Patent Application
20240016979
Embodiments of the disclosure concern at least the fields of cardiology and medicine.
Myocardial infarction (MI) associated heart failure is the leading cause of death in the U.S.(1) MI occurs when blood flow to the heart from coronary arteries is occluded, causing ischemia and subsequent myocardial tissue death. Myocardial tissue is unable to effectively regenerate following MI, thus leading to scar formation, left ventricular remodeling, and eventual heart failure.(2,3) Several tissue engineering strategies have been developed to prevent scarring and promote cardiac regeneration, including cell-based therapies, porous scaffolds, cardiac patches and hydrogels.(4-9) Among them, an injectable natural or synthetic hydrogel is minimally invasive and promising for in situ cardiac tissue repair for infarcted hearts.(10)
Decellularized mammalian Extracellular matrix (ECM), as a natural material, has been extensively investigated in regenerative medicine.(11-18) ECM hydrogel retains the full biochemical complexity and inherent bioactivity of the native matrix, which could facilitate tissue regenerative capability.(9, 19, 20) Although certain properties of ECM hydrogels are widely conserved regardless of source tissue, some characteristics vary markedly and are influenced by source species, source tissue and processing methods. In particular, human amniotic membrane (hAM) can be easily obtained and processed from the placenta without ethical concerns. The matrix derived from human amniotic membrane has various components and bioactivities, which allow such matrix to be widely applied in corneal transplantation, retinal regeneration, liver regeneration, and wound healing.(21-24) Due to its anti-inflammatory effects, anti-fibrotic effect, and angiogenic potentials,(25-28) materials derived from hAM could beneficial for restoring cardiac function following MI.
Methods and materials that can utilize injectable hydrogel matrices to restore cardiac function following myocardial infarction are needed. Embodiments of the invention meet this need.
As discussed below, we have developed an injectable human amniotic membrane (hAM) matrix that enhances cardiac regeneration following myocardial injury (MI). The invention disclosed herein provides human amniotic membranes isolated from human placenta and engineered to form a thermo-responsible, injectable hydrogel at temperature ranges that fall within human body temperature. Embodiments of the invention include such amniotic membrane hydrogel matrix compositions and methods of making and using them. The disclosure provided herein teaches the engineering of these injectable hAM matrices and then their efficacy in promoting wound healing and tissue regeneration. Embodiments of the invention are useful for attenuating degenerative changes in cardiac function following MI, material properties which have broad applications in tissue regeneration.
The invention disclosed herein has a number of embodiments. Embodiments of the invention include, for example, methods of making human amniotic membrane hydrogel matrix compositions. Typically, these methods comprise combining a human amniotic membrane with agents selected to remove cells and nucleic acids from the human amniotic membrane; and then processing the human amniotic membrane hydrogel matrix so as to form either a powder, or alternatively a liquid/gel composition that is suitable for in vivo use. These methods can include, for example, rinsing and/or dialyzing the decellularizing human amniotic membrane so as to remove agents selected to remove cells and nucleic acids; lyophilizing the rinsed and/or dialyzed decellularized human amniotic membrane; forming a dry powder from the lyophilized human amniotic membrane so that a powder composition is formed. Typically, the methods further include solubilizing this dry powder to form a solution; adjusting the pH of the solubilized dry powder solution; and then lyophilizing the pH adjusted solubilized dry powder solution, so that the human amniotic membrane hydrogel matrix powder composition is formed. Typically in these methods, the human amniotic membrane hydrogel matrix composition is formed using methodological steps and or reagents selected so that the glycosoaminoglycan content of the human amniotic membrane hydrogel matrix composition is at least 80% (e.g. about 85%) of the glycosoaminoglycan content of the native human amniotic membrane; and/or the human amniotic membrane hydrogel matrix composition exhibits a collagen content that is at least 40% or (e.g. about 45%) of the collagen content of the native human amniotic membrane.
Typically, the methods of making the human amniotic membrane hydrogel matrix composition further comprise resuspending the human amniotic membrane hydrogel matrix powder composition in an aqueous solution so as to form a liquid/gel injectable human amniotic membrane hydrogel matrix. In illustrative embodiments of the invention, the composition forms a thermo-responsible, injectable hydrogel at temperature ranges that fall within body temperature. Typically, for example, the injectable human amniotic membrane hydrogel matrix composition is designed to form a viscous liquid at temperatures from 0° C. to 20° C.; and to form a gel at 37° C. In embodiments of the invention, the injectable human amniotic membrane hydrogel matrix composition can further comprise additional constituents such as crosslinking agents, and/or pharmaceutical excipients such as those selected from the group consisting of: a preservative, a tonicity adjusting agent, a detergent, a viscosity adjusting agent, a sugar and a pH adjusting agent; and/or a therapeutic agent such as a wound healing agent, an anti-fibrotic agent, an anti-inflammatory agent, a hemostatic agent, or a chemotherapeutic agent.
Embodiments of the invention include compositions formed by the methods disclosed herein. Embodiment of the invention include, for example, compositions of matter comprising a human amniotic membrane hydrogel matrix, wherein the human amniotic membrane hydrogel matrix is decellularized, and is in either a powder form, or is in solution. Typically, in these embodiments, the human amniotic membrane hydrogel matrix comprises a glycosoaminoglycan content that is at least 80% of the glycosoaminoglycan content of a native human amniotic membrane; and/or the human amniotic membrane hydrogel matrix comprises a collagen content that is at least 45% of the collagen content of the native human amniotic membrane; and/or at least 95% of native DNA in the human amniotic membrane hydrogel matrix has been removed. In certain embodiments of the invention, the hAM hydrogel gel exhibits certain selected material properties, for example at 37° C., the composition exhibits a shear modulus of 1 PA-20 kPA, for example about 5 PA to about 10 PA (e.g., about 7.5±2.4 PA) at a frequency of about 1 Hz. Typically, the injectable human amniotic membrane hydrogel matrix compositions form a viscous liquid at temperatures from 0° C. to 20° C.; and form a gel at 37° C.
Yet another embodiment of the invention is a method of delivering a liquid/gel hAM composition disclosed herein to a preselected site such as a site or trauma or injury in vivo. These method comprise: disposing the composition in the form of a liquid (typically at a temperature between 0° C. to 20° C.) in a vessel having a first end comprising an opening and a second end; applying a force to the second end of the vessel, wherein the force is sufficient to force the liquid through the first end of the vessel; and delivering the composition out of the vessel through the opening and to the preselected site (e.g. a site having a temperature of about 37° C. where the liquid composition will then form a gel). In typical embodiments of the invention, the site is at an in vivo location, for example one where an individual has experienced cardiac injury.
Related embodiments of the invention include methods of using the hAM compositions disclosed herein to promote wound healing and tissue regeneration (e.g., cardiac regeneration). For example, embodiments of the invention include methods of inhibiting fibrosis at a site of cardiac injury in an individual, the methods comprising: disposing a liquid/gel hAM composition disclosed herein at the site of the cardiac injury such that the composition modulates cardiac remodeling, so that fibrosis is inhibited.
In certain embodiments of the invention, the modulation of cardiac remodeling comprises an inhibition of negative ventricular remodeling; and/or a decrease in myocardial infarction size. Optionally in these methods, the composition further comprises additional constituents such a pharmaceutical excipient, a therapeutic agent and/or human cells.
Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.
The figures show illustrative aspects and embodiments of the invention.
In the description of embodiments, reference may be made to the figures which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized, and structural changes may be made without departing from the scope of the present invention.
Ischaemic heart disease represents the leading cause of death worldwide. Heart failure following myocardial infarction (MI) is associated with severe fibrosis formation and cardiac remodeling. Recently, injectable hydrogels have emerged as a promising approach to repair the infarcted heart and improve heart function through minimally invasive administration. As discussed below, we have developed a novel injectable human amniotic membrane (hAM) matrix to enhance cardiac regeneration following MI. Briefly, human amniotic membrane was isolated from human placenta and engineered to form a thermo-responsive, injectable gel around body temperature. Ultrasound-guided injection of the hAM matrices of the invention into rat MI hearts significantly improved cardiac contractility, as measured by ejection fraction (EF), and decreased fibrosis. These results demonstrate the feasibility of engineering an injectable hAM matrix and its efficacy in attenuating degenerative changes in cardiac function following MI, material properties which have broad applications in tissue regeneration.
Here we report methods for making and using a decellularized hAM matrix. In vitro cell culture shows that cell proliferation and other biological activities of the hAM matrices disclosed herein are preserved. Additionally, as disclosed herein, when a thermo-responsive hAM matrix hydrogel is injected into the rat MI hearts, this composition demonstrates a number of beneficial effects, for example the enhancement of cardiac ejection fraction as well as the reduction of fibrosis.
Embodiments of the invention include, for example, methods of making human amniotic membrane hydrogel matrix compositions. Such methods typically comprise combining a human amniotic membrane with agents selected to remove cells and nucleic acids from the human amniotic membrane; and then processing the human amniotic membrane hydrogel matrix so as to form either a powder, or alternatively a liquid/gel composition that is suitable for in vivo use. These methods can include, for example, rinsing and/or dialyzing the decellularizing human amniotic membrane so as to remove agents selected to remove cells and nucleic acids; lyophilizing the rinsed and/or dialyzed decellularized human amniotic membrane; forming a dry powder from the lyophilized human amniotic membrane so that a dry powder composition is formed. Typically, the methods further include solubilizing this dry powder to form a solution; adjusting the pH of the solubilized dry powder solution; and then lyophilizing the pH adjusted solubilized dry powder solution, so that the human amniotic membrane hydrogel matrix powder composition is formed. Typically in these methods, the human amniotic membrane hydrogel matrix composition is formed using methodological steps and or reagents selected so that the glycosoaminoglycan content of the human amniotic membrane hydrogel matrix composition is at least 10%, 20%, 30%, 40% 50%, 60%, 70% or 80% of the glycosoaminoglycan content of the native human amniotic membrane; and/or the human amniotic membrane hydrogel matrix composition exhibits a collagen content that is at least 10%, 20%, 30%, 40% or 45% of the collagen content of the native human amniotic membrane. In certain embodiments of the invention, the human amniotic membrane hydrogel matrix comprises a glycosoaminoglycan content that is not more than 90% or 95% of the glycosoaminoglycan content of a native human amniotic membrane. In some embodiments of the invention, the human amniotic membrane hydrogel matrix comprises a collagen content that is not more than 50% or 60% of the collagen content of the native human amniotic membrane.
Typically, the methods of making the human amniotic membrane hydrogel matrix composition further comprise resuspending the human amniotic membrane hydrogel matrix powder composition in an aqueous solution so as to form a liquid/gel injectable human amniotic membrane hydrogel matrix. In illustrative embodiments of the invention, the composition forms a thermo-responsible, injectable hydrogel at temperature ranges that fall within body temperature. Typically, for example, the injectable human amniotic membrane hydrogel matrix composition is designed to form a viscous liquid at temperatures from 0° C. to 20° C.; and to form a gel at 37° C. In embodiments of the invention, the injectable human amniotic membrane hydrogel matrix composition can further comprise additional constituents such as crosslinking agents, and/or pharmaceutical excipients such as those selected from the group consisting of a preservative, a tonicity adjusting agent, a detergent, a viscosity adjusting agent, a sugar and a pH adjusting agent; and/or a therapeutic agent such as a wound healing agent, an anti-fibrotic agent, an anti-inflammatory agent, a hemostatic agent, or a chemotherapeutic agent; and/or mammalian cells.
Embodiments of the invention include compositions formed by the methods disclosed herein. Embodiment of the invention include, for example, compositions of matter comprising a human amniotic membrane hydrogel matrix, wherein the human amniotic membrane hydrogel matrix is decellularized, and is in either a powder form, or is in solution. Typically in these embodiments, the human amniotic membrane hydrogel matrix comprises a glycosoaminoglycan content that is at least 10%, 20%, 30%, 40% 50%, 60%, 70% or 80% of the glycosoaminoglycan content of a native human amniotic membrane; and/or the human amniotic membrane hydrogel matrix comprises a collagen content that is at least 10%, 20%, 30%, 40% or 45% of the collagen content of the native human amniotic membrane; and/or at least 85%-95% of native DNA in the human amniotic membrane hydrogel matrix has been removed.
Certain embodiments of the invention comprise a (hAM) hydrogel composition, wherein: the human amniotic membrane comprises a glycosoaminoglycan content comprising from about 70% to about 90% of the glycosoaminoglycan content found in native human amniotic membranes; the human amniotic membrane comprises a collagen content comprising from about 30% to about 60% of the collagen content found in native human amniotic membranes; and the human amniotic membrane comprises less than 5% or less than 10% of the DNA content found in native human amniotic membranes. Such human amniotic membrane hydrogel compositions of the invention have a number of unexpected and desirable material properties including, for example, an ability to form a viscous liquid at temperatures from 0° C. to 20° C.; and to form a gel at 37° C. In certain embodiments of the invention, such hAM hydrogel compositions exhibit other desirable material properties, including for example at 37° C., a shear modulus of 1 PA-20 kPA, for example about 5 PA to about 10 PA (e.g., about 7.5±2.4 PA) at a frequency of about 1 Hz (see, e.g.,
Yet another embodiment of the invention is a method of delivering a liquid/gel hAM composition disclosed herein to a preselected site such as a site or trauma or injury in vivo, the method comprising: disposing the composition in the form of a liquid (typically at a temperature between 0° C. to 20° C.) in a vessel having a first end comprising an opening and a second end; applying a force to the second end of the vessel, wherein the force is sufficient to force the liquid through the first end of the vessel; and delivering the composition out of the vessel through the opening and to the preselected site (e.g. a site having a temperature of about 37° C. where the liquid composition will then form a gel). In typical embodiments of the invention, the vessel is a catheter, and the site is at an in vivo location, for example one where an individual has experienced cardiac injury.
Related embodiments of the invention include methods of using the hAM compositions disclosed herein to promote wound healing and tissue regeneration (e.g., cardiac regeneration). For example, embodiments of the invention include methods of inhibiting fibrosis at a site of cardiac injury in an individual, the methods comprising: disposing a liquid/gel hAM composition disclosed herein at the site of the cardiac injury such that the composition modulates cardiac remodeling, so that fibrosis is inhibited. In certain embodiments of the invention, the modulation of cardiac remodeling comprises an inhibition of negative ventricular remodeling; and/or a decrease in myocardial infarction size. Optionally in these methods, the composition further comprises additional constituents such a therapeutic agent and/or human cells.
In embodiments of the invention, the injectable human amniotic membrane hydrogel matrix composition can further comprise additional constituents such as crosslinking agents (e.g. citric acid derivatives and the like), and/or pharmaceutical excipients such as those selected from the group consisting of: a preservative, a tonicity adjusting agent, a detergent, a viscosity adjusting agent, a sugar and a pH adjusting agent; and/or a therapeutic agent such as a wound healing agent, an anti-fibrotic agent, an anti-inflammatory agent, a hemostatic agent, or a chemotherapeutic agent; and/or mammalian cells (e.g. lymphocytes or stem cells). For compositions suitable for administration to humans, the term “excipient” is meant to include, but is not limited to, those ingredients described in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st ed. (2006) (hereinafter Remington's). In addition, the constituents of the compositions, their relative concentrations, and certain methodological steps can be selected to modulate hydrogel characteristics such as the gelation dynamics of the compositions of the invention (e.g., the relative concentrations of hAM within the compositions, the amounts of crosslinking agents within the compositions, the temperature of the compositions and the like).
Further aspects and embodiments of the invention are disclosed in the following sections.
Decellularization of Human Amniotic Membrane (hAM)
Full term human placentas with intact hAM were obtained from healthy donors undergoing caesarean section at the University of California, San Francisco or University of California, Davis Hospitals. The hAM was then identified and separated from the placenta and weighed. The tissue was subsequently frozen at −80° C. for 48 hours and thawed prior to decellularization. The membrane was thoroughly rinsed with water and soaked in 1 N NaCl for 15 minutes. Membranes were then treated in a solution containing 8 mM CHAPS (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate), 25 mM EDTA, and 1M NaCl at room temperature with moderate agitation. The CHAPS solution was removed and replenished every 2 hours, for 6 hours total. For comparison purposes, 8 mM SDS (sodium dodecyl sulfate) was used instead of CHAPS. Following CHAPS or SDS treatment, all membranes were subsequently washed with Tris-buffered saline (TBS, 3 times, 20 minutes each). To remove remaining nucleic acid material, membranes were incubated in a 90 U/ml benzonase solution (Sigma Aldrich Inc., St. Louis, MO) for 15 hours at 37° C. To enhance nuclease activity, benzonase was prepared in 50 mM Tris-HCL pH 8.0, with 0.1 mg/ml BSA, and 1 mM MgCl2. Following benzonase treatment, hAMs were washed repeatedly in TBS, followed by repeated washes with distilled water. hAMs were further decellularized and disinfected using a 0.1% peracetic acid, 4% ethanol and water. After repeated washing in TBS, and distilled water, hAMs were dialyzed against water at 4° C. for 4 days to remove excess reagents.
Following dialysis, portions of the decellularized hAM were snap-frozen in OCT compound (Sakura Finetek, Torrance, CA) and sectioned for histological analysis. Intact and untreated hAM was also frozen and sectioned for comparison. Sections were stained with DAPI or Hematoxylin & Eosin to visualize nuclei and matrix components respectively.
Decellularized hAMs were lyophilized overnight and subsequently ground into a dry powder using a motorized grinder. The decellularized hAM powder was solubilized by digestion in 0.2 mg/ml pepsin and 0.1N HCL for 48 hours at room temperature. A ratio of 20 mg hAM matrix per 1 ml pepsin/HCL solution was used. After 48 hours, the solution pH was adjusted to 8.0 using 10 N NaOH and 10×PBS. The pH adjusted matrix was then re-lyophilized overnight, ground into powder form, and stored at 4° C. Prior to experiments, the hAM matrix was sterilized in ethylene oxide gas overnight. To induce gelation, hAM matrix was dissolved in PBS (20 mg/ml) and allowed to gel at 37° C.
The remaining DNA content in the injectable hAM matrix was quantified using a Quant-iT™ PicoGreen® Assay (Invitrogen, Grand Island, NY). A DNeasy Blood & Tissue Kit® (Qiagen, Valencia, CA) was used to isolate DNA prior to quantification using the assay. The DNA content of CHAPS processed hAM matrix, and SDS processed hAM matrix is presented as a percentage of native, untreated hAM tissue.
Glycosaminoglycans (GAGs) are often reported to be lost following decellularization of tissue. For this reason, we quantified the GAG content of both CHAPS processed hAM matrix and SDS processed hAM matrix. GAG content was quantified using a colorimetric 1,9 dimethylmethylene blue (DMMB) assay as described previously.(29) This reagent specifically binds to the sulfate and carboxyl groups of sulfated GAGs to cause a metachromatic shift. This shift in absorption can then be quantified via spectroscopy at 530 nm. Lyophilized powder native and decellularized hAM tissue was first digested using proteinase K (1 mg proteinase K/80 mg hAM matrix). DMMB reagent (16 μg/mL) was then allowed to react with the samples. GAG content in each sample was then immediately quantified by measuring the absorbance at 530 nm and comparing to values obtained using known GAG concentrations. Final GAG content is given as a percentage of GAG content in the native tissue. The hAM matrix processed using CHAPS contained the highest amount of preserved GAG content. For this reason, the CHAPS hAM matrix was used in all in vitro and in vivo experiments.
The collagen content of the decellularized hAM matrix was measured by a hydroxyproline assay kit (Cell Biolabs Inc) by following the vendor's protocol as previously described.(30) Briefly, hAM powder was hydrolyzed by 12N hydrochloric acid for 3 hours at 120° C. to free hydroxyproline. The acid-hydrolyzed samples were further dried under vacuum evaporation at 80° C. for 40 minutes. Then chloramine T mixture was added to convert the hydroxyproline to a pyrrole. Finally, Ehrlichs Reagent was added to the solution to react with the pyrrole and produce a chromophore. The absorbance of the samples was measured at 540 nm and quantified by a standard curve of hydroxyproline. The collagen content was calculated by using the estimation that hydroxyproline makes up 13.5% of collagen.
Mass spectrometry was performed in order to survey the overall protein composition of the injectable hAM matrix. Samples were digested with trypsin and desalted with C18 spec tips prior to analysis. Proteins were separated and analyzed at the Vincent J. Coates Proteomics/Mass Spectrometry Laboratory by using multidimensional protein identification technology (MudPIT) with two-dimensional HPLC separation-tandem mass spectrometry. Peptide identification and data analysis were performed using SEQUEST.
Oscillatory rheology analysis was performed on a MCR 301 rheometer (Anton Paar). hAM matrix (400 μl) was allowed to gel for 1 hr at 37° C. on the rheometer between parallel plates with a 25-mm top probe at 0.81 mm gap width in a humidified chamber. Frequency sweep was performed from 10-0.1 Hz at 1% strain amplitude. Shear modulus was reported as the average of 5 storage modulus measurements taken at 1 Hz.
In vitro Characterization of Cell Growth on Injectable hAM Matrix
In vitro biocompatibility was examined by investigating the effect of injectable hAM matrix on the proliferation of bovine arterial endothelial cells (BAECs). BAECs were harvested from fresh bovine aortas as previously described using collagenase and gentle scrapping with a policeman.(31) All cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin antibiotic mix. Cell culture was maintained in a humidified incubator at 37° C.
For proliferation experiments, the surfaces of a 24-well tissue culture plate were coated with either 1 mg/ml rat tail collagen type-1 (BD Biosciences) or 5 mg/ml injectable hAM matrix. BAEC were seeded on coated surfaces at low density (1000 cells/cm2), medium density (5,000 cells/cm2), and high density (10,000 cells/cm2). After 48 hours, BAEC proliferation on hAM matrix and collagen type-1 surfaces was then measured and compared using a Click-iT® EdU Alexa Fluor® 488 Detection Kit (Life Technologies Inc). Samples were counterstained using DAPI. Cell proliferation is given as the percent of EdU positive cells.
In addition, cell biocompatibility and viability were evaluated. Live/dead staining (Live/Dead staining Kit, Thermo Fisher Scientific) was employed to show the cell biocompatibility on either hAM matrix or collagen-I at days 2 and 6. ECs were seeded at 5000 cells/cm2. Fluorescent images were taken using Zeiss Axio Observer Z1 inverted microscope to visualize stained cells. Furthermore, the cell viability was quantitatively measured by PrestoBlue® assay (Thermo Fisher Scientific).
All surgical procedures were approved by the Committee for Animal Research of the University of California, San Francisco. The ischemia-reperfusion model used in this study has been previously used extensively as a model for MI.(32,33) Female Sprague-Dawley rats (225-250 g) were anesthetized with ketamine (90 mg/kg) and xylazine (10 mg/kg). The chest was opened by a median sternotomy, and a single stitch of 7-0 Ticron suture (Tyco Healthcare, Norwalk, CT) was introduced around the left anterior descending (LAD) coronary artery and tightened to occlude for 30 min before reperfusion and closing the thoracic cavity. The animals were allowed to recover for 2 days after ischemia-reperfusion prior to treatment.
In vivo hAM Matrix Injection Studies
At 2 days post-MI, rats in the study group were randomized into hAM matrix (n=5) or PBS control groups (n=5). The animals were anesthetized using 2% isoflurane at 2 liters/minute and positioned on the echo-guided ultrasound apparatus. Echocardiography was performed prior to injection. Echocardiography directed intramyocardial injection of either 500 μL of sterile phosphate buffered saline (PBS) (UCSF Cell Culture Facility, San Francisco, CA) containing no hAM or 10 mg of hAM using a 27-gauge needle was delivered to the superior pole, center, and inferior pole of the infarct region as visualized by hyper-echoicity on ultrasound as previously described (Vevo 660, Visual Sonics, Toronto, Canada).(34) The successful injection was confirmed visually by changes in ultrasound signal in the vicinity of the syringe.
Functional Recovery of LV function after hAM Matrix Injection
Transthoracic echocardiography was performed immediately prior to injection, and 5 weeks after injection, Echocardiograms were used to calculate ejection fraction and fractional shortening before and after treatment. Ejection fraction was calculated as described previously.(35) A parasternal long-axis B-mode image was acquired in order to identify the maximum LV length. Three short-axis B-mode images were also acquired at basal, midventricular, and apical LV levels. Results are presented as the percent change in ejection fraction and percent change in fractional shortening. Using frames from the long axis images, as well as maximum and minimum cross-sectional areas in the heart cycle, LV end-systolic volume (LVESV) and LV end-diastolic volume were calculated. LV Ejection Fraction (LVEF) was calculated as follows, LVEF=((LVEDV−LVESV)/LVEDV)×100. Results are given as the percent change in LVEF 5 weeks post-injection. Left ventricular inner dimensions at systole (LVIDs) and diastole (LVIDd) were calculated using the leading-edge method, as prescribed by the American Society of Echocardiography. Fractional shortening, FS was calculated as follows, ((LVIDd−LVIDs)/LVIDd)×100. Results are presented as the percent change in FS 5 weeks post-injection.
Five weeks post-treatment, the rats perfused with 3M KCl prior to sacrifice to arrest the hearts in the diastolic phase. The hearts were harvested, rinsed in cold saline, blotted-dry and fresh frozen in Tissue Tek O.C.T. freezing medium (Sakura Finetek, Torrance, CA). The hearts were sectioned into 10 μm slices. Sequential slides spanning the LV region were stained with Masson's-trichrome stain and were used for morphological assessment of the infarct size as previously described.(36) Briefly, Masson's-trichrome staining images were used to evaluate the infarct size with Image J software. The infarct size (% LV) was calculated by dividing the collagen deposited area to the entire left ventricle area near the midsection of the infarct area. Three sections for each sample were measured.
All experiments are performed in triplicate unless otherwise mentioned. In vitro and in vivo data are presented as mean±standard deviation. All data were compared with one-way ANOVA tests. Holm's t-test was performed to evaluate significant differences between pairs. A p-value of less than 0.05 was considered statistically significant.
Characterization of Injectable Human Amniotic Membrane (hAM) Matrix
An injectable hAM matrix was prepared through decellularization of hAM tissue (
The decellularized tissue was sectioned and stained for nuclei using either DAPI or hematoxylin & eosin (H&E) stain. Both CHAPS and SDS decellularization processes were successful, as both tissues groups appeared devoid of cells (
In order to quantify the efficiency of decellularization protocols, remaining DNA content within the decellularized hAM matrix was quantified (
The composition of the injectable hAM matrix was characterized by the mass spectrometry analysis (Table 1). This identified and confirmed some major matrix components, including different types of collagen, fibronectin, laminin, keratin, fibrillin and growth factor receptors.
To assess the biocompatibility of the injectable hAM matrix, the proliferation of BAECs on hAM matrix was measured. As a positive control of biocompatibility, the proliferation of BAECs on type-1 collagen was also measured. At low density (1000 cells/cm2), medium density (5,000 cells/cm2), and high density (10,000 cells/cm2), there was no significant difference in proliferation on hAM matrix-coated surfaces, when compared to proliferation on Collagen-1 surfaces with similar cell densities (
In addition, the cell biocompatibility of hAM matrix-coated surface was measured by live/dead cell staining at days 2 and 6 (
In vivo studies were performed by injecting hAM matrix into the acute infarcted myocardium. After 5 weeks following injection, hAM matrix-treated group showed significantly higher LVEF than PBS-treated group (57±7.1% for hAM vs 34±3.5% for PBS) (
Fractional shortening also improved for animals receiving hAM matrix following acute MI (
After 5 weeks, the MI rat hearts were harvested and cryosectioned for histological analyses. The cardiac fibrosis for hAM matrix or PBS treated hearts was evaluated with Masson's trichrome staining. Consistent with the functional analysis, there was a significant decrease in MI size as a percentage of the total LV in the hAM matrix treated group compared to the PBS group (
Heart failure following MI is a progressive process, consisting of several stages. During the acute phase, cardiomyocyte death occurs and is followed by macrophage, monocyte, and neutrophil migration. The inflammatory response continues through the subacute phase, until cellular components begin to be replaced by dense collagen fibrils. During the chronic phase, infarct expansion occurs, leading to dilation and continued LV remodeling.(37,38) The injectable hydrogel, as a scaffold-based method, has shown great potential to treat MI by providing mechanical support and increasing myocardial thickness to prevent negative ventricular remodeling, which is advantageous as a minimally invasive and localized treatment(10). For example, the use of alginate-based hydrogel, Algisyl-LVR, has shown encouraging preclinical results to improve myocardial functionality.(39,40) The goal of this study was to develop an injectable ECM derived from human amniotic tissue and investigate its potential to promote recovery following myocardial infarction. To date, this study is the first to report the development of an injectable scaffold derived from decellularized amniotic membrane. Previous studies have utilized decellularization methods to fabricate injectable scaffolds from urinary bladder matrix, myocardial matrix, and lipoaspirate matrix.(41) Our hypothesis is that hAM matrix may promote scarless wound healing similar to other fetal tissue, which allows regenerative remodeling. It is possible that some scar-inducing components in missing in hAM matrix or hAM matrix has anti-scar molecules, which needs further investigation.
The composition of ECM is unique to the organ/tissue from which it is derived. Likewise, decellularization methods must be tailored and made unique according to the tissue of interest. Through experimentation of various decellularization techniques, we have identified a process that is capable of preserving extracellular components, most specifically, the natural GAGs, from amniotic membrane. This process is highly efficient in removing cellular contents, as seen in the removal of greater than 96% of native DNA content. For future studies, it will be helpful to determine if higher levels of decellularization are needed to yield non-immunogenic matrix products.
Additionally, it is valuable to identify and quantify the protein components of the decellularized matrix by more advanced mass spectrum analysis to understand the material properties and biological effects.(42,43)
Through in vitro cell studies on bovine aortic endothelium, we found that the injectable hAM matrix was non-cytotoxic and did not affect cell proliferation. In the future, it will be helpful to determine the effects on cell proliferation, attachment, and migration for other cardiac-specific cell lines. In addition, it will be helpful to determine if the extracted hAM matrix is capable of affecting myofibroblast differentiation, an event central to the process of scar formation.
To investigate the effect of hAM matrix injection on functional LV recovery following myocardial infarction, an acute rodent rat MI model was applied. We found that hAM matrix injection contributed to a reduction in MI scar formation and an improvement in LV function. Significant LV ejection fraction improvement was seen when each animal was used as its own control and with the hAM matrix group was compared to the control group. Due to the diversity in physiological response across various stages after MI, we chose to examine the effects of hAM matrix injection on cardiac function during the acute MI, 2 days after an MI. Hydrogel injection at 2-3 days post-MI has been commonly performed for acute MI therapy as a model to demonstrate therapeutic effects.(36, 44) In the future, it will be valuable to evaluate the therapeutic effects of hAM matrix in chronic MI models, which will facilitate the translation of this technology into clinical applications.
Future studies can further elucidate the potential mechanism of LV functional improvement from hAM matrix injection for example comparisons of embodiments of the inventions to the effects of other matrix materials. By adding additional matrix groups to the study, it can be confirmed for example, whether LV functional improvement from hAM matrix injection is due to the unique biochemical environment of the amniotic membrane, or if LV improvement is due to a mechanical constrainment of LV dilation.
All publications mentioned herein (e.g., Henry et al., Advanced Healthcare Materials. 2020 January; 9(2): e1900544, and those identified above) are incorporated by reference to disclose and describe aspects, methods and/or materials in connection with the cited publications. Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
This application claims the benefit under 35 U.S.C. Section 119(e) of co-pending and commonly-assigned U.S. Provisional Patent Application Ser. No. 63/129,013, filed on Dec. 22, 2020, and entitled “AMNIOTIC MEMBRANE FOR MYOCARDIAL REPAIR” which application is incorporated by reference herein.
This invention was made with government support under Grant Numbers HL117213, HL121450 and HL094162, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/64825 | 12/22/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63129013 | Dec 2020 | US |
