL), to obtain a thick sheet of nanofiber mesh.
- the residual solvent from the meshes was allowed to evaporate by placing them in a dessicator overnight.
- the morphology of the nanofiber meshes was examined using a Scanning Electron Microscope (SEM; Hitachi HTA, Pleasanton, CA) after gold coating using a sputter coater (Quorum Technologies, East Granby, CT).
- the diameter of the fibers were quantified by analyzing the SEM images (at 7000magnification) using a custom MATLAB ® (The MathWorks Inc., Natick, MA) code.
- the nanofiber meshes, as fabricated above, were used to create tubular implants.
- Rectangular samples measuring 13 x 19 mm were cut from a mesh. In some samples, perforations spaced approximately 1.5 mm apart were made in the mesh using a 1 mm diameter biopsy punch (Miltex Inc., York, PA). The rectangular mesh samples were wrapped around a steel mandrel (McMaster-Carr) to form a tube having a diameter of approximately 5 mm and 13 mm length. The overlapping edges of the mesh were secured together by using UV glue (DYMAX Corporation, Torrington, CT), which was cured with a LED spot curing lamp (DYMAX Corporation).
- UV glue DYMAX Corporation, Torrington, CT
- the nanofiber mesh tubes were then rinsed twice in 70% alcohol (VWR, West Chester, PA), and sterilized by submerging in 200 proof ethanol (Sigma-Aldrich) and allowing the ethanol to evaporate overnight. After the samples had dried completely, they were pre-wetted with sterile 70% ethanol for 30 minutes. After aspirating the 70% ethanol, the mesh tubes were rinsed three times with excess phosphate-buffered saline (PBS; Mediatech Inc., Manassas, VA), and placed in ⁇ MEM (Invitrogen) until implantation. Preparation of alginate hydrogel with and without growth factors. Briefly, irradiated
- RGD-modified alginates were prepared by subjecting MVG sodium alginate (FMC Biopolymer) to a 5 Mrad dose of gamma irradiation. This reduces the molecular weight of the polymer leading to a faster degradation rate, which makes it more appropriate for in vivo studies.
- the irradiated alginates were then covalently coupled with G 4 RGDASSP peptide sequences (Commonwealth Biotechnologies, Richmond, VA) at a density of 2 sequences per polymer chain using standard carbodiimide chemistry.
- the resulting RGD-alginates were sterile filtered, lyophilized and stored at -20 0 C.
- the RGD-alginates were reconstituted in ⁇ -MEM to obtain a 2.5% (w/v) solution.
- Lyophilized rhBMP-2 (R&D Systems, Minneapolis, MN) was reconstituted in 0.1% rat serum albumin (RSA; Sigma- Aldrich) made in 4 mM HCl, at a concentration of 200- ⁇ g/mL.
- the alginate solution was then mixed with either the rhBMP-2 solution at a ratio 5:1 (700- ⁇ L alginate solution @2.5% (w/v) with 175- ⁇ L rhBMP-2 @200- ⁇ g/mL or 0.1% RSA).
- hydrogels were prepared without rhBMP-2 by substituting the rhBMP-2 solution with the carrier (0.1% RSA) alone.
- the alginate solutions were allowed to gel in the syringes for 30 minutes at room temperature and then transferred to 4°C.
- the hydrogels were kept at 4°C overnight and used in surgery the following day. Aseptic conditions were maintained in all the above steps, including handling of the exterior of the syringe.
- rhBMP-2 release kinetics.
- RGD-alginate solutions containing rhBMP-2 were cross- linked with a calcium sulfate slurry as above, and immediately injected into custom designed molds containing 4 mm diameter wells.
- the alginate solutions were allowed to gel for 30 minutes at room temperature, producing cylindrical plugs measuring 4 mm in diameter and 8 mm in length. Each cylindrical alginate plug contained 500-ng rhBMP-2. Following a brief rinse in 0.1 M CaCl 2 (Sigma- Aldrich), the samples were incubated at 37°C in 1-mL PBS containing calcium and magnesium ions. At specific time points through day 21, the entire buffer solution was collected and replaced with fresh 1-mL PBS. On days 0 and 21, alginate plugs were dissolved by immersing in 8-mL and 2-mL, respectively, of 2% (w/v) sodium citrate (Sigma-Aldrich) for 30 minutes at room temperature. The amount of rhBMP-2 present in the collected PBS and sodium citrate solution was quantified using an ELISA kit (R&D Systems), following the manufacturer's instruction.
- Nanofiber mesh tubes were placed around the adjacent bone ends such that the tube lumen contained the defect and there was an overlap of 2.5 mm with the native bone ends at each end of the tube.
- 125- ⁇ L pre-gelled 2% alginate with or without 5- ⁇ g rhBMP-2 was injected in the tube lumen using a 22g needle (Jensen Global Inc.). As it is a soft hydrogel, the pre-gelled alginate is ejected from the needle in a continuous thin filament shape and fits compactly inside the tube.
- the tubes used for one of the groups had 1 mm diameter perforations to enhance vascular invasion during the repair process.
- Perforated mesh with alginate containing rhBMP-2 The groups were assigned to the right and left limbs to evenly distribute pairs of groups and obtain a balanced experimental design. After surgery, the animals were allowed to recover and move freely. For pain relief, the animals were injected with 0.03 mg/kg buprenorphine subcutaneously every 8 hours for the first 48 hours and 0.01 mg/kg buprenorphine for the next 24 hours. Radiographs and in vivo micro-computed tomography ( ⁇ CTj images were obtained at 4 and 12 weeks after surgery to evaluate bone healing. The rats were euthanized at 12 weeks and femora were extracted for mechanical testing. Histological analysis was performed on femora extracted at 4 and 12 weeks.
- 2-D radiographs and 3-D in vivo ⁇ CT imaging were taken to qualitatively assess bone regeneration and defect bridging.
- two-dimensional radiographs Faxitron MX-20 Digital, Faxitron X-ray Corp., Wheeling, IL
- in vivo ⁇ CT was performed at the same time points.
- the rats were anesthetized by isoflurane and placed in an in vivo ⁇ CT system (Viva-CT, Scanco Medical, Bassersdorf, Switzerland).
- the femoral defect region was scanned at a 38.5- ⁇ m voxel resolution, a voltage of 55-kVp and a current of 109- ⁇ A.
- the radiotranslucent polysulfone plate does not interfere with ⁇ CT scanning and therefore allows longitudinal evaluation of bone ingrowth.
- VOI volume of interest
- the density map was calculated in the segmented bone volume, and presented as a pseudo color-scaled image. Torsional testing. The freshly extracted femora at 12 weeks were wrapped in gauze moistened with PBS, and stored at -20 0 C. Just before testing, samples were thawed in PBS and the majority of soft tissues adjacent to the bone removed. The ends of the femur were embedded in end blocks using Wood's metal (Alfa Aesar, Wood Hill, MA) and aligned using a custom fixture. The polysulfone plate was then detached from the metal plates to enable loading of the bone.
- Wood's metal Alfa Aesar, Wood Hill, MA
- the potted femur was loaded into holding brackets mounted on a Bose ElectroForce system (ELF 3200, Bose EnduraTEC, Minnetonka, MN) fitted with a 2 Nm torsional load cell.
- the samples were rotated to failure at a rate of 3° per second under displacement control, and the torque and rotation were recorded. Maximum torque was calculated by locating the failure torque, which occurred within the first 15° for bridged defects. Samples that did not bridge displayed a gradual increase in torque and the absence of a sharp failure point, due to soft tissue stretching. For these samples, the failure torque was measured in the first 60° to avoid analyzing the forces generated due to the stretching of soft tissues. Stiffness was calculated by finding the slope of the straight line fitted to the linear portion of the torque-rotation plot before failure.
- Histological analysis One representative sample from each group was selected for histological evaluation at 4 and 12 weeks.
- the extracted femora were fixed in 10% neutral- buffered formalin for 48 hours. They were dehydrated in a series of alcohol solutions of increasing concentrations, infiltrated with methyl methacrylate (MMA), and embedded by polymerizing the MMA.
- vascularity during bone regeneration was investigated at 3 weeks post-surgery by using a modified version of the ⁇ CT-based angiography technique developed in our laboratory.
- a 25 gauge catheter was introduced into the abdominal aorta and 250 units (0.25 mL of 1000 units/mL) heparin were injected.
- the rat hind limb vasculature was cleared with PBS, fixed with 10% neutral buffered formalin and cleared again with PBS using a peristaltic pump (Masterflex, Cole-Parmer).
- the rats were euthanized by an overdose of isoflurane before the formalin perfusion.
- a radiopaque, lead chromate based contrast agent (Flow Tech, Carver, MA) was then injected and allowed to polymerize for at least two hours.
- the femur along with its musculature was excised carefully, fixed in 10% neutral buffered formalin for 48 hours, and decalcified for 2 weeks using a formic acid based solution (CaI-Ex II, Fisher Scientific).
- the samples were rinsed in PBS and stored in 10% neutral buffered formalin until imaging. They were imaged in a ⁇ CT system (Viva-CT, Scanco Medical) at a 21.5- ⁇ m voxel size.
- Two VOIs were defined to analyze the vessels inside the defect and adjacent to the defect. The images were globally thresholded based on X-ray attenuation to segment the contrast-filled vasculature from surrounding tissues.
- Nanofiber mesh tube characterization and placement The nanofibers obtained by electrospinning were observed to be smooth and bead-free (Figure 21A). The fibers ranged in diameter from 51 nm to 974 nm with 82% of the fibers between 50 nm and 150 nm. The mean and the median fiber diameter were found to be 154 nm and 107 nm respectively. After 5 hours of electrospinning, the mesh was found to be approximately 300-400 ⁇ m thick. This thickness was sufficient to provide a bending stiffness that prevented collapse of the mesh in solution. The thick nanofiber meshes were able to be wrapped tightly around a steel mandrel, and glued to form a tube ( Figures 21B and C).
- Radiographs Two-dimensional radiographs were taken at 4 and 12 weeks for qualitative assessment of bone healing (Figure 22). Radiographs at the early time point of 4 weeks indicated that Groups I & II (Table 1) specimens had small amounts of bone formation, originating from the cut native ends and extending somewhat along the periphery. Group I samples were implanted with a nanofiber mesh tube alone, whereas Group II contained, in addition, alginate hydrogel inside the mesh tube. On the other hand, samples from Groups III and IV, in which 5- ⁇ g rhBMP-2 was delivered within alginate, demonstrated significant infiltration of mineralized tissue throughout the defect. Group IV specimens that were implanted with the perforated mesh tube exhibited the most robust mineralization.
- Group IV demonstrated the highest bridging rate (5/8) at the 4 week time point, whereas the remaining 3/8 defects were nearly bridged.
- Group III had none bridged, but 3/6 defects were nearly bridged.
- Groups I and II had still not achieved osseous union in any specimen, with most of the bony tissue formed on the periphery. In contrast, all specimens in Groups III and IV were completely bridged with densely packed bone.
- Group IV implanted with the perforated mesh, had significantly more (b; p ⁇ 0.05) bone formation than Group III, which contained the mesh tubes without holes.
- Group III which contained the mesh tubes without holes.
- Group III 37.65 ⁇ 2.22 mm 3
- Group IV (20.02 ⁇ 2.96 mm 3 ).
- Groups I (3.96 ⁇ 1.40 mm 3 ) and II (2.09 ⁇ 0.80 mm 3 ) had significantly less bone accumulation during the same period.
- the density of the newly formed bone within the defect was also calculated at 4 and 12 weeks (Figure 23C).
- Groups I and II contained higher density bone than Groups III and IV (b and c respectively; p ⁇ 0.05).
- Group IV samples demonstrated a density higher than Group III, at both 4 and 12 weeks (b; p ⁇ 0.05). There was a significant increase in density with time for all groups from 4 to 12 weeks.
- the newly formed bone was observed to be a combination of immature woven bone and mature lamellar bone. There was good continuity of the newly mineralized matrix with the native bone ends.
- Group IV demonstrated the presence of a higher amount of lamellar bone, better integration at the native bone interface and development of marrow-like tissue. The dark areas correspond to undegraded alginate and the partially degraded mesh.
- the higher magnification images of Groups III and IV indicated the presence of osteocytes embedded in lacunae and osteoblasts lining the new bone surfaces (Figure 25B). Histological analysis performed at 4 weeks revealed no evidence of cartilage tissue formation or endochondral ossification, indicating direct, intramembranous bone formation within the alginate gel (data not shown).
- the density maps obtained from the ⁇ CT indicate good correlation with histology sections (data not shown).
- Group IV appeared to contain higher density mineralized tissue, which was distributed in a tubular pattern, similar to that of native cortical structure.
- ⁇ CT-based angiography Animals implanted with the rhBMP-2 containing Groups III and IV were euthanized at 3 weeks post-implantation, and their hind limb vasculature perfused with a radiopaque contrast agent. The femur and the surrounding soft tissues were imaged using ⁇ CT to quantify vascular ingrowth at an early time point preceding bone regeneration. Contours were drawn to define two VOIs.
- the first VOI included only the volume inside the defect region, whereas the second contained both the defect and the periphery of the defect, termed the total VOI ( Figure 26).
- the analysis of the vasculature revealed the presence of vessels, both inside and outside the defect. The majority of the vascularity was observed in the periphery, as indicated by the significantly larger vessel volume in the total VOI (a; p ⁇ 0.001). There were no significant differences in vascular volume between Groups III and IV, in either the defect or the total VOI.
- This Example demonstrates a novel growth factor delivery technique for the functional repair of large bone defects using an electrospun nanofiber mesh tube and alginate hydrogel.
- Tubular scaffolds constructed from nanofiber meshes were placed around segmental defects.
- Alginate hydrogel containing 5- ⁇ g rhBMP-2 was injected into the tubes and constrained within the defect site by the mesh tube. The above results demonstrate that this technique results in substantial bone formation and complete defect bridging.
- Samples implanted with both perforated mesh tube and rhBMP-2 containing alginate had statistically equivalent biomechanical properties to those of intact age-matched femora, indicating functional restoration of the limb function.
- scaffolds proposed for bone reconstruction are structural scaffolds designed to support in vivo loads and provide a three-dimensional framework for cell attachment. They are examples of “hard scaffolds”, usually made from slowly hydrolyzing polymers or ceramics with unpredictable degradation. Though they provide a structure for tissue growth, it is difficult to fine-tune their degradation rate to match the rate of tissue formation.
- the use of structural scaffolds also precludes the use of the intramedullary pin for limb fixation, a technique frequently used by orthopedic surgeons.
- the regular geometric shape of these scaffolds made them unsuitable to be placed inside fractures, which usually have irregular edges.
- Thin scaffold membranes have also been used for bone repair in a procedure termed guided bone/tissue regeneration.
- the membranes are positioned on the periosteal surface to provide a structure for bone formation. It has been argued that while 3-D scaffolds support the ingrowth of cells and tissue, the 2-D membranes may also protect the defect from soft tissue ingrowth and guide cell migration from the periosteum. Since the membranes are placed on the periphery of the defect, they retain space for bone deposition throughout the defect. However, when a large mass of bone is lost, repopulating the entire defect with cells would be a challenge due to the presence of a large void. Hydrogels are a class of highly hydrated materials that enable cellular and tissue infiltration with relative ease.
- Alginate hydrogels are an example of such a "soft scaffold” that can deployed using minimally invasive procedures, conform to the shape of the defect and be manipulated by cells during tissue regeneration. In addition, they can be used for sustained delivery of osteoinductive growth factors, a typical requirement for healing large defects.
- the primary concern with hydrogels is their inadequate mechanical stiffness, which causes them to deform easily under load.
- nanofiber mesh tubes prevent soft tissue invagination into the defect and create a space for tissue regeneration. In addition, they potentially guide the migration of progenitor cells along the periosteal surface, and retain the osteogenic factors within the defect site.
- nanofiber mesh tubes alone or in presence of alginate hydrogel without rhBMP-2, were not sufficient to bridge the 8 mm segmental defects in rat femora. Without the presence of the osteoinductive protein, the center was only sparsely populated by cells and bony tissue capped the ends of the defect. The above results illustrate higher bone formation with the sustained release of rhBMP-
- the perforations may enhance invasion of vascularity or improve osteoprogenitor cell migration from the surrounding soft tissues into the defect region.
- rhBMP-2 The lack of differences in vascular regrowth due to the perforations suggests that the acceleration of bone formation was due to improved osteoprogenitor cell migration into the defect.
- the current clinical technique for rhBMP-2 delivery involves soaking a collagen sponge with rhBMP-2 solution, which primarily relies on the adsorption of the protein to collagen.
- Numerous sustained delivery systems are being currently developed from natural and synthetic materials for reducing the high rhBMP-2 dose required clinically.
- the hybrid alginate/nanofiber mesh system creates an environment conducive for bone regeneration.
- the 5- ⁇ g dose utilized in this study is in the lower range of what has been reported (2-20 ⁇ g) in similar models. For example, in a 8 mm rat segmental defect model, 20- ⁇ g of rhBMP-2 delivered on inactive demineralized bone matrix was required for defect bridging.
- Nanofiber mesh tube fabrication and alginate preparation Nanofiber meshes were made by electrospinning and formed into tubes as described in Example 4. Briefly, a 12% (w/v) solution of poly ( ⁇ -caprolactone) (PCL) was made by dissolving the polymer in a 90:10 volume ratio of hexafluoro-2-propanol:dimethylformamide. The following parameters were used during electrospinning: flow rate: 0.75 mL/hr; voltage: 13 kV; collector distance: 17 cm; needle gauge: 22. Fibers were collected for 6 hours to obtain nanofiber meshes having a thickness of approximately 300-400 ⁇ m.
- PCL poly ( ⁇ -caprolactone)
- Perforations measuring 1 mm in diameter and spaced approximately 1.5 mm apart were made in rectangular 13 x 19 mm mesh samples using a biopsy punch.
- the mesh samples were wrapped around a steel mandrel and glued using UV glue (DYMAX Corporation, Torrington, CT) to form hollow tubular implants having a diameter of approximately 5 mm and 13 mm length.
- Alginate hydrogels encapsulating 33.33 ⁇ g/mL rhBMP-2, were prepared in 1 mL syringes at a concentration of 2% (w/v) by crosslinking the alginate with calcium sulfate slurry (Chapter 5).
- the hydrogels were made in the form of cylindrical plugs ( ⁇ 5 mm; 9 mm length) by using a custom built mold.
- the collagen scaffolds were obtained from a fibrous collagen sheet (average pore size 61.7 ⁇ m, 93.7% pore volume, Kensey Nash, Exton, PA).
- Group II implants consisted of a cylindrical scaffold ( ⁇ 5 mm; 9 mm length) made from alginate containing 5 ⁇ g rhBMP-2.
- perforated nanofiber mesh tubes were placed around the defect, and injected with 5 ⁇ g rhBMP-2-containing alginate using a 22g needle.
- Radiographs and ⁇ CT images of 8-10 samples per group were obtained at 4 and 12 weeks to assess bone formation. Radiographs were also taken at 2 weeks to assess early differences between groups.
- ⁇ CT data was processed to obtain mean density, connectivity density and temporal changes in volume of the regenerated bone. Based on the two dimensional scan slices, an appropriate threshold was selected at each time point to detect new bone formation.
- Torsional testing was performed on extracted femurs at 12 weeks using a Bose ElectroForce system (ELF 3200, Bose EnduraTEC, Minnetonka, MN) to test their biomechanical properties. Maximum torque and failure angle were measured at the failure point from the torque-rotation data, and the work to failure was calculated using these values. Torsional stiffness was calculated by fitting a straight line to the linear portion of the curve before failure. Histological analysis was also performed at 12 weeks by embedding decalcified femurs in glycol methacrylate (GMA) and staining 5 ⁇ m sections with H&E. Data analysis. Data are presented as mean ⁇ standard error of mean (SEM).
- the central VOI corresponds to an interior cylindrical volume that captures the defect.
- the total VOI contains the entire volume of mineralization, in and around the defect, including the irregular bone formation outside the defect.
- the alginate delivery groups (Groups II and III) had significantly more bone formation than the collagen delivery groups (Group I) ( Figure 29A).
- the central VOI analysis at 12 weeks revealed a significant increase in bone formation within the defect region associated with use of the nanofiber mesh tube (Group III).
- This Example quantitatively compares a novel hybrid BMP delivery system to the current clinical standard for challenging bone defect repair.
- rhBMP-2 delivered at the same dose via alginate hydrogel resulted in enhanced bone formation as early as four weeks.
- the addition of a perforated nanofiber mesh to spatially retain the BMP hydrogel resulted in an increased mineral accumulation between 4 and 12 weeks, leading to enhanced bone ingrowth into the defect region at 12 weeks, compared to all other groups.
- biomechanical function was significantly improved following treatment with the hybrid nanofiber mesh/hydrogel delivery system, compared to collagen matrix delivery.
- the amount and distribution of newly formed bone was influenced by the BMP delivery method. At two weeks, there appeared to a greater amount of mineral deposition with the collagen matrix delivery (Group I), compared to the alginate delivery (Groups II and III). However, by 4 weeks, the alginate groups demonstrated higher bone volume than the collagen matrix delivery, and this trend continued at 12 weeks. These results indicate that the kinetics of bone repair is dependent on the delivery system. This suggests that, whereas a majority of rhBMP-2 was released from the collagen matrix by 2 weeks, protein retention at the defect site beyond 2 weeks was improved with alginate hydrogel delivery. This is consistent with other studies that report that less than 5% of rhBMP-2 is retained within a collagen sponge by 2 weeks in vivo.
- the nanofiber mesh tube may function by constraining and therefore compacting the BMP-containing hydrogel, which could improve the local retention at later time points.
- the density of the mineralized tissue was lowest in Group III. This suggests that new, immature bone is still being laid down in Group III specimens at later time points, which lowers the overall bone density in this group.
- the density obtained from the ⁇ CT is the actual density of the bone tissue, and not the apparent density of the entire bone. T his would explain the lower tissue density in Group III specimens, even though the regenerated bone in these specimens appeared denser in the radiographs.
- Connectivity density is a parameter used to analyze trabecular bone, and is a measure of the number of trabeculae in a unit volume.
- Connectivity density is a parameter used to analyze trabecular bone, and is a measure of the number of trabeculae in a unit volume.
- BMPs the direct mineralization induced by BMPs is initiated at discrete locations, which then develop connections as the mineralization progresses.
- the connectivity obtained from the ⁇ CT analysis of regenerating bone is a measure of these connections.
- Group III specimens demonstrated the highest connectivity density, compared to the other two groups, indicating a more interconnected bone structure, perhaps because of the spatial guidance provided by the alginate hydrogel and the containment effect of the nanofiber mesh tube.
- Containment due to the mesh tube may have facilitated connection of the mineralized nodes formed during intramembranous ossification. There were no differences in connectivity density at 12 weeks, and the values were uniformly low. This could be due to the fact that as the defect fills with bony tissue, the discrete bone initiation centers merge to form a continuous volume, which would reduce the connectivity density.
- Example 3 The results of this Example demonstrate that delivery of rhBMP-2 via the hybrid nanofiber mesh/alginate system results in improved bone repair compared to delivery on a collagen matrix, the technique currently used by orthopaedic surgeons. Delivery of BMPs on collagen matrix has been shown to result in significant initial burst release, in the range of 40- 90%. In the in vitro release of Example 3, approximately 10% of the encapsulated rhBMP-2 remains attached to alginate after 3 weeks, though the amount released in the media was negligible at this time point. This suggests that the protein binds to the alginate matrix and may be available to invading progenitor cells at later time points, which would mean that the function of the alginate hydrogel is more to improve long term availability than sustained release.
- binding of the protein by alginate may be advantageous by protecting it from degradation.
- the nanofiber mesh tube may aid in containing the exogenous growth factor within the defect region.
- the delivery of BMPs on collagen matrix has been tested in a number of animal models of large bone defect repair; however high protein doses are typically required. For instance, it has been reported that delivery of 10 ⁇ g rhBMP-2 (double the dose used in this study) resulted in bony bridging of a 6 mm rat segmental defect, which is less challenging than the 8 mm defect utilized in this Example.
- the injection of 5 ⁇ g rhBMP-2 in this Example is equivalent to a 20 ⁇ g/kg dose, which is around 1/5* the clinical dose used in humans.
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- 2009-11-24 WO PCT/US2009/065754 patent/WO2010060090A1/en active Application Filing
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CN107260293A (zh) * | 2011-03-29 | 2017-10-20 | 史密夫和内修有限公司 | 具有髓内支撑物的骨折固定系统 |
CN107260293B (zh) * | 2011-03-29 | 2021-06-29 | 史密夫和内修有限公司 | 具有髓内支撑物的骨折固定系统 |
Also Published As
Publication number | Publication date |
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EP2367595A4 (de) | 2014-11-19 |
US20100168771A1 (en) | 2010-07-01 |
US9452049B2 (en) | 2016-09-27 |
EP2367595A1 (de) | 2011-09-28 |
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