Patent Grant
8894707
1. Technical Field
This document relates to methods and materials involved in tendon or ligament tissue engineering. For example, this document provides methods and materials for generating a composite of acellular tendon or ligament allograft slices seeded with cells and bundled into a graft for tissue repair.
2. Background Information
Tendon injuries are very common and place a large burden on the US economy. It has been estimated that more than 32 million patients sustain traumatic and overuse injuries to tendons and ligaments, at a cost of roughly $30 billion per year. Functional restoration of the injured tendon is still a great challenge. Similarly, ligaments, which are poorly vascularized, do not heal well and usually must be replaced after significant damage has occurred.
This document provides methods and materials related to tendon or ligament tissue engineering. For example, this document provides methods and materials related to engineering tendon or ligament tissue for human, equine, or other mammalian clinical uses. As described herein, allogenic decellularized fresh tissue is in many ways an ideal scaffold environment for cell seeding, adhesion, and survival, containing native collagen, with normal tissue structure and organization. As described herein, this document provides methods and materials for generating composite tissue engineered tendons and composite tissue engineered ligaments from tendon and ligament allograft slices, respectively. In some cases, the composite of acelluar tissue slices can be seeded with autologous stem cells and/or bundled into a thicker composite for surgical grafting or implantation. This document also provides methods and materials for using a composite tissue engineered tendon or ligament for regenerating, repairing, or replacing damaged tendon or ligament. As described herein, this document provides, for example, methods and materials by which a multilayer composite of tendon or ligament allograft slices can be fabricated to serve as a scaffold for tendon or ligament regeneration that possesses the basic structure of the tendon or ligament, a native extracellular matrix, and the capability of cell seeding and cellular ingrowth. The methods and materials provided herein can allow fabrication of a wide array of tendon and ligament substitutes, varying in size and strength.
In general, one aspect of this document features a composition comprising, or consisting essentially of, multiple layers of tendon or ligament portions. The tendon or ligament portion can be obtained from a mammal and decellularized to remove a mammal's cells from the portion. The composition can comprise tendon portions. The composition can comprise ligament portions. The composition can have portions that are decellularized to lack a mammal's cells. The composition can further comprise one or more layers. The composition can further comprise an extracellular matrix of tendon or ligament portions. The mammal can be a human. The composition can be seeded with a plurality of cells. The cells can be autologous stem cells. The autologous stem cells can be derived from bone marrow, tendon, ligament, muscle, adipose, or synovial tissue. The composition can be coated with a lubricating surface. The composition can further comprise one or more growth factors. The growth factors can be GDF5, TGF-β, VEGF, PDGF, BMPs, or any combination thereof. The composition can be reinforced by adding one or more layers of man-made material.
In another aspect, this document features a method for providing a tendon or ligament tissue implant to a mammal. The method comprises, or consists essentially of, implanting a composition into said mammal. The composition can comprise multiple layers of tendon or ligament portions. The portions can be from a mammal and decellularized to remove a mammal's cells from a portion. The method can further comprise providing an implant for repairing tendon or ligament tissue damage. The method can further comprise providing an implant for replacing damaged tendon or ligament tissue of a mammal with an implant. The multiple layers of tendon or ligament portions of a composition can be sutured to form a roll. The roll can be implanted into tendon or ligament tissue of a mammal.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
This document relates to methods and materials involved in tendon or ligament tissue engineering. As described herein, this document also provides methods and materials for generating composite tissue engineered tendons and composite tissue engineered ligaments from tendon and ligament allograft slices, respectively.
This document provides methods and materials for a composition of multiple layers of tendon or ligament tissue portions. Any appropriate human cadaver tendon or ligament tissue portions can be used. Tendon or ligament portions can be harvested from any suitable body parts. For example, tendon or ligament portions can be harvested from the leg, arm, back, hand, or foot. In some cases, tendons harvested for the methods and materials described herein can be, for example, the Achilles tendon, infraspinatus tendon, tibialis anterior tendon, peroneus longus tendon, peroneus medius tendon, extensor digitorum longus tendons, extensor hallucis longus tendon, flexor digitorum longus tendon, or patellar tendon. In some cases, ligaments harvested for the methods and materials described herein can be, for example, the ulnar collateral ligament, radial collateral ligament, medical collateral ligament, lateral collateral ligament, anterior cruciate ligament, posterior cruciate ligament, anterior or posterior talofibular ligaments, calcaneofibular ligament, talocalcaneal ligament, or posterior talocalcaneal ligament. Harvested tendon and ligament portions can be of any suitable size and shape. For example, tendon and ligament tissue portions can be rectangular, square, circular, oval, or other shapes, or include the entire tendon or ligament, as needed. Tendon and ligament tissue harvested for use in the methods and materials provided herein can be used as freshly harvested tissue, or can be stored (e.g., frozen at −80° C., freeze-dried).
Tendon and ligament tissue harvested for use in the methods and materials provided herein can be decellularized to remove the native cells. Any appropriate method can be used to remove native cells from tendon and ligament tissue while maintaining morphological integrity of the tissue portions and preserving extracellular matrix (ECM) proteins. For example, decellularization methods can include subjecting tendon and ligament tissue to repeated freeze-thaw cycles using liquid nitrogen. The tissue can also be treated with a nuclease solution (e.g., ribonuclease, deoxyribonuclease) and washed in sterile phosphate buffered saline with mild agitation. Mild, non-ionic detergents such as TRITON® X-100 (Union Carbide Corp.) or anionic detergents such as sodium deoxycholate, or sodium dodecylsulfate can be used to aid decellularization. The extent of decellularization can be assayed by hematoxylin and eosin staining or Movat-Pentachrome staining Electron microscopy can be used to assay for morphological integrity of the decellularized tissue portions. Conservation of extracellular matrix (ECM) proteins can be assayed by immunohistochemical staining against laminin and collagens I and IV.
This document also provides methods and materials for combining tendon and ligament portions with other portions to form a composite of any size or shape. For example, a tendon tissue portion can be combined with additional tendon tissue portions to form a multilayered composite of tendon tissue portions. Similarly, a ligament tissue portion can be combined with other ligament tissue portions to form a multilayered composite of ligament tissue portions. Tendon or ligament portions can be combined by stacking multiple tissue layers to form a composite. In some cases, tendon or ligament portions can be rolled together using different weaving techniques to form a tissue composite. In some cases, tendon or ligament portions can be combined by weaving tissue portions together to form a tissue composite. A tissue composite of tendon or ligament portions can be provided as a bundle or cut into slices of varying thicknesses. For example, a tissue composite of tendon or ligament portions can be cut length-wise to produce slices having a thickness of about 100 or more micrometers (e.g., about 100-500 μm, 100-400 μm, 100-300 μm, 100-200 μm, 200-500 μm, 300-500 μm, or 400-500 μm). In some cases, a tissue composite of tendon or ligament portions can be embedded in a compound (e.g., Tissue-Tek® optimal cutting temperature compound (Sakura Finetek USA, Inc., USA)) to facilitate cutting. For example, a microtome (e.g., cryostat) can be used to cut an embedded composite into slices with thicknesses of about 100, 200, 300, 400, or 500 μm. In some cases, tendon or ligament composites can be manually cut by into slices.
This document also provides methods and materials for seeding tendon and ligament portions with cells. Any appropriate cell type, such as naïve or undifferentiated cell types, can be used to seed the tissue portions or composite tendon and ligament tissue. For example, stem cells from any tissue source (e.g., skin, bone, synovium, adipose tissue, bone marrow, peripheral blood, umbilical cord blood, or muscle) can be used to seed tendon and ligament tissue. In some cases, autologous cells can be used to seed a tendon or ligament portion. In such cases, tendon or ligament portions and cells are obtained for transplantation into the same individual. Any appropriate method for isolating and collected cells for seeding can be used. For example, bone marrow stromal cells can be readily harvested from bone marrow. Peripheral blood stem cells can be collected from the blood through a process known as apheresis. Isolated cells can be rinsed in a buffered solution (e.g., phosphate buffered saline) and resuspended in a cell culture medium. In some cases, cells can be collected and cryopreserved for later seeding of tendon or ligament portions. For example, bone marrow and other cell types can be frozen for prolonged periods (e.g., cryopreserved).
Standard cell culture methods can be used to culture and expand the population of cells. The cells can be differentiated into tenocytes by stimulation with cytokines such as TGF-β. Such differentiation can be monitored by the expression of tendon-specific markers such as tenomodulin, thrombospondin-4, and scleraxis. Upon differentiation, the cells can be contacted with the tendon or ligament to seed tendon and ligament tissue portions. To facilitate seeding, the portions can be cut into slices roughly 300 micrometers in thickness. Portions of such a thickness can retain the strength and architecture of the native tendon or ligament, while amplifying the surface area and minimizing the needed depth of penetration of the cells being seeded. In some cases, portions can be cut into slices having other thicknesses (e.g., about 100-500 μm, 100-400 μm, 100-300 μm, 100-200 μm, 200-500 μm, 300-500 μm, or 400-500 μm) Tissue portions or composites of tendon or ligament tissue can be seeded with cells in vitro. Various seeding densities can be used. For example, cell densities from 0.2×106 to 0.2×107 cells/composite can be used. In some cases, the seeded tissue or composite tissue can be incubated for a period of time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, or more days) post-seeding to improve fixation and penetration of the cells in the composite. For example, the seeded tissue or composite tissue can be incubated under conditions appropriate to promote cell growth and differentiation. In some cases, seeded tissue or composite tissue can be incubated at about 37° C. with 5% CO2 at 100% humidity. Histology and cell staining can be performed to assay for seeded cell propagation. Any appropriate method can be performed to assay for seeded cell differentiation. For example, quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) can be performed to detect and measure expression levels of markers of tenocyte differentiation (e.g., tenomodulin, scleraxis), gelatinase (e.g., MMP2), and collagenase (e.g., MMP13). Any appropriate RT-PCR protocol can be used. Briefly, total RNA can be collected by homogenizing a biological sample (e.g., tendon sample), performing a chloroform extraction, and extracting total RNA using a spin column (e.g., RNeasy® Mini spin column (QIAGEN, Valencin, Calif.)) or other nucleic acid-binding substrate. In some cases, in situ hybridization can be performed on tendon or ligament portions to detect the expression of cell differentiation markers (e.g., alpha-smooth muscle actin and desmin) or markers of tendon or ligament proliferation and/or differentiation (Type I collagen, Type III collagen, tenascin C, and scleraxis).
In some cases, tendon and ligament tissue composites can be coated with formulations to ensure or reduce adherence of the composite to target tissues. For example, tendon or ligament tissue composites can be coated with lubricating agents such as hyaluronic acid or lubricin to reduce adhesion on gliding surfaces. In some cases, tendon or ligament tissue composites can be coated with autologous perisosteum or synovium to facilitate adherence to attachment sites (e.g., tendon or bone). In some cases, tendon and ligament tissue composites can be reinforced by adding one or more layers of man-made material, such as silk fabric, to increase the strength and stiffness of the composites. In some cases, tendon and ligament tissue composites can be impregnated with growth factors to stimulate differentiation of the seeded cells. For example, tendon and ligament tissue composites can be impregnated with growth factors or neuropeptides such as transforming growth factor beta (TGF-β), growth differentiation factor-5 (GDF-5), or substance P. Other growth factors appropriate for the methods and materials provided herein can include, for example: platelet derived growth factor (PDGF), basic fibroblast growth factor (b-FGF), insulin like growth factor (IGF), epidermal growth factor (EGF), bone morphogenetic protein (BMP), and vascular endothelial growth factor (VEGF).
This document also provides methods and materials for repairing and replacing damaged tendon and ligament tissue. As described herein, tendon or ligament composites can be surgically implanted in a mammal to replace or repair damaged tissue. For example, a tendon tissue composite seeded with autologous stem cells can be implanted into a mammal at the site of tendon tissue damage. In some cases, a ligament tissue composite seeded with autologous stem cells can be implanted into a mammal ay the site of ligament tissue damage. Tendons that can be repaired or replaced by the methods described herein can include, for example, the Achilles tendon, infraspinatus tendon, tibialis anterior tendon, peroneus longus tendon, peroneus medius tendon, extensor digitorum longus tendons, extensor hallucis longus tendon, flexor digitorum longus tendon, or patellar tendon. Ligaments that can be repaired or replaced by the methods described herein can include, for example, the ulnar collateral ligament, radial collateral ligament, medical collateral ligament, lateral collateral ligament, anterior cruciate ligament, posterior cruciate ligament, anterior or posterior talofibular ligaments, calcaneofibular ligament, talocalcaneal ligament, or posterior talocalcaneal ligament.
In some cases, a tendon or ligament composite can be surgically implanted into a mammal. For example, a tendon or ligament composite can be surgically implanted to reconstruct, for example, a defect in a knee ligament or shoulder rotator cuff tendon. In some cases, a tendon or ligament composite can be surgically implanted to replace a damaged finger tendon or to lengthen a tendon graft.
Any appropriate method(s) can be performed to assay for tendon or ligament tissue repair or for the replacement of damaged tendon or ligament tissue. For example, methods can be performed to assess tissue healing, to assess functionality of repaired or replaced tendons or ligaments, and to assess cellular ingrowth. As described previously, histology and cell staining can be performed to detect seeded cell propagation. In some cases, physiological tests, including ultrasound assessment of tendon integrity and motion, can be performed to assess movement and functionality of tendons or ligaments following surgical implantation of tendon or ligament composites.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Twenty mixed-breed dogs weighing between 25 and 30 kg were used for bone marrow and tendon tissue harvesting. Immediately prior to sacrifice, the dogs were anesthetized with intravenous ketamine (13 mg/kg) and diazepam (6 mg/kg) and maintained under anesthesia with 1.5% isoflurane. A total of 4.0 mL of bone marrow was aspirated from the medial aspect of the proximal tibiae using an 18G needle and 20 mL syringe (BD, Franklin Lakes, N.J.) containing 1.0 mL of heparin solution (Heparin sodium injection, Baxter Healthcare Corporation, Deerfield, Ill.). The dogs were then euthanized by an overdose of pentobarbital, and the infraspinatus tendons of both shoulders were harvested. The infraspinatus tendon was exposed by removing the deltoid muscle, and the tendinous portion between the insertion to the bone and the muscle-tendon junction was harvested. The size of harvested tendon was roughly 25×10 mm, and rectangular in shape. The harvested tendons were frozen at −80° C. until processing. Both the bone marrow and infraspinatus tendons were harvested under sterile conditions. The donor of the BMSC was not matched with the donor of the tendon in making the composite of BMSC and tendon slices for this in vitro experiment.
The heparinized bone marrow extract was added to 5.0 mL phosphate buffered saline (PBS), and centrifuged at 1500 rpm (380 g) for 5 minutes at room temperature. The bone marrow pellet was resolubilized in 10 mL of minimal essential medium (MEM) with Earle's salts (GIBCO, Grand Island, N.Y.), 10% fetal bovine serum (GIBCO), and 5% antibiotics (Antibiotic-Antimycotic, GIBCO). The cells from one dog were divided into four equal aliquots, placed in 100-mm culture dishes and incubated at 37° C. with 5% CO2 and 95% air at 100% humidity. After 5 days, the medium and any floating cells were removed and new medium was added to the remaining adherent cells. These adherent cells were defined as bone marrow stromal cells (BMSC). The medium was then changed every other day until the cells reached confluence. The cells were then released with trypsin-EDTA solution (0.25% trypsin, 0.1% EDTA in HBSS, Mediatech Inc., Manassas, Va.) to produce a cell suspension, and centrifuged at 1500 rpm for 5 minutes to remove the trypsin-EDTA solution. The concentrated cell suspension from each dog was then gathered in one tube. The cells were counted with a hemacytometer, and the concentration of the cell suspension was adjusted to 5.0×106 cells/mL by adding additional medium.
Frozen infraspinatus tendon was thawed at room temperature. The infraspinatus tendons were trimmed into segments roughly 25×10 mm in size. The tendon segments were immersed in liquid nitrogen for 2 minutes and then thawed in saline solution at 37° C. for 10 minutes. This procedure was repeated five times. Following washing in phosphate buffered saline (PBS) without EDTA (3×30 minutes), the tendon segments were incubated in 20 mL of nuclease solution from bovine pancreas, 1.5 Units/mL, Roche Diagnostic, Indianapolis, Ind.) for 12 hours at 37° C. Finally, the infraspinatus tendon segments were rinsed for 30 minutes in PBS (50 mL) at room temperature with gentle agitation. The rinsing was repeated three times. The tendon segments were then frozen to −80° C. and fixed to the cutting base plate of a cryostat (Leica CM1850, Germany) with O.C.T. compound (polyvinyl alcohol and polyethylene glycol, Tissue-Tek., Sakura Finetek USA, Inc., Torrance, Calif.). The excess O.C.T. compound around the tendon was removed by a scalpel. The tendon segments were then sliced at a thickness of 50 μm and the slices were placed in a 100-mm culture dish. Ten slices were placed on each dish. The slices were thawed on the dish in an incubator at 37° C. with 5% CO2 and 95% air at 100% humidity for 10 minutes. The tendon slices were then washed three times with 10 mL of PBS. At this point, the sliced acellular tendon segments were ready to seed with BMSC.
The concentrated bone marrow stromal cell (BMSC) solution (5.0×106 cells/mL, 10 mL/dish) was added to the sliced tendon scaffold dish and cultured at 37° C. with 5% CO2 and 95% air at 100% humidity for 2 days. The slices were then carefully detached with forceps and bundled together on a new dish. The ends of the bundled slices were fixed with 3-0 Ethilon suture (nylon suture, Ethicon Inc., Piscataway, N.J.), and a 1.2 g sterilized stainless steel clip (Alligator clip, Mueller Electric Company, Cleveland, Ohio) was attached at one end of the sutured bundle. The composite was then suspended in a 15 mL conical tube and immersed in the same medium described above. The clip served as a weight, preventing the composite from floating in the medium. The composites were incubated for 2, 7, or 14 days. As a control, tendon slices without cells were bundled and maintained in medium for the same time periods. The medium was changed every other day.
BMSC from 2 dogs were stained with the fluorescent marker PKH26-GL (PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling, Sigma, Saint Louis, Mo.) before seeding on the tendon slices, following the manufacturer's instructions. This fluorescent marker has been used for cell tracking in the studies using bone marrow stromal cells. Prior to bundling (i.e., after 2 days in culture), single tendon slices with labeled BMSC were examined by a laser scanning confocal microscope (LSM310, Zeiss) without fixation. For the evaluation of the composites incubated for 7 and 14 days, frozen sections of the composite were made at a thickness of 50 μm, and then examined under the confocal microscope. The fluorescent image of the composite after incubation was combined with the image under the ultraviolet light, with the tendon slices observed as blue under the ultraviolet light.
The 7 and 14 day incubation composites from 2 dogs were used for the histological assessment. The tendon slices without BMSC incubated in the medium for 2 days were also used to evaluate whether any host cells remained after repeated freeze-thaw cycles. These samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned longitudinally at a thickness of 5 μm. Hematoxylin and eosin staining was performed.
Histology showed that the seeded cells aligned between the collagen fibers of the tendon slices. The BMSC labeled with PKH26 were observed as red under the confocal laser microscope (
A quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to measure the gene expression levels of tenomodulin (a marker of tenocyte differentiation), collagen type I and III, MMP2 (gelatinase), and MMP13 (collagenase). RNA was extracted by TRIZOL® reagent (monophasic solutions of phenol and guanidine isothiocyanate, Invitrogen Corporation, Carlsbad, Calif.). cDNA was synthesized using 1st Strand cDNA Synthesis Kit (Roche) with random primers. The sequences of the primers are shown in Table 1. The expression level was normalized to that of GAPDH. All mRNA expressions were confirmed by melting curve analysis using LIGHTCYCLER® system (Roche). Eight samples from each group, including the BMSC solution before seeding, the composites incubated for 2, 7, and 14 days, and the intact infraspinatus tendons were used for assessment of gene expression.
Gene expression data are shown in
In vivo assays were performed using a rabbit model. Two weeks following transplantation of a BMSC-seeded tendon tissue composite to replace the middle potion of the patellar tendon, it was evident that BMSCs had successfully survived (
The maximum failure load and the linear stiffness of the composite were measured with a custom-made microtester, which was composed of a linear servo motor (MX 80 Daedal, Irwin, Pa., USA) and a load cell with the accuracy of 0.01N (MDB-5, Transducer Techniques, Temecula, Calif., USA). The composites with BMSC from 8 dogs after 7 and 14 days incubation were used. Both sides of the tendon, at a length of 10 mm, were clamped with a custom designed holder and 0.1N of preload was applied (
The results of the maximum failure load and the linear stiffness were analyzed by two-way ANOVA, and the results of the gene expressions were analyzed by the Kruskal-Wallis test and Mann-Whitney test, with Bonferroni post hoc adjustment. The non-parametric analysis was used because the gene expression results had large standard deviations, which are not suitable for parametric analysis. All results with p<0.05 were considered significant.
As demonstrated in
Engineered tendon scaffolds require appropriate mechanical strength, pore size and porosity for temporary mechanical support, cell infiltration and survival. Tendon extracellular matrix (ECM) components play important roles in modulating the biological activities of tendon fibroblasts or tendon stem cells. In order to develop a scaffold to possess the sufficient mechanical strength, efficient cell seeding area, and natural ECM micro-architecture of native tendons, it was proposed that the scaffold be made from acellular tendon slices.
The mechanical characteristics of native tendon slices were determined to identify the minimum thickness of tendon slice that maintains the fundamental mechanical characteristics of native tendon. It was hypothesized that there would be mechanical alterations with varying thicknesses of native tendon slices. Eight hind limbs were obtained from four dogs that were euthanized for studies approved by the Institutional Animal Care and Use Committee. Three bundles of each Achilles tendon (AT) were dissected from each hind limb. Each bundle was embedded in Tissue-Tek® optimal cutting temperature compound (Sakura Finetek USA, Inc., USA) and sliced with the thickness of 100, 200, 300, 400, or 500 μm in succession on a cryostat (Leica CM1850, Germany) (
Typical stress-strain curves for the AT bundle and representative slices are shown in
The tendon is a fibrous connective tissue, consisting predominantly of type I collagen fibers. The collagen fibrils follow a natural periodic crimp, which is planar with respect to the alignment of the tendon structure. According to hierarchical arrangement of the tendon, the dimension of fibers with crimp waveform is approximately 20 μm, and the dimension of mature fascicles is roughly 200 μm. Slice thickness less than 200 μm would be likely to damage the crimp structure and fascicles, and result in structural weakening. In contrast, the slices with thickness of 300 μm or more should preserve most of the crimp structure and collagen fibril integrity. These data confirmed this hypothesis, as the slices with thickness of 300 μm or more exhibited a standard stress-strain curve similar to the native AT bundles.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application is a National Stage application under 35 U.S.C. 371 and claims benefit under 35 U.S.C. 119(a) of International Application No. PCT/US2010/025870, having an International Filing Date of Mar. 2, 2010, which claims the benefit of priority to U.S. Provisional Application Ser. No. 61/158,091, filed on Mar. 6, 2009. The disclosures of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2010/025870 | 3/2/2010 | WO | 00 | 9/2/2011 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2010/101883 | 9/10/2010 | WO | A |
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| Number | Date | Country | |
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| 20110319993 A1 | Dec 2011 | US |
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| 61158091 | Mar 2009 | US |
