WO2015171880A1 - Compositions de modulation de l'angiogenèse à libération prolongée et méthodes pour l'induction et la modulation de l'angiogenèse - Google Patents

Compositions de modulation de l'angiogenèse à libération prolongée et méthodes pour l'induction et la modulation de l'angiogenèse Download PDF

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WO2015171880A1
WO2015171880A1 PCT/US2015/029666 US2015029666W WO2015171880A1 WO 2015171880 A1 WO2015171880 A1 WO 2015171880A1 US 2015029666 W US2015029666 W US 2015029666W WO 2015171880 A1 WO2015171880 A1 WO 2015171880A1
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Prior art keywords
angiogenesis
microparticles
human
placental
extract
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PCT/US2015/029666
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English (en)
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Peter S. MCFETRIDGE
Marc C. MOORE
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The University Of Florida Research Foundation, Inc.
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Priority to US15/309,306 priority Critical patent/US10300091B2/en
Priority to CA2948348A priority patent/CA2948348A1/fr
Priority to JP2016566709A priority patent/JP2017520517A/ja
Priority to KR1020167034051A priority patent/KR20160147055A/ko
Priority to AU2015255907A priority patent/AU2015255907A1/en
Priority to EP15789993.1A priority patent/EP3139937A4/fr
Publication of WO2015171880A1 publication Critical patent/WO2015171880A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3808—Endothelial cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/602—Type of release, e.g. controlled, sustained, slow
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/62—Encapsulated active agents, e.g. emulsified droplets

Definitions

  • Angiogenesis enables the formation of blood vessels in physiological and pathological states ranging from wound healing to cancer.
  • Angiogenesis modulation is both location and stimuli dependent, and each instance may involve a unique combination of regulatory molecules.
  • embodiments of the present disclosure provide a human placental extract compositions comprising the human placental extract, sustained-release angiogenesis-modulating compositions, methods of inducing angiogenesis, and anti-inflammatory compositions and methods.
  • the present disclosure provides a composition including a human placental extract coupled to biodegradable microparticles, such that the human placental extract is released from the
  • the human placental extract is obtained from a human placental sample where blood and solids have been substantially removed from the extract and the extract includes placental proteins including cytokines and growth factors that were present in the placental sample.
  • Embodiments of methods for inducing angiogenesis of the present disclosure include contacting cells with a sustained release angiogenesis-modulating composition, where the sustained release angiogenesis-modulating composition includes biodegradable microparticles coupled to the human placental extract of the present disclosure such that the human placental extract is released from the microparticles over a period of time after exposure of the microparticles to cells in vitro or in vivo.
  • the method further includes coupling the sustained release angiogenesis- modulating composition to a biomaterial to induce vascularization of the biomaterial.
  • the present disclosure also provides methods of reducing inflammation in a subject by administering the human placental extract of the present disclosure or the sustained release/human placental extract composition of the present disclosure to a subject in need of treatment for inflammation.
  • FIGS. 1 A-1 K illustrate the formation of a human placental extract (hPE) (also referred to herein as a human placenta matrix or hPM), characterization of hPE thin films, and characterization of angiogenic networks formed on hPE thin films.
  • FIG. 1 A illustrates an embodiment of steps for obtaining hPM by homogenization of placental ECM followed by urea solubilization and dialysis.
  • FIGS. 1 B-1 C are SEM images showing the surface morphology of the hPE
  • FIGS. 1 D-1 E are SEM images of vasculogenic network formation when HUVECS were seeded at 4x10 4 cells/cm 2 onto hPm thin films and cultured for 3 days.
  • FIG. 1 F illustrates Rhodamine Phalloidin ("red”-shown as grey branching pathways) and DAPI ("blue”-shown as lighter gray spots within branching pathways) showing branched cell filopodia during angiogenic sprouting after 1 day on placenta extract.
  • FIG. 1 G illustrates Rhodamine Phalloidin ("red”-shown as grey branching pathways) and DAPI ("blue”- shown as lighter gray spots within branching pathways) showing a maturing angiogenic network with extensive cell cording after 3 days.
  • FIG. 1 G illustrates Rhodamine Phalloidin ("red”-shown as grey branching pathways) and DAPI (“blue”- shown as lighter gray spots within branching pathways) showing a maturing angiogenic network with extensive cell cording after 3 days.
  • FIG. 1 H shows Calcein ("green”-shown as grey branches) and DAPI ("blue”— shown as lighter grey spots within branches) stained HUVECs during the initial stages cell cording and angiogenic network formation after 1 day on placenta extract.
  • FIG. 1 1 illustrates DAPI ("blue”— shown as grey dashed pathway) staining showing cell cording of HUVECS after 3 days on placenta extract.
  • FIG. 1 J illustrates HUVECs seeded onto a tissue culture plate at 4x10 4 cells/cm 2 and cultured in endothelial cell medium for 3 days.
  • FIG. 1 K illustrates formation of angiogenic networks by HUVECs seeded at 4x10 4 cells/cm 2 onto placenta extract that was adhered to the surface of a tissue culture plate at 100 ⁇ _ PE/cm 2 and then cultured in endothelial cell medium for 3 days.
  • FIGS. 2A-2C illustrates biochemical analysis of hPE and genetic analysis of HUVECs seeded on hPE.
  • FIG. 2A is a bar graph illustrating cytokines analysis as performed using a sandwich-based human angiogenesis antibody array; data was normalized on a scale ranging from negative control values (0 %) to positive control values (100 %) (data are representative of three biological replicates).
  • FIGS. 2B and 2C are a bar graphs illustrating the normalized spectral abundance factor (%) of immune related (2B) and angiogenesis related (2C) BM related proteins as determined using LC- MS/MS.
  • Fibrinogen normalized spectral abundance factor value is given as the sum of FGA and FGG values
  • Laminin is given as the sum of LAMA2, LAMA4, LAMA5, LAMB1 , LAMB2, LAMB3, and LAMC1 values.
  • FIG. 2D illustrates genetic analysis performed on HUVECS seeded for 3 days onto 100 ⁇ _ PE/cm 2 at a density of 80,000 cells/cm 2 .
  • Data are representative of four biological replicates. P-values are calculated using a Student's t-test of the replicate 2 ⁇ (- Delta Ct) values for each gene in the control group and treatment groups.
  • FIGS. 3A-3D illustrate in vitro angiogenic networks formed on hPE and Matrigel coated tissue culture flasks.
  • FIG. 3A illustrates calcein stained HUVECS on hPE and Matrigel at variable cell seeding densities after 1 d, 3d, and 5d.
  • the rate of angiogenic network maturation defined as the time until maximum number of tubules/mm 2 , was modulated in hPE samples by varying cell seeding densities.
  • Quantitative analysis revealed that at 40,000 cells/cm 2 angiogenic networks took until day 3 to reach their maximum tubule density (tubules/mm 2 ), but at 80,000 cells/cm 2 networks reach their maximum tubule density in 1 day (data not shown).
  • FIG. 3B illustrates that WPMY-1 myofibroblasts did not have angiogenic formations when seeded on placenta extract (FIG. 3B.i) but did when seeded on Matrigel (FIG. 3B.N).
  • FIG. 3C illustrates quantitative image analysis (masking) to determine angiogenic network parameters including mean tubule length (mm), tubule density, number of branch points, number of meshes, and tubule width.
  • 3C is a series of bar graphs showing a comparison of hPE and Matrigel® induced vasculogenic network parameters, showing that by day 1 , in samples seeded at 80,000 cells/cm2, Matrigel samples had reached their maximum mean tubule length, tubule density, branch points, and number of meshes, with apoptotic ball formation by day 3, while the hPM network parameters were more stable over 5 days of culture.
  • FIGS. 4A-4C illustrate screening of anti-antiangiogenic tumor suppressive protein
  • FIG. 4A illustrates HUVECS seeded onto hPE, Matrigel, and control culture flasks (not coated) for 1 day with TSP-1 added to the culture media were then stained using Calcein AM. Scale Bars a, 200 ⁇ .
  • the graph of FIG. 4B shows, in hPE-coated flasks, mean total tubule length [mm] and mean number of branch points both decreased linearly with increasing TSP-1 concentrations.
  • FIG. 4C illustrates a comparison of normalized percent reduction of angiogenic network coverage area; hPE-coated culture plates had significantly higher sensitivity to TSP-1 concentration than Matrigel-coated culture plates, with R 2 values being 0.97 and 0.36, respectively.
  • FIGS. 5A-5C illustrate in vitro angiogenesis on 3D tissue constructs.
  • FIG. 5A is a schematic drawing illustrating placental derived cells, scaffolds, and cytokines, to induce angiogenesis in vitro in a hPE-soaked (human umbilical vein) bioscaffold after seeding and culturing for 3 days.
  • FIG. 5B illustrates HUVEC seeded tissue scaffolds without hPE soaking did not form angiogenic networks.
  • FIG. 5C shows a series of representative images of hPE-soaked bioscaffolds illustrating occurrences of both sprouting and intussusceptive mechanisms of angiogenesis after 3 days of culture.
  • intussusceptive angiogenesis were observed (FIGS. 5C.iii.-5C.iv.), whereas at a density of 60,000 cells/cm 2 (FIGS. 5C.v.-5C.vi.) angiogenic tubules formed via intussusception.
  • FIGS. 6A-6B represent illustrations of in vivo angiogenesis in hPE-incubated bioscaffolds.
  • FIG. 6A is a schematic drawing illustrating decellularized HUV scaffolds incubated in PE, Matrigel, or phosphate buffered saline (control) for 2 hr prior to implantation into a rat model between the fascia and muscle layers.
  • FIG. 6B shows a series of images illustrating scaffolds removed for analysis after 5 d implantation. Significantly more fibrotic capsule formation occurred in control and Matrigel- incubated bioscaffolds in comparison to hPE incubated scaffolds (FIGS. 6B.i.-6B.iii.).
  • FIG. 6B.iv.-6B.vi. Brightfield images taken through the frontal plane of the semi-translucent bioscaffold sheets show that in comparison to controls, Matrigel and hPE-incubated scaffolds (FIGS. 6B.iv.-6B.vi.) had significantly improved capillary network formation, with the most mature capillary beds in hPE scaffolds, showing formation of vascular structures with connected arteriole to capillary to venule blood flow (FIG. 6B.vi. (circled in dashed line)). Hematoxylin and Eosin staining revealed that hPE-incubated scaffolds (FIGS 6B.vii.-6B.vi.) had the most scaffold remodeling in comparison to control and Matrigel scaffolds. Control scaffolds (FIG.
  • Matrigel-incubated scaffold had slightly less cell migration from the ablumen surface of the HUV in comparison to hPE-scaffolds (FIGS. 6B.vii.-6B.ix.); when compared to controls, Matrigel-incubated scaffolds also had less uniform cell distribution and less scaffold remodeling than hPE-incubated scaffolds, which had new collagen fiber orientation and a more uniform cell distribution.
  • FIGS. 7A-7E illustrate an embodiment for formation of angiogenic networks on human umbilical vein scaffolds (HUV) cultured using dynamic cell-culture conditions.
  • HUV human umbilical vein scaffolds
  • FIGS. 7A and 7B tubular HUV scaffolds were incubated in placenta extract for 2 hours before cell-seeding, and constructs were cultured for 5 days in a dual-perfusion bioreactor under standard cell culture conditions. Cells remained on the lumen of the scaffold and did not migrate (FIG. 7C). Cell-cording, an initial stage of tubule formation, was sporadic (FIG. 7D and 7E).
  • FIG. 8 illustrates an original image (on the left) and a zoom of its area (on the right) of tubes and cell network of growth on PE.
  • the zoomed image shows how each tubule (dotted line) was identified and measured.
  • the circlets on the right image represent the branch points, which are identified by numbered dots on the image, pointed by an arrow.
  • Each hexagonal arrangement of the tubules identifies a mesh (dotted circle).
  • FIGS. 9A-B are a series of images (FIG. 9A) and a bar graph (FIG. 9B) illustrating the angiogenic potential of PE.
  • the panels in FIG. 9A show cell morphologies of microvessel tubules formed by HUVECs seeded onto PE and cultured for 1 , 3 and 5 days compared with HUVECS seeded onto a tissue culture plate.
  • the capillary network started forming at day 1 and it reached a more mature configuration at day 3 (see enlargement, bottom right). On day 5 the network regressed: the number of meshes decreased, and some isolate cells (white dots) were present.
  • FIG. 9B shows the morphological and topographic features of tubule-like network formed on PE after 1 , 3 and 5 days of culture. Tubule length, number of BPs and of meshes between day 1 , 3 and 5 were compared. A statistical difference (indicated with asterisks) in all three parameters was found between cells of Day 1 and of Day 5. The statistical analysis was performed with a double tailed t-test with unequal variance at p ⁇ 0.05.
  • FIGS. 10A-9B illustrates the cell morphologies of a microvessel network formed by HUVECs seeded onto PE and cultured for 5 days compared with HUVECs seeded onto a tissue culture plate (control). Controls are shown in the inset images in the top right corners of the images in FIG. 10A ; from top left: only one inoculation of PE on day 1 , two inoculations on day 1 and 3 and three inoculations on day 1 , 3 and 4. It is possible to notice that increasing the number of inoculations (from 1 to 3), the capillary network evolved to a more mature and long lasting configuration. Tubule length, number of BPs and of meshes between day 1 and day 5 are compared in the histogram in FIG.
  • FIG. 1 1 shows a series of images illustrating HUVECs cultured for three days with PE stored in a humidified 6% C02 incubator at 37°C from 1 up to 20 days. The numbers on each image indicates the incubation time (in days). PE maintained its capability to induce angiogenesis even after 15 days of incubation.
  • FIGS. 12A-12B illustrate the characteristics of gelatin microparticles.
  • I n F I G . 1 2A , th e top row shows SEM images of a blank particle with a size of 40 ⁇ approximately. It has a regular shape and a smooth surface.
  • the bottom row of Fig. 12A shows SEM images of a PE- loaded particle. The change in the surface morphology and the increase in size are likely due to PE sorption.
  • the graph in Fig. 12B shows the size distribution analysis of gelatin microparticles by light microscopy.
  • FIGS. 14A-14B illustrate protein release kinetics of blank and loaded microparticles.
  • FIG. 14B illustrates in vitro difference in percent of release between loaded and blank microparticles. The first peak indicates the release of PE from the surface of the particles whereas the second one is likely due to bulk erosion.
  • FIG. 15 illustrates delivery of PE using gelatin microparticles.
  • the images show the response of HUVECs, seeded with a density of 20,000 cell/cm 2 , to PE-loaded microparticles after 5 days of culture (D5) compared with HUVECs seeded with blank microparticles (control).
  • the arrows point cells in different conditions: illustrating the difference in shape. After 5 days of culture with PE-loaded particles some sprouts were present but network formation was not observed. The bigger spheres are microparticles.
  • FIGS. 16A-16G illustrate the effect of a continuous delivery of hPE on HUVECs.
  • FIG. 16A illustrates the control with HUVECs cultured at 20,000 cells/cm 2 in Angiogenic media;
  • FIG. 16B-16D illustrate HUVECs with, respectively, a single inoculation of hPM occurring on day 1 (FIG. 16B), two inoculations on day 1 and 3 (FIG. 16 C), and three inoculations on days 1 , 3, and 5 (FIG. 16D).
  • FIGS. 16E-16G are bar graphs representing the quantification of angiogenesis performed on the images in FIGS. 16B-16D after the staining. Results are shown as the average between
  • FIGS. 17A-17F illustrate evaluation of microparticles.
  • FIGS. 17A-17C illustrate optical microspoce images for three protocols evaluated: single (2 min) homogenization (FIG. 17A), dual (2 min/1 min) homogenization (FIG. 17B), and dual (2 min/20sec) homogenization (FIG. 17C).
  • the histograms of FIGS. 17D-17F illustrate the distribution of the size in microns for the particles formed in each of the 3 protocols, respectively (each batch prepared in triplicate).
  • FIGS. 18A-18F are scanning electronic microscope (SEM) images of PLGA microparticles loaded with hPM for each of the three protocols: single (FIGS. 18A, 18D), 2/1 dual (FIGS. 18B, 18E), and the 2/20 dual (FIGS. 18C, 18D).
  • FIGS. 18A-18C illustrate the microparticle shape
  • FIGS. 18D-18F illustrate the surface porosity.
  • FIGS. 19A and 19F provide a table (FIG. 19A) of loading efficiency and a graph (FIG. 19B) of the evaluation of the release rate from the particles for each protocol (P2: single, P3: 2/1 dual, and P4 2/20 dual).
  • the curves represent the cumulative release rate up to 21 days for each protocol.
  • FIG. 20 illustrates SDS page analysis of supernatant of microparticles.
  • the images is of the gel after staining showing qualitative protein content in supernatant of BSA-loaded and hPM-loaded microparticles and of the relative initial content loaded during microparticle preparation.
  • FIG. 21 is a series of images illustrating endothelial cells stained with Calcein AM after 7, 14, 21 , and 28 days of culture to evaluate the difference between angiogenic network stability when pure hPM is added to Alginate matris (top row) vs. sustained release of hPM from PLGA microparticles embedded in the matrix (second row).
  • the bottom row shows two controls: one with embedded blank PLGA microparticles in alginate matrix and the second with no microparticles.
  • the HUVECS maintained a generally circular shape throughout all time points for both controls.
  • Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, biochemistry, molecular biology, biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
  • polypeptide and protein refer to a polymer of amino acids of three or more amino acids in a serial array, linked through peptide bonds.
  • polypeptide includes proteins, protein fragments, protein analogues, oligopeptides, and the like.
  • polypeptides contemplates polypeptides as defined above that are encoded by nucleic acids, produced through recombinant technology (isolated from an appropriate source such as a bird), or synthesized.
  • polypeptides further contemplates polypeptides as defined above that include chemically modified amino acids or amino acids covalently or non-covalently linked to labeling ligands.
  • polynucleotide oligonucleotide
  • nucleic acid sequence include, but are not limited to, coding sequences (polynucleotide(s) or nucleic acid sequence(s) which are transcribed and translated into polypeptide in vitro or in vivo when placed under the control of appropriate regulatory or control sequences); control sequences (e.g., translational start and stop codons, promoter sequences, ribosome binding sites,
  • polyadenylation signals polyadenylation signals, transcription factor binding sites, transcription termination sequences, upstream and downstream regulatory domains, enhancers, silencers, and the like); and regulatory sequences (DNA sequences to which a transcription factor(s) binds and alters the activity of a gene's promoter either positively (induction) or negatively (repression)).
  • regulatory sequences DNA sequences to which a transcription factor(s) binds and alters the activity of a gene's promoter either positively (induction) or negatively (repression)
  • gene refers to nucleic acid sequences (including both RNA or DNA) that encode genetic information for the synthesis of a whole RNA, a whole protein, or any portion of such whole RNA or whole protein.
  • a “gene” typically refers to a hereditary unit corresponding to a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a characteristic(s) or trait(s) in an organism.
  • gene product refers to RNAs or proteins that are encoded by the gene.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (e.g., not worsening) of disease, delaying or slowing of disease progression, substantially preventing spread of disease, amelioration or palliation of the disease state, and remission (partial or total) whether detectable or undetectable.
  • stabilization e.g., not worsening
  • substantially preventing spread of disease amelioration or palliation of the disease state
  • remission partial or total
  • “treat”, “treating”, and “treatment” can also be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • prophylactically treat or “prophylactically treating” refers completely, substantially, or partially preventing a disease/condition or one or more symptoms thereof in a host.
  • delaying the onset of a condition can also be included in “prophylactically treating”, and refers to the act of increasing the time before the actual onset of a condition in a patient that is predisposed to the condition.
  • administration is meant introducing a compound of the present disclosure into a subject; it may also refer to the act of providing a composition of the present disclosure to a subject (e.g., by prescribing).
  • organism refers to any living entity in need of treatment, including humans, mammals (e.g., cats, dogs, horses, mice, rats, pigs, hogs, cows, and other cattle), birds (e.g., chickens), and other living species that are in need of treatment.
  • the term “host” includes humans.
  • the term “human host” or “human subject” is generally used to refer to human hosts.
  • the term “host” typically refers to a human host, so when used alone in the present disclosure, the word “host” refers to a human host unless the context clearly indicates the intent to indicate a non-human host.
  • Hosts that are "predisposed to" condition(s) can be defined as hosts that do not exhibit overt symptoms of one or more of these conditions but that are genetically, physiologically, or otherwise at risk of developing one or more of these conditions.
  • expression describes the process undergone by a structural gene to produce a polypeptide. It is a combination of transcription and translation. Expression generally refers to the "expression” of a nucleic acid to produce a polypeptide, but it is also generally acceptable to refer to "expression" of a polypeptide, indicating that the polypeptide is being produced via expression of the corresponding nucleic acid.
  • Angiogenesis is a physiological process involving the growth of new blood vessels.
  • Angiogenesis is an important part of biological processes, such as growth and development, wound healing, embryogenesis, and the like. Excessive angiogenesis can occur when diseased cells produce abnormal amounts of angiogenic growth factors, overwhelming the effects of natural angiogenesis inhibitors. Imbalances between the production of angiogenic growth factors and angiogenesis inhibitors can cause improperly regulated growth or suppression of vascular vessels. Angiogenesis-dependent or related diseases result when new blood vessels either grow excessively or insufficiently.
  • the angiogenesis related disease can include diseases such as, but not limited to, cancer, precancerous tissue, tumors, cardiac infarction, and stroke.
  • Excessive angiogenesis can include: cancer, diabetic blindness, age-related macular degeneration, rheumatoid arthritis, psoriasis, and more than 70 other conditions.
  • Insufficient angiogenesis can include: coronary artery disease, stroke, and delayed wound healing, and is also a factor in tissue engineering as discussed in greater detail in the present disclosure.
  • modulate and/or “modulator” generally refers to the act of directly or indirectly promoting/activating/inducing/increasing or interfering with/inhibiting/decreasing a specific function and/or trait in a cell/organism.
  • a modulator may increase or decrease a certain activity or function relative to its natural state or relative to the average level of activity that would generally be expected.
  • Modulation includes causing the overexpression or underexpression of a peptide (e.g., by acting to upregulate or downregulate expression of the peptide), or it may directly interact with the subject peptide to increase and/or decrease activity.
  • Modulation also includes causing the increase or decrease of a specific biological activity or biological event, such as angiogenesis or biological events related to angiogenesis
  • upregulate refers to the act of increasing the expression and/or activity of a protein or other gene product.
  • Downregulation refers to decreasing the expression and/or activity of a protein or other gene product.
  • isolated cell or population of cells refers to an isolated cell or plurality of cells excised from a tissue or grown in vitro by tissue culture techniques.
  • a cell or population of cells may refer to isolated cells as described above or may also refer to cells in vivo in a tissue of an animal or human.
  • tissue generally refers to a grouping of cells organized to cooperatively carry out a biological function and/or serve a biological purpose, such as forming all or part of an organ in an organism (e.g., connective tissue, endothelial tissue). While a “tissue” generally includes a grouping of similar cells, or cells of all the same type, a tissue may also include cells of more than one type where the group of cells as a whole serve a common purpose.
  • biocompatible refers to the ability to co-exist with a living biological substance and/or biological system (e.g., a cell, cellular components, living tissue, organ, etc.) without exerting undue stress, toxicity, or adverse effects on the biological substance or system.
  • a living biological substance and/or biological system e.g., a cell, cellular components, living tissue, organ, etc.
  • bioscaffold refers to any biocompatible substrate (naturally derived or synthetic) with sufficient structural stability to support the growth of a living biological substance (e.g., living cells).
  • the biocompatible scaffold material is a naturally derived substrate (e.g., procured from a living organism, but that may have undergone additional processing and treatment; or produced from materials derived from a natural source), such as, but not limited to, decellularized human umbilical vein scaffolds,
  • the bioscaffolds of the present disclosure have a three-dimensional structure (rather than a planer, 2-dimensional structure) to support three-dimensional growth of living cells.
  • biodegradable refers to a material that, over time in a natural environment (e.g., within a living organism or living culture), dissolves, deteriorates, or otherwise degrades and loses its structure integrity and ceases to exist in its original structural form.
  • biodegradable materials dissolve/degrade over a period of time within a host organism.
  • engineered indicates that the engineered object is created and/or altered by man.
  • An engineered object may include naturally derived substances, but the object itself is altered in some way by human intervention and design.
  • test compound may include peptides, peptidomimetics, small molecules, nucleic acid sequences, or other compounds that may have an effect on a living cell or organism.
  • the "test compound” may be a compound, such as a chemical or peptide that is suspected of having a modulating effect on a biological activity, function or response to another compound.
  • a “test compound” may be a compound suspected of having a modulating effect on angiogenesis, such as increasing angiogenic activity, decreasing angiogenic activity, and/or modulating the effect of a different angiogenesis modulator.
  • the term “removed” or “substantially removed” indicates that an amount of a substance or compound has been separated from another composition, but does not require that absolutely all traces of the removed substance be absent from the remaining composition, such that the removed substance is completely undetectable.
  • a composition or substance can be about 99% free, about 95% free, or about 90% free of the "removed” component, or any percentage or range within the exemplary percentages, given above).
  • the embodiments of the present disclosure encompass methods and compositions for inducing angiogenesis and methods and compositions for modulating angiogenesis, and methods of making compositions for modulating angiogenesis.
  • the present disclosure also includes methods of identifying modulators of angiogenesis and assays for identifying modulators of angiogenesis.
  • Embodiments of the present disclosure further include methods and compositions for delivering compositions for modulating angiogenesis.
  • the present disclosure includes a placental extract that can be used to induce and/or modulate angiogenesis in vitro and/or in vivo in a tissue construct and/or in natural tissue and methods and compositions for delivering a placental extract to cells in a tissue construct and/or natural tissue.
  • the present disclosure also includes a placental extract that can be used in an assay to identify compounds that modulate angiogenesis.
  • the present disclosure includes a composition of delivery vehicle loaded with placental extract for controlled release of the extract to in vivo or in vitro cell populations to induce
  • Angiogenesis is a complex process that is both location and stimuli dependent, and in each instance the capacity to modulate these processes may involve a complex combination of regulatory molecules. Control of vessel formation is further complicated by different mechanisms of formation, with the two most understood being intussusception and sprouting. Intussusception is characterized by the insertion of interstitial cellular columns into the lumen of preexisting vessels, and sprouting is characterized by endothelial cells sprouting toward an angiogenic stimulus in tissue previously devoid of microvessels. Many molecules have been found to modulate angiogenesis, with more likely to be discovered. This diversity of angiogenesis inducers has driven the continued search and
  • angiogenesis modulators for use in studies of vascular development, drug screening, and regenerative medicine therapies.
  • Standard in vivo angiogenesis models include the rabbit corneal neovascularization assay, the in vivo/in vitro chick chorioallantoic membrane assay, and the rat mesentery window assay.
  • in vitro angiogenesis models are chosen to better control complex biological phenomena; however, this often limits studies to a limited number of molecular species, e.g.,. VEGF.
  • the outcomes of using a single molecule (or several) for this complex cascade maybe limiting in itself, where a more complex or multifactorial 'mix' may be needed promote competent vascularization.
  • the murine derived basement membrane matrix (BMM) or 'Matrigel' assay has been the preferred model, as it brings a degree of in vivo complexity to an in vitro model and results appear to be more comparable to in vivo results. It is not suitable for clinical use, however, due to its derivation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells and that it requires the sacrifice of large numbers of animals 11 .
  • EHS Engelbreth-Holm-Swarm
  • a number of in vitro human-derived modulators have been used to model angiogenesis. Historically, these have been based on single modulators (FGF, TGF- ⁇ , VEGF) and lack the variety of cytokines and chemical gradients that are native in vivo 12 .
  • the present disclosure provides methods to induce and modulate angiogenesis in vitro and in vivo.
  • this model enables modulation of the rate of microvessel network maturation as well as selectively modeling sprouting and intussusceptive angiogenesis.
  • the human placental extract (PE) was shown to significantly enhance capillary formation while eliminating fibrosis using dosed collagen based bioscaffolds.
  • the present disclosure describes such methods to induce and modulate angiogenesis in vitro and in vivo using a complex set of tunable, fully-human biomolecules derived from the human placenta.
  • the approach uses directed fractionation and separation techniques to derive a complex of active human biomolecules isolated from the human placenta.
  • the methods and compositions of the present disclosure enable modulation of the rate of microvessel network maturation as well as selectively modeling sprouting and intussusceptive angiogenesis.
  • the methods and compounds of the present disclosure also induce and modulate angiogenesis in both polymeric and ex vivo derived tissue scaffolds.
  • the human placental extract of the present disclosure was shown to significantly enhance capillary formation while eliminating fibrosis using dosed collagen based bioscaffolds.
  • Sustained delivery of growth factors effecting angiogenesis is also a challenge facing successful modulation of angiogenesis to promote vascularization for tissue engineering approaches.
  • the present disclosure also provides methods and compositions for controlled release of the compositions of the present disclosure for modulating angiogenesis both in vitro and in vivo.
  • the present disclosure provides a composition and methods for induction and/or modulation of angiogenesis that includes a human placental extract (PE or hPE).
  • the PE is made by obtaining a sample from a human placenta, removing blood from the placental sample to produce a crude placental extract (crude PE), mixing the crude PE with urea or other protein solubilization agent to solubilize the proteins present in the extract, removing remaining solids from the crude extract; dialyzing the urea-placental extract mixture to remove a substantial amount of the urea from the mixture to produce the human PE.
  • the human PE is a matrix-like compound, and is sometimes referred to herein as a human placental matrix (hPM).
  • the process to make the human PE is performed at temperatures between about -86 °C and about 5 °C.
  • the human placental extract is made at temperatures at or below about 4°C.
  • the process of removing blood from the placental sample to make a crude placental extract includes homogenizing the human placenta sample with a buffer, centrifuging the homogenized sample, and discarding the supernatant containing blood. This process can be repeated multiple times (e.g., 2, 3 or more times) until substantially all of the blood has been removed from the sample (e.g., the sample is about 99% free of blood, about 95% free of blood, about 90 percent free of blood, etc.) to produce a crude PE.
  • the buffer is a Sodium
  • the proteins in the crude placental extract are solubilized by mixing the crude placental extract with a protein solubilization agent.
  • the protein solubilization agent can be any compound or mixture of compounds capable of solubilizing (e.g., denaturing) proteins without permanently destroying the proteins or otherwise permanently rendering them inactive (e.g., the solubilization should reversibly denature the proteins, such that the proteins are capable of refolding, such as upon removal of the protein solubilization agent).
  • the protein solubilization agent can be, but is not limited to, urea, guanidine-HCI, or other similar compounds.
  • the protein solubilization agent is urea
  • the crude extract is mixed with a urea composition by homogenizing the crude extract with urea.
  • the urea is mixed with the crude extract for a period of time between about 12 and about 36 hours.
  • the urea is mixed with the crude extract for about 24 hours.
  • the urea solution is a urea buffer having about 0.5M concentration of urea or greater.
  • the urea is about 2M or greater, about 4M urea, or greater, up to about 15M.
  • the urea solution can have a concentration of about 0.5M to about 15M.
  • the protein solubilization agent is guanidine-HCI having a concentration of about 0.5M to about 15M. In embodiments the guanidine-HCL has a concentration of about 6M.
  • solids are removed from the solubilized protein-crude extract mixture (e.g., urea-crude extract mixture).
  • the solids are removed by centrifuging the PE mixture and discarding the pellet (containing the solids). This step can be repeated multiple times.
  • the PE mixture e.g., the supernatant
  • the dialysis solution is TBS.
  • the dialysis solution is changed after a period of time (e.g., 1 hour, 2 hours, 3 hours, etc.) and dialysis is repeated a number of times (e.g., 2, 3, 4, etc.) to remove substantially all urea from the PE (e.g., the placental extract is about 99% free of urea, about 95% free of urea, etc.).
  • the PE may be centrifuged again to remove remaining solids (e.g., polymerized proteins, and the like).
  • the remaining PE is a clear to pinkish viscous substance. Additional details about embodiments of the process of the present disclosure of making the placental extract of the present disclosure can be found in the Examples below.
  • embodiments of the present disclosure also include a PE made by the methods of the present disclosure.
  • the present disclosure includes a PE made by removing blood from a sample obtained from a human placenta sample to produce a crude PE; mixing the crude placental extract with a protein solubilization agent (such as, but not limited to urea, guanidine-HCI, etc.) to solubilize proteins in the crude extract; separating solid materials from the solubilized protein- PE mixture; and performing dialysis on the PE mixture to remove the protein solubilization agent (e.g., urea) from the mixture to produce the human PE.
  • a protein solubilization agent such as, but not limited to urea, guanidine-HCI, etc.
  • the present disclosure thus includes a human placental extract including an extract obtained from a human placenta (e.g., from a human placental sample) having the blood and solids substantially removed and retaining (some or all) of the placental proteins that were present in the placental sample.
  • the placental proteins include cytokines and growth factors.
  • the PE includes many proteins including many cytokines and growth factors.
  • the extract includes at least 20 different cytokines. In some embodiments it contains up to 40 different cytokines. Other embodiments include at least 50 cytokines.
  • Some cytokines that can be present in the PE of the present disclosure include those listed in the example below. For instance, some of the cytokines that can be present in the PE of the present disclosure include, but are not limited to, angiogenin, Acrp30Ag, IGFBP-1 , NAP-2, and Fas/TNFGSF6, and RANTES, and MIF.
  • the cytokines and growth factors and other placental compounds present in the placental extract of the present disclosure can induce angiogenesis in a culture of endothelial cells, a tissue, a tissue construct, an engineered bioscaffold, and the like.
  • the placental extract of the present disclosure can induce angiogenesis in vitro and in vivo.
  • the placental extract of the present disclosure is capable of stimulating growth of endothelial cells.
  • the human PE of the present disclosure is capable of modulating angiogenesis.
  • the PE of the present disclosure stimulates increased angiogenic growth of endothelial cells (e.g., tubule and network formation) and decreased angiogenic-type growth of myofibroblasts (e.g., tubule formation) as compared to BMM.
  • the PE of the present disclosure also stimulates different growth and/or differentiation patterns for various cell lines (e.g., stem cells, smooth muscle cells, etc.) as compared to BMM, such that the growth/differentiation patterns of such cells are distinguishable from growth with BMM.
  • the PE of the present disclosure is also capable of upregulation of various genes in endothelial cells in comparison to endothelial cells grown in the absence of the PE.
  • Some such genes include angiogenesis related genes, extracellular matrix remodeling genes, and vascular development genes.
  • Some angiogenesis related genes include, but are not limited to: ANGPTL4, CXCL3, human growth factor (HGF), ANGPT2, PGF, TYMP, VEGFA, HIF1A, and FGF1.
  • Some extracellular matrix remodeling genes that can be induced by the placental extract of the present disclosure include, but are not limited to: MMP2, MMP9, COL4A3, and LAMA5.
  • Vascular development genes include, but are not limited to: CDH2, HAND2, LECT1 , and MDK.
  • the present disclosure also includes methods for inducing angiogenesis in a cell culture, wherein the method includes growing endothelial cells in the presence of a human placental extract of the present disclosure.
  • the cell culture is grown in the presence of a placental extract of the present disclosure obtained from a human placenta sample that was treated to remove blood and solids, mixed with urea, and dialyzed to remove urea, wherein the placental extract comprises placental proteins including cytokines and growth factors.
  • the endothelial cells are human endothelial cells; in yet other embodiments, the cells are human umbilical vein endothelial cells (HUVECs).
  • the cells are seeded at a density of at least about 40,000 cells/cm 2 . In embodiments they are seeded at a density of at least about 80,000 cells/cm 2 . In embodiments, the cell cultures can be grown on a plate containing growth media and the placental extract of the present disclosure.
  • the present disclosure also include methods for inducing vascularization of a biomaterial in vivo including incubating a biomaterial in a composition including the human placental extract of the present disclosure and implanting the biomaterial in the host.
  • the biomaterial includes naturally derived materials and/or cells.
  • the biomaterial includes an engineered bioscaffold including human derived substrate material.
  • the engineered bioscaffold includes human umbilical vein scaffold.
  • the human umbilical vein scaffold is decellularized.
  • the biomaterial is seeded with endothelial cells, such as, but not limited to, human endothelial cells (e.g., HUVECs).
  • the biomaterial includes an engineered scaffolding material including a human umbilical vein scaffold seeded with HUVECs).
  • the HUVECs are seeded on the bioscaffold at a cell density of at least about 40,000 cells/cm 2 . In embodiments they are seeded at a density of at least about 80,000 cells/cm 2 .
  • the biomaterial is incubated in the placental extract for at least about 2 hours.
  • the present disclosure also includes methods of vascularizing biomaterials, including but not limited to, engineered biomaterials, naturally derived biomaterials, and other biomaterials to be implanted in a host.
  • treatment of biomaterials with the placental extract of the present disclosure can also be used to pre-treat biomaterials for use in-vivo to aid in bio-acceptance, reduce inflammation, reduce rejection and scarring, etc.
  • the placental extract of the present disclosure and compositions including the placental extract of the present disclosure can be used to "dose" any number of biomaterials in order to improve the outcome of such implant.
  • the present disclosure also includes specifically engineered biomaterials, such as implantable, engineered bioscaffolds including a human derived substrate material incubated in a composition including a human placental extract of the present disclosure.
  • the bioscaffolds of the present disclosure can be implanted in a mammal, such as a human.
  • Bioscaffolds of the present disclosure can include any biomaterial suitable for implantation in a host. Examples of bioscaffolds for use in the present disclosure include, but are not limited to, engineered bioscaffolds including tissue, matrix materials, any number of naturally derived biomaterials, and the like.
  • the bioscaffolds are 2D or 3D bioscaffolds.
  • the bioscaffolds includes human derived substrate material.
  • the bioscaffold includes decellularized human umbilical vein scaffold.
  • the bioscaffold is seeded with cells, such as, but not limited to human cells, human endothelial cells (e.g., human umbilical vein endothelial cells (HUVECs)), stem cells, other pluripotent cells, and the like.
  • human endothelial cells e.g., human umbilical vein endothelial cells (HUVECs)
  • stem cells other pluripotent cells, and the like.
  • the bioscaffolds of the present disclosure incubated in the placental extract of the present disclosure induce more vascularization (e.g., angiogenesis) and less fibrosis that bioscaffolds incubated in the angiogenesis inducing compound BMM or a control compound.
  • the bioscaffolds incubated in the placental extract of the present disclosure also had a higher ratio of immune suppressive and pro-angiogenic positive macrophages (e.g., CD205(M2)) versus proinflammatory positive macrophages e.g., (CD86(M1 )) as opposed to bioscaffolds incubated in BMM or a control.
  • the placental extract of the present disclosure induces angiogenesis in cell culture it provides a good assay for identifying and screening for angiogenesis modulators.
  • the present disclosure also includes methods and assays for identifying angiogenesis modulators.
  • a method includes growing a culture of human endothelial cells in the presence of a compound including a human placental extract of the present disclosure and contacting the human endothelial cell culture with a test compound. Since the placental extract induces angiogenesis in the cell culture, if angiogenesis is less than or more than expected, the test compound can be identified as an angiogenesis modulator. Thus, the method also includes determining an amount of angiogenesis in the culture and identifying the test compound as an angiogenesis modulator when the amount of angiogenesis in the cell culture is greater or less than the amount of angiogenesis is a culture growth in the absence of the test compound.
  • an increase in the amount of angiogenesis relative to a culture grown in the absence of the test compound indicates the test compound induces angiogenesis.
  • a decrease in the amount of angiogenesis relative to a culture grown in the absence of the test compound indicates the test compound inhibits angiogenesis.
  • a screen of the compound Thrombospondin-1 (TSP-1 ) according to the methods of the present disclosure identified the compound as an inhibitor of angiogenesis.
  • the present disclosure also provides assays for screening test compounds to identify modulators of angiogenesis including a culture of endothelial cells grown in the presence of a human placental extract of the present disclosure. The assays of the present disclosure can be used with the methods of the present disclosure to identify modulators of angiogenesis.
  • compositions and methods for sustained release of the PE of the present disclosure are also provided in the present disclosure.
  • Embodiments of the present disclosure include a composition including the human PE (hPE) of the present disclosure coupled to biodegradable microparticles to provide a sustained- release/human PE composition.
  • the PE loaded biodegradable microparticles provide a sustained release angiogenesis-modulating composition.
  • the sustained-release angiogenesis-modulating composition includes, a human PE of the present disclosure and biodegradable microparticles, where he human PE is coupled to the microparticles such that the human PE is released from the microparticles.
  • the human PE is obtained from a human placental sample and having the blood and solids substantially removed from the extract, where the extract includes placental proteins that were present in the placental sample, including cytokines and growth factors.
  • the human PE used in the sustained-release angiogenesis- modulating composition can be any embodiment of the human PE as described above.
  • the human PE is released from the microparticles after exposure to cells in vivo or in vitro.
  • the biodegradable microparticles when placed in a host in vivo or in contact with cell culture or cell seeded bioscaffold in vitro, the biodegradable microparticles begin to degrade and release the PE to the surrounding cells, tissues, etc. over time.
  • the biodegradable microparticles release an initial "burst" of the PE and then slowly release PE over a sustained period of time (e.g., several days, weeks, etc.).
  • the release profile of the microparticles can be controlled by varying the size of the microparticles and the degree of crosslinking.
  • the microparticles are crosslinked and the degree of crosslinking can be controlled to modify the release parameters of the particles.
  • the biodegradable microparticles are made of gelatin. Further details about embodiments of gelatin biodegradable microparticles are described in Example 2, below.
  • the biodegradable microparticles include a mixture of different sizes of microparticles. It is believed that including various sizes of microparticles provides sustained release of PE since different size particles release PE at different rates.
  • the biodegradable microparticles are poly(lactic-co-glycolic acid) (PLGA) microparticles.
  • PLGA poly(lactic-co-glycolic acid)
  • the hPE is loaded into (e.g., encapsulated) in the PLGA
  • the PLGA microparticles loaded with the hPE of the present disclosure have an average particle size of about 10 to about 1000 ⁇ .
  • the PLGA microparticles are made by an oil in water emulsion process including a dual homogenization step.
  • the PLGA microparticles are made by mixing a PLGA oil solution with a first water solution (W1 ) including the hPE of the present disclosure, preparing a first emulsion by homogenizing the PLGA and W1 in a first homogenization step, and adding the first emulsion to a second water solution (W2) including a solvent (e.g., and alcohol, such as, but not limited to, polyvinyl alcohol) in water to form a second emulsion (a water-in-oil-in-water emulsion), and then the solvent is evaporated.
  • a second homogenization step is used to homogenize the secondary emulsion.
  • the first homogenization is from about 1 to about 2 minutes. In some embodiments, no other homogenization step is used. In other embodiments, a second
  • hPE loaded PLGA microparticles made with a single homogenization step have size of about 100 to about 1000 ⁇ in diameter. In embodiments where the single homogenization step is about 2 min, the resulting microparticles have sizes ranging from about 50 to about 500 ⁇ in diameter, with an average of about 260 to about 290 ⁇ in diameter.
  • hPE loaded PLGA microparticles made with a second homogenization step of about 1 min range in size from about less than 20 to about 100 ⁇ in diameter, with an average of about 36 to about 40 ⁇ in diameter.
  • hPE loaded PLGA microparticles made with a second homogenization step of about 20 sec range in size from less than 20 to about 200 ⁇ , with an average of about 85 to about 95 ⁇ in diameter.
  • the present disclosure also includes methods of using the sustained release angiogenesis- modulating composition described above for sustained release of PE.
  • methods include using the sustained release composition to obtain sustained release of human PE in vivo or in vitro, such as, but not limited to, sustained release of human PE to a biomaterial (e.g., cell culture, tissue, tissue construct, bioscaffold, etc.) in vivo or in vitro, over time.
  • a biomaterial e.g., cell culture, tissue, tissue construct, bioscaffold, etc.
  • a method of the present disclosure includes contacting a cells (e.g., cells in culture, cells in vivo, cells in a biomaterial or cells in contact with an engineered biomaterial, etc.) with a sustained release angiogenesis-modulating composition described above including biodegradable microparticles coupled to a placental extract of the present disclosure, such that the human placental extract is released from the microparticles into the biomaterial over a period of time after exposure of the microparticles to the cells.
  • the cells are endothelial cells, such as, but not limited to, human umbilical vein endothelial cells (HUVECs).
  • the sustained release angiogenesis-modulating composition is coupled to a biomaterial.
  • the biomaterial is a cell culture.
  • the biomaterial is a tissue construct and/or tissue matrix including a cell culture.
  • the biomaterial is an alginate matrix including cells (e.g., human cells, e.g., HUVECs) embedded in the alginate matrix and the sustained release angigogeneisis-modulating composition (e.g., hPE loaded biodegradable microparticles) is also embedded or contacted with the alginate matrix.
  • the biomaterial coupled to the sustained release angiogenesis-modulating composition is implanted in a subject and induces vascularization of the biomaterial.
  • the biomaterial is an engineered bioscaffold including human derived substrate material, such as, but not limited to, decellularized human umbilical vein scaffold seeded with human endothelial cells.
  • human derived substrate material such as, but not limited to, decellularized human umbilical vein scaffold seeded with human endothelial cells.
  • the subject is a mammal; in embodiments, the subject is a human.
  • Other variations of the method of inducing angiogenesis with the sustained release angiogenesis-modulating composition of the present disclosure are possible, and exemplary embodiments of the method are described in greater detail in the examples below.
  • compositions of the present disclosure also include anti-inflammatory compositions including the human PE or the sustained-release/human PE composition of the present disclosure.
  • Methods of the present disclosure also include methods of treating (e.g., reducing, ameliorating, counteracting, preventing, etc.) inflammation in a subject, or a tissue of a subject by exposing a subject or a tissue to the human PE or the sustained-release human/PE composition of the present disclosure.
  • ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and subrange is explicitly recited.
  • a concentration range of "about 0.1 % to about 5%” should be interpreted to include not only the explicitly recited concentration of about 0.1 wt% to about 5 wt%, but also include individual concentrations (e.g., 1 %, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1 .1 %, 2.2%, 3.3%, and 4.4%) within the indicated range.
  • the term "about” can include traditional rounding according to significant figures of the numerical value.
  • the present example describes methods to induce vascularization using a complex human placental extract (PE).
  • the PE is derived from the human placenta and is capable of inducing angiogenesis in 2D and 3D in vitro models, as well as in vivo within bioengineered tissue implants.
  • This example also describes using the placental extract to positively screen thrombospondin-1 as an angiogenesis inhibiting protein with increased sensitivity relative to current in vitro models.
  • this model allows for modulation over the rate and type (intussusceptive vs. sprouting) of angiogenesis and presents many advantages over conventional approaches as well as broad applications in the fields of regenerative medicine and pharmaceutics.
  • Mass transfer limitations within tissues represent one roadblock to producing effective biomaterials. Even if this can be temporarily overcome to allow improved cell migration within a human bioscaffold, the creation of an effective vasculature remains the primary goal to provide long- term nutrient delivery to thick, cell-dense materials.
  • new blood vessels are predominately produced through the physiological process of angiogenesis, 47 which ultimately leads to the formation of nutrient rich vascular networks.
  • angiogenesis can be induced in a human umbilical vein (HUV) vascular graft and lead to a long-term nutrient delivery system.
  • UUV umbilical vein
  • the present example provides a human placenta extract (hPE) that is capable of inducing angiogenesis in 2D and 3D in vitro models, as well as in vivo within bioengineered tissue implants.
  • hPE human placenta extract
  • the PE is a complex of active human biomolecules, and the present example demonstrates that, in addition to inducing in vivo and in vitro angiogenesis in the ex vivo derived human umbilical vein vascular graft, this model enables modulation over the rate and stage of angiogenesis.
  • This example also demonstrates that the PE enhances capillary formation while also reducing fibrosis using dosed collagen based bioscaffolds.
  • Placental extract derivation Full-term placentas were collected from UF Health Shands Hospital (Gainesville, FL) within 12 hours of birth. The umbilical cords and fetal membranes were removed and the placenta was dissected into 2 cm cubes and frozen. 12 hours after progressive freezing to -86 °C at a rate of -1 °C/min, the placental cubes were transported to a cold room maintained at 4 °C where the rest of the procedures were completed. Once at 4 °C, 100 grams of the tissue was mixed with 150 mL cold 3.4 M NaCI buffer (198.5 g NaCI, 12.5 ml 2M tris, 1.5 g EDTA, and 0.25 g NEM in 1 L distilled water).
  • the NaCI buffer/tissue mix was homogenized into a paste using a Tissuetek Homogenizer at 3200 RPM, then centrifuged at 7000 RPM for 15 minutes and separated from the supernatant. This NaCI washing process was repeated two additional times, discarding the supernatant each time to remove blood.
  • the pellet was homogenized in 100 mL of 4M urea buffer (240 g urea, 6 g tris base, and 9 g NaCI in 1 L distilled water), stirred on a magnetic stirplate for 24 hours, and then centrifuged at 14000 RPM for 20 minutes (Sorvall RC6+ Centrifuge, Thermo Scientific, NC, USA). The supernatant was removed and dialysed using 8000 MW dialysis tubing (Spectrum Laboratories, Inc., CA, USA) placed in 1 L of TBS (6 g tris base and 9 g NaCI in 1 L distilled water) and 2.5 ml of chloroform for sterilization. The buffer was replaced with fresh TBS 4 more times, each at 2 hour intervals.
  • 4M urea buffer 240 g urea, 6 g tris base, and 9 g NaCI in 1 L distilled water
  • Biomolecular composition analysis Relative cytokine levels were determined using a sandwich immunoassay array from RayBiotech, Inc. (Human Cytokine Antibody Array C Series 1000, Inc, GA, USA). Chemilumenescence was detected using a Foto/Analyst Luminaryfx Workstation (Fotodyne Incorporated, Wl, USA) and the signal intensities were measured using TotalLab 100 software (Nonlinear Dynamics, Ltd, UK). The relative abundance of basement membrane
  • Biomolecules was performed by MSBioworks (Ann Arbor, Ml) using nano LC/MS/MS with a Waters NanoAcquity HPEC (Waters, Milford, MA) system interfaced to a Orbitrap Velos Pro (ThermoFisher, Waltham, MA). Proteins were identified from primary sequence databases using Mascot database search engine (Boston, MA).
  • RT-PCR analysis of cells from hPL-induced angiogenic networks Relative angiogenic gene expression was determined using 384-well RT 2 Human Angiogenesis RT 2 Profiler PCR Arrays (PAHS-024A, Quiagen, CA, USA). ECs were detached from culture plates using Accutase (Innovative Cell Technologies, San Diego, CA) and immediately stored in 100 ⁇ of RNA/afer. RNA was extracted using the RNeasy Mini Kit (Qiagen, CA, USA), and genomic DNA was digested using an RNase-Free DNase kit (Quiagen, CA, USA).
  • RNA was reverse transcribed to cDNA using the RT 2 First Strand Kit (SA Biosciences, TX, USA) with incubation at 42°C for 15 minutes followed by incubation at 95°C for 5 minutes to stop the reaction.
  • cDNA was mixed with RT 2 SYBR Green Mastermix (SA Biosciences, TX, USA) and loaded into 384-well Human Angiogenesis PCR Arrays.
  • Endothelial cells were derived from human umbilical veins (collected from UF Health Shands Hospital, Gainesville, FL) by detachment from the vessels walls using a 1 mg/ml solution of bovine Type-I Collagenase in phosphate buffered saline (Gibco, Invitrogen, NY, USA).
  • the primary derived human umbilical vein endothelial cells (HUVEC) were used between passages 1-3 for all experiments.
  • For proliferation cells were cultured using complete VascuLife Basal media (VascuLife VEGF Medium Complete Kit, Lifeline, MD, USA).
  • endothelial cell media was prepared using VascuLife Basal media with 25 ml of glutamine, 0.5 ml of hydrocortisone, 0.5 ml of ascorbic acid, 10 ml of FBS, and 1.25 ⁇ of bFGF to 500 mL of (VascuLife VEGF Medium Complete Kit, Lifeline, MD, USA).
  • Human myofibroblasts (CRL 2854) were used between passages 5 and 10 (ATCC, Manasses, VA) and cultured using 10% FBS supplemented low-glucose DMEM.
  • placenta extract-derived angiogenesis assays Preparation of placenta extract-derived angiogenesis assays. Unless otherwise stated, 32 ⁇ of placental extract was thawed and pipetted into each well of a 96 well plate. The extract was evenly coated onto the bottom of each well using an orbital shaker at 30 RPM for 1 minute. The coated plate was then incubated at 37°C for 30 minutes. HUVEC were then plating by direct pipetting at 20000 cells/cm 2 , 40000 cells/cm 2 , or 80000 cells/cm 2 . Multiple time points were investigated at each concentration including at days 1 , 3, and 5. Thrombospondin-1 was tested as an angiogenesis inhibiting drug using final concentrations 0, 5, 10, 20, and 35 ⁇ g ⁇ L diluted in endothelial cell media
  • Branch points were assigned manually as the positions at every node where branches meet or tubules sprout, and tubule length was assessed by determining the curve length from branch point to connected branch point.
  • Tubule width measurements were carried out in three different zones per tubule, with two zones each 10 ⁇ from the start and end and one zone in the middle of the curve length. The percent area of coverage was determined by processing the images using the imageJ function "binary»convert to mask” followed by measurement of the "mean.” In TSP-1 experiments, final values were normalized to no dose samples, calculated as the percentage of "1 " values relative to the total count of pixel values, and given as "% area coverage".
  • microvessel networks grown on glass slides were fixed in 2.5% glutaraldehyde, washed in PBS, fixed in 1 % osmium tetroxide solution, and progressively dehydrated in 25%, 50%, 75%, 85%, 95%, and 3x100% ethanol solutions. Smaples were then critical point dried, coated with gold/palladium, and imaged using a Hitachi S-4000 FE-SEM.
  • Placentas Human umbilical vein scaffold derivation and placental extract incubation. Placentas were collected from UF Health Shands HospitalFlorida (Gainesville, FL) and HUVs were dissected using an automated method as previously described. 32 Dissected HUV samples were decellularized in a 1 % SDS (Thermo Scientific, Rockford, IL) solution at a solvent/tissue mass of 20:1 (w:v).
  • Samples were decellularized on an orbital shaker plate at 100 rpm for 24 hours and then rinsed with PBS prior to incubation overnight at 37 ° C in a 70 U/mL DNase I solution (Sigma-Aldrich, St. Louis, MO) in PBS. Sample were terminally sterilized using a 0.2% peracetic acid/ 4% ethanol (Sigma- Aldrich, St. Louis, MO) solution for 2 hours and finally pH balanced (7.4) using PBS.
  • scaffolds were cut into 1.5 cm x 1.5 cm x 0.075 cm sheets, prefrozen to - 85 C, and then lyophilized using a Millrock bench top manifold freeze dryer (Kingston, NY) for 24 hours at -85 C under 10 mT vacuum. Immediately prior to cell seeding, scaffolds were soaked for 2 hours in hPE, Matrigel, or PBS (control) and seeded.
  • fibrotic capsules were dissected with a scalpel and the HUV samples were placed onto glass slides.
  • Top- down images of the semi-translucent scaffold sheets were taken using an Imager M2 light microscope (Zeiss, Oberkochen, Germany) with an Axiocam HRm digital camera (Zeiss, Oberkochen, Germany).
  • tissue samples were embedded in Neg-50 frozen section medium, sectioned into 7 ⁇ sections (Microm HM550 cryostat, Thermo Scientific, Waltham, MA), and stained using standard hematoxylin and eosin (H&E) staining (Richard-Alan Scientific, Kalamazoo, Ml).
  • the hPE derivation technique utilized a urea step to linearize and solubilize molecules. This was followed by dialysis separations to remove urea and allow the biomolecules to refold into their original conformations (FIG. 1 A). All steps of the derivation were performed in a cold room at 4°C. The final solution of PE was translucent, highly viscous, and consisted of biomolecules between 8 kD to 868 kD.
  • the hPE could be used to soak biomaterials or made into a thin-film for tissue culture assays. Reproducibility of hPE was assessed by analysis of standard deviation of the total protein content in n 1 ⁇ 4 3 batches of hPE (with each batch created using equal masses of tissue from 3 separate donors) and shown to have a have a similar reproducibility and protein content to Matrigel®. Scanning electron microscopy images show the surface morphology of hPE (FIG. 1 B-1 C) and angiogenic network formation when HUVECS were seeded at 4 x 10 4 cells/cm 2 onto hPE thin films and cultured for 3 days (FIG. 1 D-1 E).
  • Angiogenic potential of the human placental extract was initially characterized by seeding primary human umbilical vein endothelial cells (HUVEC) onto tissue culture plates (TCP) coated with the hPE. Early stage cell cording and sprouting were visible within 1 hour of cell seeding (data not shown), and angiogenic networks continued to mature until experimental termination at 3 d (FIG. 1 F-1 G). The length of individual cell cords (multicellular) increased significantly from day 1 (FIG. 1 H) to day 3 (FIG. 11) of seeding. After 3 days of culture, cells had formed extensive angiogenic networks relative to control samples (FIG. 1J, 1 K).
  • angiogenesis related cytokines were detected in the placental lysate (FIG. 2A).
  • the most prevalent angiogenesis related chemokine was angiogenin, which is a potent stimulator of new blood vessel formation 16 .
  • Significant pro-angiogenic chemokines including, but not limited to, hepatocyte growth factor (HGF), fibroblast growth factor-4 (FGF4), leptin (LEP), ICAM-1 , ICAM-2 and TIMP-2 were also detected.
  • LC-MS/MS showed the presence of immune-related proteins including annexins (ANXA1 , ANXA2, ANXA4, and ANXA5), neutrophil defensin (DEFA1 ), interleukin enchacer-binding factors (ILF2 and ILF3), IL27, ITBG1 , and MRC1 (FIG. 2B).
  • annexins ANXA1 , ANXA2, ANXA4, and ANXA5
  • DEFA1 neutrophil defensin
  • ILF3 interleukin enchacer-binding factors
  • IL27 IL27
  • ITBG1 MRC1
  • Angiogenesis related basement membrane (BM) proteins were also detected using LC- MS/MS, including laminin (LAMA2, LAMA4, LAMA5, LAMB1 , LAMB2, LAMB3, and LAMC1 ), fibronectin (FN1 ), heparin sulfate (HSPG2) and type-4 collagen (COL4A1 , COL4A2, and COL4A3) (FIG. 2C), each of which has been shown to play key roles in angiogensis. 17-20
  • HUVEC gene analysis further affirmed the angiogenic nature of placenta extract.
  • RT-PCR analysis showed that endothelial cells seeded on hPE for 3 d expressed a wide range of essential pro-angiogenic genes including hepatocyte growth factor, epidermal growth factor, and placental growth factor (FIG. 2D).
  • Additional upregulated genes include MMP2 and MMP9, which are proteolytic enzymes that aid in the degradation of the surrounding extracellular matrix in order to facilitate the migration of the endothelial cells as well as other cells associated with ECM remodeling 21 .
  • Type IV collagen was also upregulated, which is associated with the formation of basement membranes in maturing microvessel systems 22 .
  • in vitro assays have little or no control over the rate and stage of angiogenesis.
  • the present data shows in vitro hPE-based angiogenesis assays can be modulated to control the maturation and morphology of angiogenic network formation by varying the initial cell seeding density. After 1 day, HUVECs seeded at density of 40,000 cells/cm 2 formed more defined tubules by comparison to seeding at a density of 80,000 cells/cm 2 , but by day 5, cells seeded at both densities had well defined tubules (FIG. 3A). These results show that the maturation stage of network formation can be controlled when cultures are exposed to hPE by varying the cell seeding density.
  • FIG. 3C Quantitative image analysis of the capillary networks (mean tubule length [mm], tubule density [#/mm 2 ], branch points [#/mm 2 ], meshes [#/mm 2 ], and mean tubule width [mm]) (FIG. 3C) showed that higher seeding densities resulted in a slower network maturation (an extended time frame to reach the highest mean tubule length and number of meshes/mm2), allowing a more detailed analysis as the time frame of network formation can be extended. Comparatively, lower cell densities resulted in a faster maturation rate that would be more conducive to rapid screening approaches to (for example) test the effectiveness of angiogenesis blockers for cancer therapies.
  • Matrigel-induced angiogenic networks were compared to hPE-induced networks (FIG. 3D). Morphologies of endothelial cell capillary networks were first analyzed by exposing cell cultures to either Matrigel or hPE using Calcein AM to determine viability and network structure. One day post seeding, Matrigel coated plates had shown HUVEC to form defined angiogenic tubule networks, but after 3 d network structures collapsed into spherical balls of apoptotic cells (FIG. 3A, 3D). While some cell death was noted in hPE induced networks no apoptotic ball formations were observed after an extended 5 d period.
  • angiogenesis driven by the human placental extract was tested for its ability to screen angiogenesis related drugs in vitro.
  • Matrigel was used as control.
  • Matrigel and hPE-based angiogenesis assays were screened against
  • TSP-1 thrombospondin-1
  • TSP-1 represents a model drug for the anti-angiogenic treatment of solid tumors 23 .
  • control HUVECs seeded directly onto tissue culture plates were not affected by TSP-1 , with cells forming typical cobblestone morphologies.
  • HUVECs cultured on hPE treated culture plates had significantly reduced angiogenic network formation.
  • FIG. 4A Results show the total tubule-length and branch points to decrease linearly as a function of TSP-1 concentration (FIG. 4B).
  • angiogenesis occurs by a variety of mechanisms, most commonly sprouting or intussusception. These data show sprouting versus intussusceptive angiogenesis can be modulated in vitro by varying cell density when incubated with hPE. At lower cell densities (2x10 4 cells/cm 2 ) network morphologies on the hPE-incubated scaffolds exhibited sprouting angiogenesis (FIG. 5C.i, 5C.ii), at intermediate densities (4x10 4 cells/cm 2 ) network morphologies exhibited a combination of sprouting and intussusceptive angiogenesis (FIG.
  • FIG. 6A Using a subcutaneous rat model (FIG. 6A) the angiogenic response to dosed scaffolds (Matrigel and hPE) was assessed 5 days post implantation. Both control and Matrigel-incubated scaffolds displayed significant fibrosis surrounding the scaffold, whereas hPE dosed scaffolds exhibited no discernible fibrosis around the implant (FIG. 6B.i-6B.iii.).
  • Fibrosis prevention in hPE samples is believed to result from immune related molecules, as detected with LC-MS/MS, including, but not limited to, anti-inflammatory Annexins (ANXA1 , ANXA2, ANXA4, and ANXA5) 27 , antimicrobial defensin peptides such as DEFA1 28 , and MRC1 , which is know to bind to potential pathogens including viruses and bacteria.
  • anti-inflammatory Annexins ANXA1 , ANXA2, ANXA4, and ANXA5
  • DEFA1 28 antimicrobial defensin peptides
  • MRC1 which is know to bind to potential pathogens including viruses and bacteria.
  • Tissue regeneration, infarct tissue and ischemic wound repair are three clinical areas where an improved strategy for wound recovery or organ replacement would have significant clinical impact.
  • the use of amniotic and chorionic membranes in a variety of applications has grown significantly over the last 5 years, with an increasing body of evidence indicating perinatal tissues hold considerable clinical promise 29"32 .
  • Results herein detail a novel approach to concentrate and deliver physiological ratios of a potent human derived stimulator of angiogenesis and tissue remodeling. These data show enhanced cellular activity toward initiating capillary formation (in vitro and in vivo), controlling EC phenotype during angiogenesis with a capacity to modulate growth or maturation dynamics, and a significant reduction in in vivo tissue fibrosis.
  • the capacity to modulate the in vitro maturation rate of capillary network formation and to control the occurrence of sprouting and intussusceptive angiogenic network morphologies may provide a useful platform to further the understanding of regulatory pathways during wound healing and organ regeneration. Based on comparisons with Matrigel, the mechanism with which hPE stimulates cells appears to be fundamentally different. SMC incubated with Matrigel initiated capillary-like formations whereas SMC exposed to the hPE retained their typical hill and valley morphology, as such the human derived hPE may provide a more representative model of physiological angiogenesis in more complex models.
  • a number of current methods are based on human-derived (recombinant) modulators that rely on single or discrete combinations of angiogenesis modulators 33 . While discrete combinations are useful to control variation and reduce the inherent complexity of multifarious approaches, they constrain the screening process and fail to represent the broad set of human in vivo molecular interactions that are likely to be critical when testing the potential of anti-angiogenic, tumor suppressive drugs.
  • hPE based models may lead to advances in the pharmaceutical industry by providing a more effective screening approach for tumor suppressive drugs.
  • TSP-1 angiogenesis inhibiting drug-concentrations
  • hPE-based models induce angiogenesis using a broad set of human-derived molecules at near physiological ratios. It is believed that regulation of only selected molecular pathways will confine attempts to discover novel anti-angiogenesis drugs as vessel formation in vivo requires the induction of multiple metabolic pathways 34, 35 . As such, a drug may modulate angiogenesis via interaction with any of these numerous pathways but may have little effect inducing competent angiogenesis when the complexity of the local environment is lacking. Results from the in vivo analysis in the present example provide further evidence that the complex PE influences numerous biochemical pathways, resulting in a broad range of effects.
  • hPE not only displayed enhanced angiogenic properties, but was also shown to have immune reductive properties, as illustrated by reduced fibrosis within hPE dosed bioscaffolds.
  • the molecular composition of hPE provides a suitable basis for the development of clinically applicable techniques to induce capillary formation without significant immunological and inflammatory reactions.
  • the hPE angiogenesis model has been validated in 2D and 3D in vitro models, as well as in vivo within bioengineered tissue implants and can be readily adapted to a variety of clinical or pharmaceutical applications. Its derivation from physiologically healthy, human vascular beds combined with its angiogenic and immune reductive properties make it unique among current angiogenesis models.
  • the data presented here have shown hPE to play a pivotal role in a number of key clinical issues where demand for alternative, more successful, approaches are a clinical priority.
  • Adair T. in Integrated systems physiology, from molecule to function to disease (Morgan & Claypool, 201 1 ).
  • Tissue engineering aims to build tissues and organs from scratch in vitro in order to transplant them into ill patients.
  • this revolutionary alternative to transplantation is subordinated to the lack of the formation of a suitable vasculature for the supply of oxygen and nutrients to cells seeded in the transplanted graft. Accordingly, an effective method to induce angiogenesis in tissue- engineered constructs is urgently needed.
  • the present disclosure provides a protocol to derive a pro-angiogenic extract from the human placenta, which was shown, as described in the example above, to induce and modulate the initial stages of angiogenesis.
  • the present example describes a 3D in vitro angiogenesis assay to promote the formation of a capillary network and to sustain it over time.
  • the angiogenic potential of the placental extract and its bioactivity over time has been analyzed.
  • biodegradable gelatin microparticles for incorporation and controlled release of the extract were prepared and their degradation kinetics were studied.
  • a 3D in vitro angiogenesis assay was developed. Placental extract loaded microparticles were embedded in a Collegen Type I hydrogel scaffold seeded with HUVECs, and evidence of initial phase of microvessel formation within the matrix was demonstrated.
  • engineering tissues capable of long term sustainability would benefit from methods to facilitate the delivery of oxygen and nutrients to cells seeded in 3D tissue constructs.
  • the placental extract and methods the present disclosure provide an approach to overcome oxygen and nutrient deficiencies that involves inducing the rapid development of a nutrient rich capillary system within the scaffold.
  • Implementing methods to supply these essential nutrients not only to the margins of the construct, but also in the center, would help to prevent the formation of the fibrotic capsule.
  • Angiogenesis is central to tissue development and maintenance and its successful modulation promotes the controlled formation of an established vascular network in implanted grafts.
  • Several biological factors and molecular pathways are involved in the regulation of this complex process which is still partially unknown.
  • angiogenesis assays that are cell culture systems which reproduce in vitro or in vivo the definitive elements of angiogenesis under simplified, defined and controlled conditions.
  • a variety of different approaches have been used to promote in vitro angiogenesis but to date there has been little success in translating them to the clinical practice.
  • a limitation of most angiogenesis models is that they are either animal-derived (e.g. Matrigel based) or entirely dependent on the use of live animals (e.g.
  • the chick chorioallantoic membrane and the rabbit corneal micropocket lack the variety of cytokines and chemical gradients that are native in vivo. As a consequence, the result of these assays has often been disappointing because of the lack of a long-lasting vessel formation. Thus, a robust in vitro model of human origin would be useful for mechanistic studies and screening angiogenesis drugs for humans.
  • hPE multi-protein human placental extract or matrix
  • hPM a viscous protein compound, rich in cytokines and angiogenesis related growth factors, that promotes angiogenesis
  • hPE can induce and modulate the initial stages of angiogenesis in vitro for a limited period of time. Additionally, hPE has also been shown to significantly reduce fibrosis.
  • the present example provides a method for the sustained delivery of hPE so that growth factors within it remain functional long enough to enable the formation of mature capillary networks.
  • microparticles were developed as delivery system due to their versatility and to their ability to efficiently encapsulate polypeptides and release them at a continuous rate for a long period of time 22 .
  • Microparticles are solid, approximately spherical particles with a size ranging from 1 to 1000 ⁇ and with a large surface-to-volume ratio 23 . They can be prepared with several different substances, both natural (e.g. starches, gums) and synthetic (e.g., polylactic and polyglycolic acid) and using different techniques such as hot melt extrusion, spry drying or solvent removal 24 .
  • the release rate of microparticles can be modulated by changing their size: smaller particles dissolve more quickly than large ones due to their increased surface-to-volume ratio. For this reason, it is possible to modulate the delivery rate by combining particles of different sizes 25 .
  • Drug or protein release from microparticles usually occurs by simple matrix bioerosion. This process involves the erosion of the particle surface which is then followed by bulk erosion and entrance of the releasing medium in the particles pores 26,27 .
  • biodegradable gelatin microparticles were prepared and used to performed a 3D in vitro angiogenesis assay.
  • Angiogenesis assays are cell culture systems that reproduce in vitro or in vivo the definitive elements of angiogenesis under simplified, defined and controlled conditions 28 . They can be two-dimensional or three- dimensional.
  • ECs develop tubular structures both on the surface of the substrate and that invade the surrounding matrix, usually constituted by a biogel 13 .
  • the present example provides an embodiment of a 3D in vitro angiogenesis assay to promote the formation of a long lasting vascular network within an implanted graft.
  • the assay is prepared by embedding Human Umbilical Vein Endothelial Cells (HUVECs) together with PE-loaded microparticles in a collagen type I matrix. The angiogenic response of HUVECs at different incubation times was then analyzed.
  • HUVECs Human Umbilical Vein Endothelial Cells
  • the cell pellet was resuspended in Media (25 ml of glutamine, 0.5 ml of hydrocortisone, 0.5 ml of ascorbic acid, 10 ml of FBS, 1.25 ⁇ _ of VEGF, and 1.25 ⁇ _ of bFGF added in 500 ml of VascuLife Basal Media) and HUVECs were counted using a hemocytometer.
  • hPM angiogenicity was assessed using an isolation methods described in the example above.
  • a vial containing the extract was thawed and pipetted (100 ⁇ _ per cm 2 ) in the wells of a 96 well plate.
  • HUVECS were prepared for plating by direct pipetting of the cell solution at 20,000 cells/cm 2 .
  • Angiogenic Media was added to each prepared sample of the plate (200 ⁇ _ per cm 2 ) and this latter was placed in a humidified 5% C0 2 incubator at 37°C.
  • hPE retains bioactivity overtime, it was stored (in an incubator at 5% C0 2 and 37°C) for varying amounts of time including 20 days, 15 days, 9 days, 7 days, 5 days, 3 days and 1 day. Then, a film hPE from each time point was coated onto a tissue culture 96 well plate and seeded with HUVECs as described above. Cells were cultured for 3 days, and angiogenic networks were qualitatatively characterized.
  • hPE angiogenicity as a function of number of innoculations.
  • the response of HUVECs to the number of hPE inoculations was also analyzed.
  • Gelatin microparticles were prepared using the method described by Tabata, et al. (incorporated by reference herein with respect to the preparation of gelatin microparticles) 33 . All the reagents used were obtained from Fisher Scientific. Briefly, a 10% wt aqueous solution of Type B gelatin was prepared by adding 1 g of gelatin to 9 ml_ of deionized water. Temperature was increased to 45°C and the solution was added dropwise via a syringe and a 21 -G needle to 375 ml of warm (45°C) olive oil under constant stirring at 400 rpm. After 10 min the emulsion temperature was decreased to 15°C and stirring was maintained for 30 min to induce gelation. 100 ml_ of chilled acetone were added and the emulsion was stirred for 1 hour.
  • Microparticles were removed by vacuum filtration, washed with acetone and dried. Once dried, they were placed in a aqueous solution containing 0.1 % wt of Tween 80 and 0.5% wt of Gluteraldehyde. The solution was constantly stirred at 125 rpm at 4°C for 15 hours to facilitate the crosslinking of the microparticles.
  • Crosslinked microparticles were collected by vacuum filtration, washed in deionized water and then agitated in 100 ml_ of 10-mM glycine aqueous solution to block any unreacted gluteraldehyde. After 1 hour, microparticles were again collected by filtration, washed in deionized water and freeze-dried. Cross-linked freeze-dried gelatin microparticles were loaded by incubating 100 ⁇ _ of pure hPM per mg of microparticles. The mixture was vortex at maximum speed and incubated overnight at 4°C to allow adsorption to occur.
  • a non-planar 3D in vitro angiogenesis assay was prepared using a collagen type I matrix in which HUVECs and PE-loaded microparticles were embedded.
  • the collagen hydrogel matrix was prepared by mixing 8 ml_ of chilled Vitrogen Collagen, 1 ml. of sterile PBS and 1.166 ml. of 0.1 M NaOH solution. A transition in the color of the solution from red to purple indicated a pH change. The pH of the solution was checked with a pH paper and adjusted to 7.4 by the addition of few drops of 0.1 M NaOH or 0.1 M of HCI solution.
  • Calcein AM staining was carried out using the Live-Dead Assay (Invitrogen-Life Technologies, NY, USA). Briefly, Calcein AM was pipetted directly in the media present in the culture well with a final concentration of 2 ⁇ g/ml. The dyed cells where incubated for 30 min at 37°C and they were then observed using an inverted fluorescence microscope (Zeiss Axiovert 200 Inverted Fluorescence Microscope). Qualitative and Quantitative Network Formation Analysis. The analysis of the cells response to experimental conditions in in vitro models of angiogenesis has been done in previous studies in several semiquantitative and quantitative methods (each of which is incorporated by reference herein) 34"37 . Both morphological (mean tubule length) and topological (number of branching points and number of meshes) parameters have been taken in to account since they allow the characterization of the spatial organization of the ECs in the capillary-like network.
  • branch points that are nodes where branches meet or from where tubules sprout, were identified and counted. Also the meshes of the cell network, identified by avascular zones surrounded by hexagonally arranged vessels 38 , were manually counted. Finally, tubule length was assessed by drawing a line (dotted white line in the zoomed image FIG. 8) along each tubule and the measure of that line was automatically calculated by the software.
  • SEM Scanning Electron Microscopy
  • Angiogenic potential of PE The data collected show that the extent of the angiogenic response of HUVECs to PE is strongly affected by incubation time and by the number of inoculations of PE given.
  • HUVECs incubated with PE formed angiogenic-like networks whose morphology varied as function of incubation time (FIG. 9A-B).
  • HUVECs did not yet form a defined network (FIG. 9A, DAY 1 ).
  • the meshes were numerous
  • the network was still visible but it started to degrade: the tubules were longer (170.56 ⁇ 16.51 ⁇ ) but less numerous and thinner. Meshes were difficult to identify and their number decreased (36.33 ⁇ 6.02). As for the BPs, their number slightly decreased (63 ⁇ 4).
  • the degradation of the network may be due to cells having already exhausted the angiogenic molecules contained in the PE. No tubule formation was observed in the control plate (top right corner in each imagine) thus indicating that the change in the cell morphology is not due to stress or other external causes.
  • FIG. 9B A semi-quantitative analysis of the spatial organization of HUVECs is presented in the histogram in FIG. 9B.
  • the bars refer to mean tubule length, number of BPs and number of meshes. It is possible to notice that a statistical difference has been found between Day 1 and Day 5 in all three parameters taken into account.
  • the bar graph in Fig. 10B presents a semi-quantitative analysis of the spatial organization of HUVECs.
  • the bars referring to mean tubule length, number of BPs and number of meshes, are divided in three groups according to the number of inoculations of PE received (1 , 2 or 3).
  • Data show an increase in mean tubule length from cells that received one inoculation (129.71 ⁇ 12.88 ⁇ ) to those that received three (153.89 ⁇ 8.54 ⁇ ), suggesting that the cells were organizing into a more mature network.
  • This finding is also supported by the observed decrease in both the number of meshes (from 76.67 ⁇ 19.74 to 43.89 ⁇ 6.52) and of BPs (from 1 10.78 ⁇ 15.40 to 80.67 ⁇ 1 1.92). From the statistical analysis performed, a difference was found in all three parameters between cells that received one inoculation and cells that received three.
  • Blank microparticles presented a smooth surface and a regular shape. After loading (bottom row) the particles were bigger and had an irregular surface. These two aspects implies: (i) adhesion of the PE to the surface (adsorption), and (ii) penetration of the PE inside the particles (absorption) 27 .
  • FIG. 13 shows the degradation profile of non-crosslinked microparticles. This experiment was performed only with blank microparticles to assess the degradation kinetics of gelatin in PBS. An initial burst was observed: after six hours the cumulative percent release was 6.45% ⁇ 0.12 (FIG. 13, inset). The burst was then followed by a slower release. After 20 days, total cumulative release was 18.65% ⁇ 0.09 (Fig. 13, main graph).
  • FIG. 14A shows in vitro degradation of blank microparticles (black, NO PE) and in vitro release of microparticles loaded with PE (grey, PE). In both cases a small initial burst was observed. After 48 hours the cumulative percent release from loaded microparticles was 1.03% ⁇ 0.08, while for the blank ones, it was 0.76% ⁇ 0.05, followed in both cases by a near-constant release. After 22 days cumulative percent release from loaded and blank microparticles was nearly the same: 4.65% ⁇ 0.07 and 4.65% ⁇ 0.1 1 , respectively, as it is highlighted also in the graph. On day 23 the release form blank microparticles was slightly greater than the one from the loaded particles (5.18 ⁇ 0.05 and 4.92 ⁇ 0.09 respectively). Base on the evidence that most of the PE has been released, the experiment was concluded.
  • FIG. 14B shows the difference in percent of release between loaded and blank microparticles. Two peaks can be observed: the first from the first hour to day 5 and the second from day 5 to day 22. The first peak indicates that the PE bonded to the particles surface was released at first and in a shorter period of time. The second peak, probably due to the erosion of the bulk of the particles, lasts longer and total cumulative release is greater. This indicates that PE is gradually released from pores or channels formed in microparticles by releasing medium.
  • 3D in vitro angiogenesis assay After having determined the optimal cell density and PE volume, the angiogenesis assay was prepared as described in the Methods, above. After 3 days of culture, no sign of tubulogenesis was present and no difference could be noticed between HUVECs seeded with PE-loaded microparticles and the control (result not shown). After 5 days of culture, the cells changed morphology and sprouts were observed, even though no capillary-like network was yet formed (FIG. 15). Sprouting is the initial phase of angiogenesis, thus the presence of sprouts after five days of culture demonstrates an angiogenic response from HUVECs 41 .
  • the amount PE released by the particles may have been insufficient to promote angiogenesis in the time frame observed. However, the formation of sprouts indicates that the PE was released and angiogenic response was initiated.
  • the release rate of the microparticles can be optimized to promote tubule formation in a shorter time frame.
  • the findings of this example reveal that the angiogenic response of HUVECs is influenced not only by the incubation time but also by the number of inoculations of PE received.
  • the capillary network started forming one day after seeding, became well- defined after three days, and started degrading after five.
  • HUVECs form a more mature capillary network when they received more than one inoculation of PE.
  • the networks did not degrade after five days as in the experiment discussed above.
  • the extract maintains its angiogenic potential until the fifteenth day of storage. Given all these characteristics of the PE, it can be considered suitable for a constant and sustained released over time.
  • a method of drug delivery for the multi-protein mixture was developed using biodegradable gelatin microparticles to further preserve bioactivity, to control and to extend the delivery of hPE, with the goal of improving its ability to induce and modulate angiogenesis
  • biodegradable gelatin microparticles were prepared as a delivery system for PE. Absorption and adsorption of hPE into the gelatin particles was assessed by SEM examination and by an analysis of in vitro release kinetics. SEM analysis showed an increase in size and changes in morphology between loaded and blank particles, which is the result of adhesion of hPE to the surface (adsorption) and/or penetration of into the particles (absorption). Release kinetics showed two peaks, with the first peak believed to be the result of release of proteins bonded on the surface of the microparticles, with this peak being smaller than the second peak and occurring over a shorter period of time. The second peak was believed to result from bulk erosion because it had higher total cumulative release over a longer period of time. Despite an initial “burst", the release kinetics was close to a zero-order profile after day 1. The analysis revealed the particles were suitable for use as a vehicle for the sustained release of the extract in a 3D in vitro angiogenesis assay.
  • biodegradable gelatin microparticles can be used as a vehicle for sustained release of the multiprotein hPE in a 3D angiogenesis assay.
  • Cell sprouting was observed after five days of culture using hPE-loaded-gelatin microparticles, but no
  • An increase in the amount of hPM released by the particles or an extension of the culture time may promote the formation of more interconnected capillary networks.
  • hPE was encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles to extend the release period. Microparticle preparation was optimized for hPE loading, morphological features (size, encapsulation efficiency, porosity) were characterized and protein release was profiled.
  • PLGA poly(lactic-co-glycolic acid)
  • human placenta was used to derive a human placental extract, referred to in the present example as ahuman placental matrix (hPM) that is capable of inducing capillary network formation and contains angiogenic and immunomodulatory proteins, as described in the examples above.
  • hPM human placental matrix
  • endothelial cells (HUVECs) seeded onto hPM were shown above to form angiogenic networks with upregulation of angiogenic genes, and in vivo the matrix was shown to induce blood vessel formation within dosed bioscaffolds, while inhibiting tissue fibrosis.
  • endothelial cells receiving multiple hPM inoculations at regular time points formed more stable, longer lasting angiogenic networks in comparison to cells receiving only a single inoculation whose networks begin to degrade after 5 days.
  • approaches for controlled delivery of the matrix over time were investigated to allow longer lasting and more stable capillary network formation.
  • hPM complex and heterogeneous nature of hPM can complicate controlled release mechanisms. For example, different charges and chain properties of proteins can have effects on loading efficiency, because of reciprocal interaction and interaction with the material used for the controlled release.
  • Both natural and synthetic microparticle materials have been investigated for their potential to encapsulate proteins. Natural materials such collagen, chitosan, and alginate offer biocompatibility and non-aggressive encapsulation technique, and they have degradation rates of between about 7 and 10 day, which allows some degree of sustained release but may limit use for longer term sustained protein release 11 , 12, 13 . Chemical or photochemical crosslinking could slow degradation of these microparticles 9, 14 .
  • PLA-copolymers have been used for protein encapsulation in biomedical applications, and are FDA approved biocompatible synthetic materials 15,16,17 . These polymers can provide a long lasting controlled release of proteins, and can be used to create composites and multi-layered microparticles 15,18 . Among these materials, PLGA or poly(lactic-co-glycolic acid) is used for controlled release of specific growth factors (for example BMP, VEGF, bFGF) 19, 20, 21 .
  • specific growth factors for example BMP, VEGF, bFGF
  • the present example describes a composition and encapsulation technique for hPM using PLGA microparticles and evaluates the effect of the controlled release of this mixture in a 3D culture of endothelial cells.
  • the PLGA synthesis protocol was optimized to suit multiprotein hPM release for applications in cell culture. Considerations included optimization of microparticle size (to be suitable for regenerative medicine applications), high loading and encapsulation efficiency, low initial burst, and controlled release profile. Additionally, it was confirmed that hPM was released from the microparticles in concentrations adequate to induce angiogenesis using endothelial cells. Following microparticle synthesis and loading, analysis was performed to evaluate if the encapsulation process was selective for hPM proteins.
  • the effect of controlled hPM release on the induction of angiogenesis was assessed using a 3D culture system with hPM-loaded microparticles embedded into an alginate-based hydrogel seeded with HUVECs. Cell behavior was assessed at specific time points during 28 days to evaluate the response between cells receiving single, direct inoculations of hPM at day 1 in comparison to cells receiving a controlled dose of hPM from PLGA microparticles throughout the entire period of culture.
  • HUVECs Effect of multiple bolus hPM innoculations. Analysis of the effect of hPM dosing profiles during angiogenic network formation was determined in vitro using HUVECs by pipetting and evenly coating wells of a 96-well tissue culture plate using an orbital shaker at 30 rpm for 1 minute. The plate was then incubated at 37°C for 30 minutes to allow the hPM to warm up. HUVECs were suspended in Angiogenic media, pipetted on the top of the hPM and then placed in a humidified 6% C0 2 incubator at 37°C. For a control, HUVECs were cultured at 20,000 cells/cm 2 in Angiogenci media.
  • hPM was added only inoculated on day 1 (day of seeding)
  • hPM was innoculated on day 1 and 3
  • hPM was innoculated on day 1 ,3 and 5.
  • Cells were cultured for 7 days and media was replaced on day 3 and 5.
  • As a control cells were directly seeded at the same density directly onto the bottom of the plate, and media was replaced every two days. Cells were stained on day 7, imaged with a fluorescence microscope and angiogenesis was quantified by analyzing images as described in the
  • PLGA Microparticle Preparation PLGA (Poly(DL-lactide-co-glycolide)) microparticles were prepared using a water-in oil-in-water emulsion (PROTOCOL 1 ) (Durect, Cupertino, CA).
  • One water solution (W1 ) was prepared using the hPM protein mixture as previously described in Example 1
  • the other water solution (W2) was prepared by dissolving 2g of polyvinyl alcohol (wt 30000-70000, 87-90% hydrolyzed, Sigma-Aldrich, St. Louis, MO) in "l OOmL of Dl water.
  • the oil solution (O) was obtained by dissolving 90 mg of PLGA in 3 mL of chloroform until the solution appeared clear.
  • W1 was added to O and homogenized at 20000 rpm for 1 minute.
  • the obtained primary emulsion was added dropwise in W2 while stirring at 300 rpm.
  • the resulting secondary emulsion was covered loosely with an aluminum foil and left to stir (300 rpm) overnight in a fume hood to let the solvent evaporate.
  • the secondary emulsion was centrifuged at 1000 rpm for 10 minutes, the supernatant removed and the microparticles washed two more times with Dl water.
  • the hardened microparticles were suspended in Dl water, freeze-dried for 48 hours and then stored at 4C until needed.
  • single protein loaded PLGA microparticles were created by substituting bovine serum albumin (BSA) to make W1 instead of the hPM (Sigma-Aldrich, St. Louis, MO).
  • BSA bovine serum albumin
  • PLGA microparticle morphological characterization Morphologic features of PLGA microparticles were evaluated using microscopy. The average size of the microparticles was evaluated using an inverted optical Leica microscope with attached color digital camera (Leica DM IL LED, Leica Microsystems Inc., IL, USA). Images taken were analyzed using the free software ImageJ 1.45s (Wayne, Rasband - National Institutes of Health, USA - http://imagej.nih.gov/ij/). The diameter of each particle was determined by manually tracing the particles followed by
  • hPM loading efficiency in PLGA microparticles was determined using a direct measurement of encapsulated protein after microparticles dissolution. It was performed by adaptation of a hydrolysis technique described in Ravi., et al., Development and characterization of polymeric microspheres for controlled release protein loaded drug delivery system. 70, (2008) and Igartua, M. et al., Stability of BSA encapsulated into PLGA microspheres using PAGE and capillary electrophoresis. International Journal of Pharmaceutics 169, 45-54 (1998), both of which are incorporated by reference herein for the hydrolysis technique.
  • HUVECs were suspended in media and gently suspended in the Alginate matrix before polymerization by pipetting of a 0.054M Calcium Chloride solution.
  • the Alginate-cell suspensions were held steady to allow 15 mintues for polymerization, then the gels were washed with PBS, culture media was added, and the plate was incubated in a humidified 6% C02 incubator at 37°C.
  • the concentration of hPM mixed in each respective gel was equal to the concentration of protein released after 21 days from the embedded microparticles (78 ⁇ g of proteins per cm 2 ).
  • the effect of sustained controlled-hPM release from microparticles was compared to bolus inoculations of hPM at 7, 14, 21 and 28 days by morphological characterization of cell networks within gels stained using Calcein AM (Life
  • Angiogenesis quantification Quantification of angiogenesis output was performed using ImageJ 1.45s (NIH, Bethesda, MD). Images at 5X magnification taken using the inverted fluorescence microscope were processed evaluating and quantifying the following parameters: number of meshes, number of branching points and length of the tubule-like structures formed by HUVEC during time as described in Example 1.
  • Significance was calculated using ANOVA tests were more than two conditions were evaluated, and specific differences were evaluated using post-hoc tests. When only two conditions were compared unpaired, two-tailed, Student's t-Test with unequal variance were used. Significance levels were set at * p ⁇ 0.05.
  • Protocol 1 PLGA microparticles resulting from protocol 1 had an average size of 447 ⁇ 32 ⁇ and a normal distribution of sizes ranging from 100 to 1000 ⁇ . Three batches of microparticles were prepared under the same conditions, and repeatable distributions could be demonstrated. The loading efficiency of hPM in the PLGA microparticles was 64 ⁇ 4 % (data not shown).
  • Control BSA-loaded PLGA microparticles had an average size of 239.60 ⁇ 9.45 ⁇ and a normal distribution of sizes from to 50 to 400 ⁇ , with 70% of microparticles in the range between 150 and 350 ⁇ .
  • the loading efficiency of BSA loaded PLGA microparticles was 76 ⁇ 3 % of the total amount of protein loaded initially.
  • In vitro release analysis showed a higher initial burst compared to hPM loaded microparticles at 20% of the total amount of BSA encapsulated, ( 51.52 ⁇ 1.76 ⁇ g mL), while 66.7 ⁇ 9.55 % ( 166.74 ⁇ 23.8 ⁇ g mL) of the protein initially encapsulated was released from PLGA microparticles after 21 days (data not shown).
  • FIGS. 18A-18F show the microparticle shape and FIGS. 18D-F show the microparticle surface porosity of particles prepared by protocols 2, 3, and 4, respectively.
  • FIG. 21 illustrates that after 7 days of culture, only a few sprouts and tubular structures were observed in HUVECs cultured in Alginate hydrogels with embedded hPM-loaded microparticles (and no mature network were observed), whereas angiogenic formations in matrices containing pure hPM were comparatively more mature in the same timeframe, with average tubule lengths of 207.90 ⁇ 15.31 ⁇ , with 1.27 ⁇ 0.84 meshes/mm 2 , and with 9.60 ⁇ 0.70 branching points/ mm 2 .
  • the angiogenic networks which formed remained stable with only minor changes in the number of branch points/mm 2 (FIG. 21 ).
  • the average tubule length was 168.88 ⁇ 10.31 ⁇
  • the average number of branching points/mm 2 was 12.93 ⁇ 1.61
  • the average number of meshes was 3.17 ⁇ 0.93 per mm 2 .
  • Protein encapsulated using PLGA microparticles is a drug delivery system for controlled release of growth factors, with a commonly employed fabrication technique being the water in oil in water method. 19, 20, 21
  • a complex human derived multiprotein mixture placental matrix or hPM
  • results show that using hPM-loaded-PLGA microparticles, the release of a multiprotein fusion containing pro-angiogenic and fibrotic proteins can be sustained and controlled overtime.
  • hPM-loaded-PLGA microparticles allow the formation of stable angiogenic networks.
  • hPM had an influence on PLGA loading and microparticle morphology which results from the complex, multiprotein composition.
  • BSA single protein
  • hPM-loaded microparticles showed an average size significantly bigger than BSA loaded ones. This is likely the result of the complex interactions between proteins in hPM and the PLGA polymer, which have a wide array of different molecular charges, pH's, and sizes in comparison to the BSA-loaded-microparticles.
  • SDS-PAGE analysis revealed that despite the low concentration of proteins released from hPM-loaded-PLGA microparticles, a broad distribution could be observed in the supernatant with a composition similar to diluted hPM.
  • encapsulation efficiency did not correlate with the size of the microparticles, but instead was affected by the length of homogenization of the secondary emulsion, which is likely the result of the formation of fragmented incomplete particles caused by interactions between the heterogeneous protein mixture and the polymer.
  • hPM-loaded-microparticles were incorporated into an alginate-based angiogenesis assay to assess endothelial cells (HUVECS) response to controlled hPM release overtime.
  • the 3D angiogenesis assay was created by embedding hPM-loaded- microparticles into alginate gel, which was chosen as the basis for the assay because its cost effective processability and overall good properties as an extracellular material.
  • HUVECS formed angiogenic tubules within the gel in regions close to the embedded hPM-loaded-PLGA microparticles, despite that the total amount of protein delivered from the microparticles (78 ⁇ g/cm 2 ) was lower than the concentration delivered in previous 2D angiogenesis studies using bolus injections (263 ⁇ g/cm 2 ).
  • the use of hPM-loaded-microparticles allowed for an extended and sustained angiogenic network formation over 28 days.
  • this example describes the optimization of a PLGA microparticle synthesis protocol for applications to deliver complex, multiprotein mixtures with a low initial burst and a high encapsulation efficiency.
  • the methods developed here were then applied to develop a novel angiogenesis assay, which allowed the formation of sustained angiogenic networks within alginate matrices.
  • the techniques developed have applications in the delivery of any complex serums and protein mixture, with applications in a wide range of tissue engineering and
  • collagen, fibrin, laminin with embedded serum-loaded PLGA microparticles to develop improved in vitro angiogenesis assays, and to gain a better understanding of the role played by the sustained serum delivery within cell seeded matrices.
  • This example describes an embodiment of a method for hPM encapsulation, and angiogenic structure formation was observed with an Alginate-based angiogenic assay. A sustained angiogenic response over an extended period of 21 days was observed within the 3D hydrogel culture system. This confirmed the effectiveness of the controlled hPM release approach to guide formation and maintenance of capillary networks.

Abstract

La présente invention concerne des compositions comprenant un extrait placentaire humain et des microparticules biodégradables, des compositions de modulation de l'angiogenèse à libération prolongée, des compositions et des méthodes permettant de libérer un extrait placentaire vers une cible pendant une période de temps, et des méthodes permettant d'induire et/ou de moduler l'angiogenèse et d'identifier des modulateurs de l'angiogenèse. La présente invention concerne également des procédés de préparation d'une composition, comprenant un extrait placentaire qui peut induire et/ou moduler l'angiogenèse.
PCT/US2015/029666 2013-04-02 2015-05-07 Compositions de modulation de l'angiogenèse à libération prolongée et méthodes pour l'induction et la modulation de l'angiogenèse WO2015171880A1 (fr)

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US15/309,306 US10300091B2 (en) 2013-04-02 2015-05-07 Sustained release angiogenesis modulating compositions and methods for induction and modulation of angiogenesis
CA2948348A CA2948348A1 (fr) 2014-05-08 2015-05-07 Compositions de modulation de l'angiogenese a liberation prolongee et methodes pour l'induction et la modulation de l'angiogenese
JP2016566709A JP2017520517A (ja) 2014-05-08 2015-05-07 持続放出性血管新生調節組成物ならびに血管新生を誘導および調節する方法
KR1020167034051A KR20160147055A (ko) 2014-05-08 2015-05-07 서방형 혈관신생 조절용 조성물 및 혈관신생 유도 및 조절을 위한 방법
AU2015255907A AU2015255907A1 (en) 2014-05-08 2015-05-07 Sustained release angiogenesis modulating compositions and methods for induction and modulation of angiogenesis
EP15789993.1A EP3139937A4 (fr) 2014-05-08 2015-05-07 Compositions de modulation de l'angiogenèse à libération prolongée et méthodes pour l'induction et la modulation de l'angiogenèse

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