Patent Application
20090191165
The present invention relates to a composition for repairing nerve damage and enhancing functional recovery of a damaged nerve, as well as methods utilizing the same.
Nerve damage is usually caused by trauma or ischemia, and is difficult to repair. A vertebrate having nerve damage may suffer from motor deficits, paralysis, or even death. Ischemia is caused by abrupt interruption of cerebral blood flow that leads to cerebral infarction and ischemic changes with long-term motor deficits (S. I. Savitz et al., 2002, Ann. Neurol. 52: 266-275). Loss of neural cells after ischemic injury to the central nervous system (CNS) makes CNS repair difficult.
Many studies showed that neural stem cells (NSCs) isolated from various rodent and human CNS areas are capable of differentiating into neural cells in the adult rodent CNS under the influence of the environment and/or exogenous growth factors (F. H. Gage, 2000, Science 287: 1433-1438; J. Price and B. P. Williams, 2001, Curr. Opin. Neurobiol. 11: 564-567). Thus, replenishment of NSCs is thought to be a potential strategy for human CNS treatment.
Stem cells derived from human bone marrow are heterogeneous in morphology. They multipotentially differentiate into osteoblasts, adipocytes, chondrocytes and muscle and can also generate neurons (E. Mezey et al., 2000, Science 290: 1779-1782; E. Mezey et al., 2003, Proc. Natl. Acad. Sci. USA. 100: 136-41369; J. R. Sanchez-Ramos et al., 2000, Exp. Neurol. 164: 247-256; D. Woodbury et al., 2000, J. Neurosci. Res. 61: 364-370). Recently, human and mouse bone marrow stem cells (BMSCs) have been reported to express neuronal progenitor marker (nestin), neuron-specific nuclear protein (NeuN), and glial acidic fibrillary protein (GFAP) after stimulation with retinoic acid and epidermal growth factor (EGF) or brain-derived neurotrophic factor (BDNF) (J. R. Sanchez-Ramos et al., 2000, supra). It has also been demonstrated that transplanted BMSCs are able to differentiate between the neuronal and glial lineages in damaged CNS (J. R. Sanchez-Ramos, 2002, J. Neurosci Res. 69: 880-893). Moreover, it has been found that the transplantation of BMSCs can improve functional recovery in rats with focal cerebral ischemia (J. Chen et al., 2000, Neuropharmacology 39: 711-716), in rats with traumatic brain injury (D. Lu et al., 2001, J Neurotrauma 18: 813-819), and in mice with Parkinson's disease (Y. Li et al., 2001, Neurosci Lett. 316: 67-70). These findings indicate the potential role of BMSCs as a useful cell source for CNS cell therapy in humans. In the aforementioned studies, the routes of administration of BMSCs are via intravenous or intracerebral injection. There has been no literature reporting the effect of topical application of BMSCs on damaged CNS areas in test animals subjected to nerve damage caused by, for example, chronic focal cerebral ischemic injury. Moreover, there remains a need for a means to effectively repair nerve damage and as a result thereof, enhance the functional recovery of damaged nerves.
The purpose of the present invention is to provide compositions and methods for effectively repairing nerve damage and as a result thereof, enhancing at least partially the functional recovery of the damaged nerve.
Accordingly, in a first aspect, the present invention relates to a fibrin glue composition for repairing nerve damage which comprises fibrin glue and an amount of bone marrow stem cells (BMSCs) effective to repair the nerve damage. In a preferred embodiment of the present invention, the fibrin glue composition may further comprise an amount of a nerve growth factor effective to repair the nerve damage.
In another aspect, the present invention relates to a fibrin glue composition for enhancing functional recovery of a damaged nerve which comprises fibrin glue and an amount of BMSCs effective to enhance at least partially the functional recovery of the damaged nerve. In a preferred embodiment of the present invention, the fibrin glue composition may further comprise an amount of a nerve growth factor effective to enhance at least partially the functional recovery of the damaged nerve.
In a further aspect, the present invention relates to a method for repairing nerve damage in a subject, the method comprising providing a fibrin glue composition comprising fibrin glue and an amount of BMSCs effective to repair the nerve damage, and topically applying the composition to the damaged nerve. In a preferred embodiment of the present invention, the fibrin glue composition may further comprise an amount of a nerve growth factor effective to repair the nerve damage.
In an even further aspect, the present invention relates to a method for enhancing functional recovery of a damaged nerve in a subject, the method comprising providing a fibrin glue composition comprising fibrin glue and an amount of BMSCs effective to enhance at least partially the functional recovery of the damaged nerve, and topically applying the composition to the damaged nerve. In a preferred embodiment of the present invention, the fibrin glue composition may further comprise an amount of a nerve growth factor effective to enhance at least partially the functional recovery of the damaged nerve.
The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.
In the drawings:
The present invention relates to a fibrin glue composition for repairing nerve damage and/or enhancing the functional recovery of a damaged nerve which comprises fibrin glue and an amount of bone marrow stem cells (BMSCs) effective to repair the nerve damage and/or enhancing at least partially the functional recovery of the damaged nerve.
According to the present invention, BMSCs are stem cells derived from human bone marrow having the potential of differentiating into neurons, and thus are considered to be the best material for regenerating neural cells. Preferably, the BMSCs used in the present invention are size-sieved stem cells (SSCs) derived from human bone marrow.
SSCs are developed based on their different sizes and specific surface markers to generate homogeneous populations by using cell sorting to avoid heterogeneous population generation of primary BMSC cultures. SSCs have greater renewal capability than a heterogeneous population of BMSCs. In a preferred embodiment of the present invention, the SSCs are isolated as a homogeneous population from human bone marrow on the basis of cell size and adherent capacity via Percoll gradient separation and a 3-μm porous sieve for discarding smaller cells as described by Hung et al. (S. C. Hung et al., 2002, Stem Cells 20: 249-258; the disclosure of which is hereby incorporated herein by reference).
According to the present invention, SSCs may be induced for neural differentiation before addition into the fibrin glue composition. The induction may be achieved by treating the SSCs with antioxidant agents such as β-mercaptoethanol and retinoic acid, which are often used in vitro to induce the neural differentiation of stem cells (S. C. Hung et al., 2002, Stem Cells 20: 522-529; the disclosure of which is hereby incorporated herein by reference). In a preferred embodiment of the present invention, the SSCs are induced by a neurotrophic factor such as glial cell line-derived neurotrophic factor (GDNF) or pituitary adenylate cyclase-activating polypeptide (PACAP), and/or dibutyryl cAMP (dbcAMP) as described in the co-pending U.S. patent application Ser. No. 10/873,640, filed Jun. 23, 2004 (Pub. No. 20050287665), the disclosure of which is hereby incorporated herein by reference.
As used herein, the term “effective amount” refers to an amount of the BMSCs, and, when optionally used, the nerve growth factor, in the fibrin glue composition of the present invention, which, when applied to a subject suffering from nerve damage, attains a desired effect, i.e., repairs nerve damage in the subject, and/or enhances at least partially functional recovery of a damaged nerve. The effective amount can be readily determined in view of the present disclosure by persons of ordinary skill in the art without undue experimentation. Preferably, effective amount of BMSCs applied to a target area in the present invention is about 105 to about 107 cells. More preferably, the effective amount is about 106 cells. Preferred and more preferred concentrations of the nerve growth factor, when it is used with the BMSCs, are set forth below.
The term “fibrin glue” as used herein refers to a biocompatible and biodegradable product formed by fibrinogen and other reagents. Fibrin glue has been used customarily as an adhesive agent in various kinds of surgery, including neurosurgery. It is commercially available under the trademark Beriplast P™ (ZLB Behring, Germany), for example.
The fibin glue used in the present invention preferably is composed of three ingredients: fibrinogen, aprotinin and a calcium source providing divalent calcium ions (such as calcium chloride or calcium carbonate). To apply the fibrin glue, fibrinogen is first mixed with aprotinin, and further mixed with the calcium source in the surgical area to form a glue cast.
To make fibrin glue comprising BMSCs, the BMSCs can be mixed with fibrinogen and aprotinin before mixing with calcium chloride in the surgical area.
In a fibrin glue composition of the present invention, the concentration of fibrinogen may preferably be in the range of about 10 mg/ml to about 1000 mg/ml, and more preferably about 100 mg/ml; the concentration of aprotinin may preferably be in the range of about 10 KIU/ml to about 500 KIU/ml, more preferably about 200 KIU/ml; and the concentration of calcium chloride used as the calcium source may preferably be in the range of about 1 mM to about 100 mM, more preferably about 8 mM.
The fibrin glue composition of the present invention may further comprise, in addition to the fibrin glue components (referred to herein as the “fibrin glue”) and BMSCs, an effective amount of a nerve growth factor. The nerve growth factor used in the composition of the present invention may be selected from, but not limited to, glial cell line-derived neurotrophic factor (GDNF), transforming growth factor-beta, fibroblast growth factor, platelet-derived growth factor, epidermal growth factor, vascular endothelial growth factor (VEGF), and neurotrophin (such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT3, NT4 and NT5). Most preferably, the nerve growth factor is glial cell line-derived neurotrophic factor (GDNF).
According to the invention, the concentration of the nerve growth factor in the fibrin glue composition of the invention may preferably be about 1 μg/ml to 1000 μg/ml, and more preferably about 50 μg/ml of the composition.
The fibrin glue composition of the invention is suitable for use in repairing all kinds of nerve damages and enhancing the functional recovery of damaged nerves in all nerve systems, including the central nervous system, peripheral nervous system, sympathetic nervous system, and parasympathetic nervous system. Preferably, the damaged nerve is in the central nervous system. In an embodiment of the present invention, the nerve damage is caused by focal cerebral ischemia (FCI). In a preferred embodiment, the nerve damage is caused by chronic focal cerebral ischemia.
As used herein, the term “repairing nerve damage” and other grammatically equivalent terms, refer to an improvement in the pathological conditions of the subject suffering from nerve damage, and the term “functional recovery of a damaged nerve” refers to restoration, at least partially, of the physical function of the damaged nerve. For example, for nerve damage caused by FCI, the reduction in total infarction volume and motor deficits are signs of the repair of the damaged nerves and restoration of the function thereof. In an animal model according to the present invention, the reduction in motor deficits comprises improvement in balance, coordination and grasping strength.
As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a damaged nerve refers to one or more damaged nerves.
As used herein, the term “subject” includes a mammal, including a pet animal, such as a cat and a dog, a laboratory animal, such as a mouse and a rat and livestock, such as a horse, a cow, etc., and preferably a human.
The fibrin glue composition of the present invention can be topically applied to damaged nerves. The topical application may be practiced during surgery that exposes the nerve to topical administration by coating the area including the nerve with the glue from a syringe.
The method according to the present invention exerts a long-term effect needed for repairing chronic nerve damage and enhancing the functional recovery of the damaged nerve. In addition, topical application of the composition of the present invention to a damaged nerve only once can sufficiently achieve the desired effect of functional recovery. Preferably, the method further comprises preparing the fibrin glue composition freshly right before use.
The composition and method of the present invention may be used to treat various nerve damage-related diseases, including, but not limited to, ischemic brain disease. Preferably, the ischemic brain disease comprises stroke, thrombosis and embolization of the middle cerebral artery (MCA).
The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation.
Animals
The following experiments conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication NO. 85-23, revised 1996). Male Long-Evans rats (National Lab. Animal Breeding and Research Center) weighing 300 g-350 g were used in the following experiments. The animals were housed in a room with controlled temperature (24±1° C.) and humidity (55±5%) under a 12:12 h light-dark cycle. They were allowed free access to food and water.
Size-Sieved Stem Cells (SSCs)
SSCs used in the following experiments were isolated from human bone marrow as previously reported (S. C. Hung et al., 2002, supra). In brief, bone marrow aspirated from the iliac crest of normal donors was washed twice with phosphate-buffered saline (PBS) and loaded onto 1.073 g/ml Percoll solution (Sigma), and then centrifuged at 900×g for 30 mins. The cells were suspended in Dulbecco's modified Eagle's medium-low glucose (DMEM-LG) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 μg/ml amphotericin B, and were plated in a 10-cm plastic culture dish comprising a plate with 3-μm pores (Transwell System, Corning) at a density of 106 mononuclear cells/cm2. SSCs that adhered to the pore-containing plate were recovered on the 7th day after initial plating, plated at about 6,000 cells/cm2, and subcultured at a ratio of 1:3 until cells reached more than 80% confluence.
Undifferentiated SSCs were cultured in DMEM-LG supplemented with 10% FBS. When SSCs grown in the serum containing medium reached 80% confluence, the SSCs were seeded at a density of 4,000 cells/cm2, treated in serum medium (ITS medium) containing GDNF (20 ng/ml, RBI, Natick, Mass., USA) for 48 hours, and then treated with serum depletion for 5 hours to 3 days to induce differentiation. ITS medium consisted of 56% DMEM-LG (Life Biotech), 40% MCDB-201 medium (Sigma), and 1×ITS medium supplement (Sigma) that contained 1 mg/ml insulin, 0.55 mg/ml human transferrin, 0.5 μg/ml sodium selenite, 10 nM dexamethasone (Sigma), and 10 μM ascorbic acid (Sigma).
Statistics
Data acquired in the following experiments are expressed as mean±standard error of mean (S.E.M.). Statistical analysis of the differences in volume of infarcts and the behavioral deficit scores of rats between control and treatment groups were carried by one-way analysis of variance (ANOVA) followed by unpaired, two-tailed t tests for combined data. A value of P<0.05 was considered to be statistically significant.
Generation of Cerebral Infarction and Infarct Volume Analysis
The technique used was a modification of the method of Huang et al. (S. S. Huang et al., 2001, Free Radic. Biol. Med. 30: 643-649, the disclosure of which is hereby incorporated herein by reference). In brief, each male Long-Evans rat was anesthetized by inhalation of a nitrous oxide/oxygen/halothane (69%:30%:1%) mixture during surgical preparation. Body temperature was maintained during surgery at 37±0.5° C. with a heating pad servo-controlled by a rectal probe. The ventral tail artery was cannulated for continuous monitoring of heart rate and mean arterial blood pressure (MABP) by a Statham P23 XL transducer and displayed on a Gould RS-3400 physiological Recorder (Gould, Cleveland, Ohio, USA) and the pH, PO2 and PCO2 in the blood were tested using blood sampling with Blood Gas Analyzer (GEM-5300 I.L. CO, USA). Measurements were performed before, during, and just after unilateral middle cerebral artery (MCA) occlusion.
Focal cerebral ischemic (FCI) infarcts were produced in the right lateral cerebral cortex in the territory of MCA. Both common carotid arteries were exposed by midline anterior cervical incision. The animal was placed in a lateral position, and a skin incision was made at the midpoint between the right lateral canthus and the anterior pinna. The temporal muscle was retracted, and a small (3-mm diameter) craniectomy was made at the junction of the zygoma and squamosal bone using a drill (Dremel Multipro+5395, Dremel com. USA) cooled with saline solution. Using a dissecting microscope (OPMI-1, ZISS, Germany), the dura was opened with fine forceps, and the right MCA was ligated with 10-0 monofilament nylon ties. Both common carotid arteries were then occluded by microaneurysm clips for 1 hr. After removing the clips, return of flow was visualized in the arteries.
One week following the FCI injury, rats were assigned, in randomized sequence, to one of four treatment groups (n=6 each): (a) the sham group: animals underwent the same described surgical procedures except MCA ligation; (b) the control group: topically applied fibrin glue; (c) the SSCs-only group: topically applied SSCs (106 cells); and (d) the SSCs (106 cells)-fibrin glue group: topically applied fibrin glue containing SSCs (106 cells). Topical application, in all instances was by coating the area including the nerve with the glue, with or without SSCs, from a syringe.
The fibrin glue (Beriplast P™, ZLB Behring, Germany) used in this experiment and customarily used as an adhesive agent in CNS tissue, was prepared before use by mixing the fibrinogen (100 mg/ml) with an aprotinin solution (200 KIU/ml). This solution was then mixed with calcium chloride (8 mM) in the surgical area to form a glue cast. The final volume of the solution locally applied in the infarcted brain tissue was 20 μl.
After focal cerebral ischemia for 1 hr and reperfusion for 5 weeks, the rats were anesthetized and killed by rapid decapitation. Brains were removed, inspected visually for the anatomy of the MCA and for signs of hemorrhage or infection, immersed in cold saline solution for 10 minutes, and sectioned into standard coronal slices (each 2-mm thick) using a brain matrix slicer (JACOBOWITZ Systems, Zivic-Miller Laboratories INC, Allison park, USA). Slices were placed in the vital dye 2,3,5-triphenyltetrazolium chloride (TTC, 2%; Sigma, USA) at 37° C. in the dark for 30 minutes, followed by 10% formalin at room temperature overnight. The outline of right and left cerebral hemispheres as well as that of infarct tissue, clearly visualizable by TTC staining (S. T. Chen et al., 1986, Stroke 17: 738-743), was outlined on the posterior surface of each slice using an image analyzer (color image scanner, EPSON), connected to an image analysis system (AIS software, Imaging research INC, Canada), which was run on a personal computer (AMD K6-2 3D 400). The area of infarction was measured by subtracting the area of the non-lesioned ipsilateral hemisphere from that of the contralateral side. Infarct volume was calculated as the sum of infarct area per slice multiplied by slice thickness. Both the surgeon and image analyzer operator were blinded to the treatment given each animal.
Reproducible brain infarcts were obtained from a territory of right MCA occlusion in the SSCs-fibrin glue group, the SSCs-only group, and the control group. The infarct volume at the end of the 5th week after FCI injury in the SSCs-fibrin glue group was significantly reduced (88.1±13.8 mm3, P<0.05) but not in the SSCs-only group (152.0±21.9 mm3), as compared with the control group (145.8±10.1 mm3) (
The results indicated that topically applied SSCs mixed with fibrin glue at infarct brain tissue after FCI injury significantly reduced the total infarcted volume by 40% and 42%, respectively, as compared to that of the control group and the SSCs-only group (
Behavioral measurements were performed by the rotarod test and the grasping power test at the end of the 1 st, 2nd, 3rd, and 4th weeks after SSCs administration on rats subjected to FCI injury.
Rotarod Test
For the rotarod test, an accelerating rotarod was used to assess motor deficit following ischemic injury in rats (R. J. Hamm et al., 1994, J. Neurotrauma 11: 187-196). The rats were placed on rungs of the accelerating rotarod and the amount of time the animals remained on the rotarod were measured. The speed was increased slowly from 4 rev/min to 40 rev/min over the course of 5 minutes. The time, in seconds, at which each animal fell off the rungs was recorded. Each animal received three consecutive trials.
Grasping Power Test
The grasping power test was a modification of the method of Bertelli and Mira (J. A. Bertelli and J. C. Mira, 1995, J. Neurosci. Methods 58: 151-155). For the assessment of grasping strength, a bar of wires was connected to an ordinary electronic balance. Both forepaws were tested, one forepaw at a time. The untested forepaw was temporarily prevented from grasping by wrapping it with adhesive tape, and the tested forepaw was kept free. The rats were allowed to grasp the bar while being lifted by the tail with increasing firmness until they loosened their grip and the grasping power was scored.
The effects on grasping power test of the four groups are shown in
To confirm the neuronal regeneration and SSCs differentiations in the brain at 5 weeks after FCI injury, RT-PCR analysis using human glycerol-3-phosphate dehydrogenase (HG3PDH) was performed.
Total RNA was prepared from the brain of half of the animals from each group using the RNeasy purification system as described by the manufacturer (Qiagen; Valencia, Calif.; http://www.qiagen.com). Total RNA (1 μg) was reversely transcribed with Moloney murine leukemia virus reverse transcriptase at 42° C. in the presence of oligo-dT primer. PCR was performed for the HG3PDH gene using specific primers designed from the published sequence of cDNA as follows: sense: 5′-GGCTGGGACTCATGGAGAT-3′ (SEQ ID NO:1), and antisense: 5′-CGGGTAAGTCGTTGAGAAAG-3′ (SEQ ID NO:2). Nested PCR was performed with the following primers: sense: 5′-TCTTGGAGAGCTGTGGTGTTG-3′ (SEQ ID NO:3), and antisense: 5′-GTTACCTGAAAGGACTGC-3′ (SEQ ID NO:4). The thermal profile was 1 minute at 95° C., 1 minute at 60° C., and 2 minutes at 72° C. The amplification cycles were all 35. To exclude possible contamination of genomic DNA, PCR was also applied to reactions without reverse transcription. The amplified complementary DNA was subjected to electrophoresis on a 3.5% polyacrylamide gel and visualized by silver staining.
HG3PDH was found in the brain of animals topically applied with SSCs (106 cells) in fibrin glue (SSCs-fibrin glue group) one week after FCI injury and five weeks of reperfusion. However, no HG3PDH was detected in the control and the SSCs-only groups (
As shown in the above examples, while topical application of the fibrin glue composition containing SSCs to ischemic brain significantly decreases the infarction volume in MCA occluded rats and also significantly improves the motor performance at the end of the 4th week after chronic FCI injury, the control group did not exhibit a neuroprotective effect in rats after chronic focal cerebral ischemia. The data indicate that the fibrin glue composition of the present invention is effective in treating the cerebral ischemia from MCA occlusion and enhancing recovery of motor function.
The following experiments were performed to investigate the therapeutic effects of a fibrin glue composition comprising not only SSCs, but also GDNF.
Infarct Volume Analysis
As described in Example 1, reproducible brain infarcts were obtained from a territory of right MCA occlusion in a GDNF-fibrin glue group, a SSCs-fibrin glue group, a GDNF-SSCs-fibrin glue group, and the control group. For the GDNF-fibrin glue group and GDNF-SSCs-fibrin glue group, 1 mg GDNF (RBI, Natick®, MA, USA) was mixed with the solution containing fibrinogen and aprotinin, with and without SSCs, respectively. The glues from the various groups were applied topically to the nerves as described above in Example 1.
Rotarod Test
The rotarod test was performed as described in Example 2.
Grapsing Power Test
The grasping power test was also performed as described in Example 2. The effects on grasping power test of (A) right forepaw and (B) left forepaw on rats after FCI injury of the five groups are shown in
The above results showed that topical application of GDNF, SSCs or GDNF-SSCs-fibrin glue is associated with improvement of grasping strength of rats after FCI injury.
Whishaw Reaching Test
To further investigate the ability of the fibrin glue composition comprising SSCs+GDNF to enhance the functional recovery of nerves damaged by FCI injury, the whishaw reaching test was performed.
The whishaw reaching test is a modification of the method of Kolb and Cioe (B. Kolb and J. Cioe, 2000, Neuropharmacology 39(5): 756-64). Rats were food deprived to 85% body weight for the training and testing. Each animal was placed in the test cages (10×18×10 cm high) with floors and fronts constructed of 2 mm bars, 9 mm apart edge to edge. A 4-cm wide and 5-cm deep tray containing 45 mg food pellets was mounted in the front of each box. The rats are required to extend a forelimb through the gap in the bars, grasp and retract the food. The tray was mounted on runners and was retracted 0.5 cm from the cage so that the rats cannot scrape the food into the cage. If the animal attempts to rake the pellet out of the tray, the pellets will fall through the gap. An attempt is scored only when the rat reaches into the tray and touches the food pellets. If the rat reaches into the tray without touching a pellet, no attempt is scored.
The effects on the whishaw reaching test on rats after FCI injury of the five groups are shown in
The publications whose disclosures were not previously specifically incorporated herein by reference are now hereby incorporated herein by reference.
It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.
This application is a division of U.S. patent application Ser. No. 11/516,378, filed Sep. 6, 2006, the disclosure of which is hereby incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11516378 | Sep 2006 | US |
| Child | 12354470 | US |
