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A polymer scaffold is provided having an extensively interconnected macroporous network with macropores having microporous struts as walls. Macropore diameter ranges from about 0.5 to about 3.5 mm. The polymer may be a biocompatible, biodegradable polymer such as poly(lactide-co-glycolide) containing 75% polylactide and 25% polyglycolide. The polymer scaffold is prepared by mixing a liquid polymer with particles, precipitating the liquid polymer with a non-solvent for the liquid polymer and dissolving the particles with a solvent to form the macroporous polymer scaffold which preferably has porosity greater than 50%. The surface of the polymer scaffold may be modified by acid or base treatment, or by collagen or calcium phosphate deposition. The polymer scaffold has utility for tissue engineering, particularly as a scaffold for in vitro and in vivo cell growth.
1. A macroporous polymer scaffold comprising porous walls that are essentially non-membranous, said porous walls consisting of microporous polymer struts defining macropores which are interconnected by macroporous passageways, said microporous polymer struts containing microporous passageways extending through said microporous polymer struts so that macropores on either side of a given microporous polymer strut are in communication through said given microporous polymer strut, said macropores having a mean diameter in a range from about 0.5 to about 3.5 mm, and said macroporous polymer scaffold having a porosity of at least 50%.
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11. A macroporous polymer scaffold comprising porous walls that are essentially non-membranous, said porous walls consisting of microporous polymer struts defining macropores which are interconnected by macroporous passageways, said microporous polymer struts containing microporous passageways extending through said microporous polymer struts so that macropores on either side of a given microporous polymer strut are in communication through said given microporous polymer strut, said macropores having a mean diameter in a range from about 0.5 to about 3.5 mm, said macroporous passageways connecting macropores having a mean diameter in a range from about 200 μm to about 2 mm, and wherein said microporous passageways have a mean diameter less than about 200 μm, and said macroporous polymer scaffold having a porosity of at least 50%.
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This patent application is a continuation-in-part patent application of U.S. patent application Ser. No. 09/191,107 filed on Nov. 13, 1998, now U.S. Pat. No. 6,379,962 B1, and which is incorporated herein by reference in its entirety.
The present invention relates to the use of a biodegradable polymer scaffold for tissue engineering applications. More particularly, the present invention relates to a novel macroporous polymer scaffold having a high level of interconnectivity between macropores.
Bone treatments for injuries, genetic malformations and diseases often require implantation of grafts. It is well known that autografts and allografts are the safest implants; however, due to the limited supply and the risks of disease transmission and rejection encountered with these grafts, synthetic biomaterials have also been widely used as implants. Complications in vivo were observed with some of these biomaterials, as mechanical mismatches (stress shielding) and appearance of wear debris lead to bone atrophy, osteoporosis or osteolysis around the implants (Woo et al., 1976; Terjesen et al., 1988).
A new approach, defined as Tissue Engineering (TE), has recently raised a lot of interest. Tissue engineering involves the development of a new generation of biomaterials capable of specific interactions with biological tissues to yield functional tissue equivalents. The underlying concept is that cells can be isolated from a patient, expanded in cell culture and seeded onto a scaffold prepared from a specific biomaterial to form a scaffold/biological composite called a "TE construct". The construct can then be grafted into the same patient to function as a replacement tissue. Some such systems are useful for organ tissue replacement where there is a limited availability of donor organs or where, in some cases (e.g. young patients) inadequate natural replacements are available. The scaffold itself may act as a delivery vehicle for biologically active moieties from growth factors, genes and drugs. This revolutionary approach to surgery has extensive applications with benefits to both patient well-being and the advancement of health care systems.
The application of tissue engineering to the growth of bone tissue involves harvesting osteogenic stem cells, seeding them and allowing them to grow to produce a new tissue in vitro. The newly obtained tissue can then be used as an autograft. Biodegradable polyesters—in particular poly(lactide-co-glycolide)s—have been used as scaffolds for tissue engineering of several different cell populations, for example: chondrocytes (as described by Freed et al. in the J. of Biomed. Mater. Res. 27:11-13, 1993), hepatocytes (as described by Mooney et al. in the Journal of Biomedical Mat. Res. 29, 959-965, 1995) and most recently, bone marrow-derived cells (as described by Ishaug et al. in the J. Biomed. Mat. Res. 36: 17-28, 1997 and Holy et al., in Cells and Materials, 7, 223-234, 1997). Specifically, porous structures of these polyesters were prepared and seeded with cells; however, when bone marrow-derived cells were cultured on these porous structures, bone in growth only occurred within the outer edge of 3-D polymeric scaffold (Ishaug et al., supra; Holy et al., supra). Thus, the polymeric scaffolds prepared in these instances were inadequate to allow for the cell growth required to render tissue suitable for implantation or for use as an autograft.
The method of producing polymer scaffolds disclosed in Thomson et al., Fabrication of Biodegradable Polymer Scaffolds to Engineer Trabecular Bone", J. Biomater. Sci. Polymer Edn. Vol. 7, No. 1 pp. 23-38, 1995 VSP, involves formation of gelatin beads, after which a polymer is then "melted" at 80� C. and 333 g pressure around the beads after which the bead/polymer composite is cooled down, and the gelatin is leached out in distilled deionized water. The polymer is forming sheets of material around the beads and is in a solid state before the leaching of the beads/particulate.
U.S. Pat. No. 5,338,772 issued to Bauer et al. is directed to an implant material which is a composite of calcium phosphate ceramic particles and a bioadsorbable polymer. In the method of preparation disclosed in Bauer, calcium phosphate powder is mixed with a polymer and the mixture is subjected to microwave energy which melts the polymer to a liquid that forms a polymer coating around the particles with polymer bridges between encased particles.
It has now been found that polymer scaffolds characterized by macropores in the millimeter size range with interconnections as seen in trabecular bone, are particularly useful for tissue engineering as they allow cell in growth which is crucial for the development of three-dimensional tissue. Such polymer scaffolds can be prepared using a novel process which combines the techniques of phase-inversion and particulate-leaching.
Accordingly, in one aspect of the present invention, there is provided a polymer scaffold comprising macropores, ranging in size between 0.5 mm to 3.5 mm, and having an interconnecting porosity similar to that found in human trabecular bone.
The present invention provides a macroporous polymer scaffold comprising microporous polymer struts defining macropores which are interconnected by macroporous passageways, including microporous passageways extending through the microporous polymer struts so that macropores on either side of a given microporous polymer strut are in communication through the given microporous polymer strut, said macropores having a mean diameter in a range from about 0.5 to about 3.5 mm, and the macroporous polymer scaffold having a porosity of at least 50%.
The present invention also provides a macroporous polymer scaffold comprising microporous polymer struts defining macropores which are interconnected by macroporous passageways, including microporous passageways extending through the microporous polymer struts so that macropores on either side of a given microporous polymer strut are in communication through the given microporous polymer strut, the macropores having a mean diameter in a range from about 0.5 to about 3.5 mm, the macroporous passageways connecting macropores having a mean diameter in a range from about 200 μm to about 2 mm, and wherein the microporous passageways have a mean diameter less than about 200 μm, and the macroporous polymer scaffold having a porosity of at least 50%.
In another aspect of the present invention, a process for making a polymer scaffold is provided comprising the steps of
mixing liquid polymer with particles to form a particulate-polymer mixture;
submerging the particulate-polymer mixture in a polymer non-solvent to precipitate said polymer producing a solidified particulate-polymer mixture; and
submerging the solidified particulate-polymer mixture into a particulate solvent for a time sufficient to dissolve the particles.
In another aspect of the invention there is provided a process for growing tissue, with pervasive distribution, in a macroporous polymer scaffold including macropores to a depth of at least 2.5 times an average macropore size in the scaffold, comprising the steps of:
synthesizing a macroporous polymer scaffold with a trabecular morphology having a porosity of at least 50%, including interrupted pore walls and polymer struts defining macropores which have a mean diameter in a range from about 0.5 to about 3.5 mm and are interconnected by macroporous passageways having a size in a range from about 200 μm to about 2 mm;
seeding the polymer scaffold with tissue cells; and
culturing said tissue cells.
In accordance with one embodiment of the invention a macroporous polymer scaffold is disclosed comprising porous walls that are essentially non-membranous. The porous walls consist of microporous polymer struts which define macropores which are interconnected by macroporous passageways. The microporous polymer struts contain microporous passageways extending through the microporous polymer struts so that macropores on either side of a given microporous polymer strut are in communication through the given microporous polymer strut. The macropores have a mean diameter in a range from about 0.5 to about 3.5 mm, and the macroporous polymer scaffold has a porosity of at least 50%.
According to another embodiment of the invention, a macroporous polymer scaffold is disclosed comprising porous walls that are essentially non-membranous. The porous walls consist of microporous polymer struts which define macropores which are interconnected by macroporous passageways. The microporous polymer struts contain microporous passageways which extend through the microporous polymer struts so that macropores on either side of a given microporous polymer strut are in communication through the given microporous polymer strut. the macropores have a mean diameter in a range from about 0.5 to about 3.5 mm. The macroporous passageways connecting macropores have a mean diameter in a range from about 200 μm to about 2 mm, and the microporous passageways have a mean diameter less than about 200 μm. The macroporous polymer scaffold has a porosity of at least 50%.
Embodiments of the present invention are described in greater detail with reference to the accompanying drawings and computer digitized micrographs, in which:
FIG. 1 is a diagramatic representation of a portion of a polymer pore system illustrating different components as defined hereinafter;
FIG. 2 is a light micrograph of the bone trabeculae in the neck of the femora showing the isotropic and anisotropic areas (Modified light micrograph from Tobin W J, in J. Bone Jt Surg 37A(1) 57-72, 1955);
FIG. 3A is a light micrograph of a polymer in accordance with the present invention (field width=1.8 cm);
FIG. 3B is a light micrograph of a 20 μcm section of the polymer scaffold of FIG. 3A (field width=3.5 mm);
FIG. 3C is a scanning electron micrograph of the pore walls of the polymer scaffold of FIG. 3A;
FIG. 4A is a chart illustrating the stress/strength curve of the polymer scaffolds when submitted to a compressive test at a rate of 1% deformation per second;
FIG. 4B is a chart illustrating the effect of polymer concentration on mechanical properties of polymer scaffolds. The Young Modulus of the first elastic region is referred to Y1 and the Young Modulus of the second elastic region is referred to Y2;
FIG. 5 is a scanning electron micrograph of the pore wall structure of a scaffold prepared with a concentration of 0.05 g/ml PLGA 75:25 in DMSO;
FIG. 6 is a scanning electron micrograph of the pore wall structure of a scaffold prepared with a concentration of 0.2 g/ml PLGA 75:25 in DMSO;
FIG. 7A is a scanning electron micrograph of PLGA 75/25 scaffolds obtained using particles less than 0.35 mm;
FIG. 7B is a scanning electron micrograph of PLGA 75/25 scaffolds obtained using particles ranging from 0.54 to 0.8 mm;
FIG. 7C is a scanning electron micrograph of PLGA 75/25 scaffolds obtained using particles ranging from 0.8 to 2.0 mm;
FIG. 8 is a scanning electron micrograph of a PLGA 75/25 membrane prepared in absence of particles;
FIG. 9A is a scanning electron micrograph of a PLGA 75/25 foam obtained at Tmix=11� C., and Tnonsolvent=0� C.;
FIG. 9B is a scanning electron micrograph of a PLGA 75/25 foam obtained at Tmix=−20� C., and Tnonsolvent=0� C.;
FIG. 9C is a scanning electron micrograph of a PLGA 75/25 foam obtained at Tmix=−20� C., and Tnonsolvent=40� C.;
FIG. 10 is a scanning electron micrograph of leaflet CaP coating a PLGA 75/25 scaffold;
FIG. 11 is a confocal micrograph of a Dex+ scaffold cultured for 42 days (field width=1.8 mm);
FIG. 12 is a UV-light illuminated light micrograph of a Dex+ scaffold stained with tetracycline (Field width=2.0 cm);
FIG. 13 is a light micrograph of an osteocalcin immunolabeled scaffold. (Field width=1.1 cm);
FIG. 14 is a light micrograph of a haematoxylin and eosin stained Dex+ cultured scaffold section. (Field width=0.8 cm);
FIG. 15 is a light micrograph of a haematoxylin and eosin stained Dex− cultured scaffold section. (Field width=0.6 cm);
FIG. 16A is a scanning electron micrograph of a prior art PLGA 75/25 membranous scaffold created with particles less than 0.35 mm;
FIG. 16B is a scanning electron micrograph of a prior art PLGA 75/25 membranous scaffold created with particles ranging in size from 0.54 to 0.8 mm.
FIG. 16C is a scanning electron micrograph of a prior art PLGA 75/25 membranous scaffold created with particles ranging in size from 0.8 to 2.0 mm;
FIG. 16D is a scanning electron micrograph of PLGA 75/25 Intermediate scaffold created with particles less than 0.35 mm;
FIG. 16E is a scanning electron micrograph of PLGA 75/25 Intermediate scaffold created with particles ranging in size from 0.54 to 0.8 mm;
FIG. 16F is a scanning electron micrograph of PLGA 75/25 Intermediate scaffold created with particles ranging in size from 0.8 to 22.0 mm;
FIG. 16G is a scanning electron micrograph of PLGA 75/25 Bone-like scaffold created with particles less than 0.35 mm;
FIG. 16H is a scanning electron micrograph of PLGA 75/25 Bone-like scaffold created with particles ranging in size from 0.54 to 0.8 mm; and
FIG. 16I is a scanning electron micrograph of PLGA 75/25 Bone-like scaffold created with particles ranging in size from 0.8 to 2.0 mm.
FIG. 1 is a diagrammatic representation of a portion of a polymer scaffold showing two macropores defined by interrupted walls and struts discussed more fully below, the macropores being interconnected with each other by a macroporous interconnection. The two macropores are also connected to the surrounding macropores by microporous passageways (also referred to as micropores). These and several other terms used in the description of the polymer scaffold produced according to the present invention are defined herebelow.
Scaffold: device designed as a cell carrier for tissue engineering or related applications. This device has preferably a porous morphology to be colonized by cells. In the present invention the scaffold has an open-pore morphology.
Macropores: voids within the polymer scaffold, delinated by interrupted walls and struts. The macropores typically have a diameter between 0.5 and 3.5 mm.
Pore walls: predominately formed from polymer struts that delineate the macropores but parts of the pore walls may be considered to be interrupted walls, i.e. small flat or planar plates. This is consistent with the definition of trabeculae by Martin R B (in CRC Critical Reviews in Biomedical Engineering, 10(3), 179-222, 1984), who described the trabeculae of bone as "a complex system of interrupted walls and struts". Webster's New World Dictionary defines trabecula as "a) a small rod, bar or bundle of fibers b) small septum of fibers forming, with others of its kind, an essential part of the framework of an organ or part".
When the polymer struts form anisotropic bundles, in which microporous interconnections separate struts from each other in the same bundle, the structure of the pore wall is defined as "lamellar". The struts may also exhibit isotropic morphologies in which the struts are widely separated from each other by mostly macroporous interconnections. Both lamellar and strut-like pore wall structures exhibit nanopores when sectioned.
Micropororous interconnections (also called micropores or microporous passageways): Voids found in lamellar pore walls. Each strut or lamellae of polymer is separated from each other by elongated, parallel pore structures called micropores. The size of these pores is less than 200 μm. Micropores contribute to the overall interconnectivity of the scaffolds.
Macroporous interconnections (also called macroporous passageways): these are passageways between lamellar arrays of pore walls, or between polymer struts. They contribute mostly to the interconnectivity of the macropores, and range in size between 200 μm and 2 mm.
Nanopores: Voids found in the bulk of the polymer. Cross-sections of bulk polymer material, either from pore wall struts or pore wall lamellar structures, exhibit rounded concavities that may, or may not, perforate the entire polymer bulk material. These nanopores may result from trapped non-solvent within the bulk of the polymer, or from autocatalytic degradation of the bulk of the polymer. Nanopores are distributed in the walls of the scaffold. They only contribute to the overall interconnectivity of the macropores when they go through the entire bulk material.
Interconnections: the flow passageways connecting the macropores with each other. The interconnections comprise the macroporous interconnections (passageways), the microporous interconnections (passageways), and the nanopores that perforate the entire bulk material defined above.
The present invention provides a macroporous polymer scaffold comprising macropores and interconnections. Macropores have a diameter in the range of 0.5-3.5 mm, and interconnections as seen in trabecular bone. The morphology of the polymer scaffolds (also referred to as foam structures) disclosed herein is based on that of trabecular bone.
Trabecular bone has been shown to be metabolically the most active site in bone (as described by Rodan G A, in Bone 13, S3-S6 1992). The specific open pore geometry of trabecular bone favorably affects bone formation and resorption, and is therefore of considerable interest in the context of bone tissue engineering: indeed, the design of an ideal scaffold for bone tissue engineering should also allow fast bone formation and resorption. The morphology of bone trabeculae has therefore served as a model to create the new polymer scaffold structures disclosed herein.
The architecture of the trabeculae of bone depends on the anatomic site where the bone is found and, to a lesser extent, on the age of the patient.
As mentioned above, Martin R B (in CRC Critical Reviews in Biomedical Engineering, 10(3), 179-222, 1984) defines the trabeculae of bone as "a complex system of interrupted walls and struts" wherein the voids found between the trabeculae are called "marrow spaces". The directions of the trabeculae are irregular; however, a global organization of the trabecular geometry is sometimes visible and follows the forces acting on the bone. Areas where trabeculae follow a given direction are anisotropic whereas areas where trabeculae are disposed randomly are isotropic (cf. FIG. 2).
Whitehouse and Dyson (supra) as well as Martin (supra) described the porosity of the trabeculae bone in the femora in great detail. Table 1.1 indicates different porosities and trabecular width determined by Whitehouse and Dyson for all areas of the femora.
The structure of trabecular bone has been investigated for trabecular width, porosity, anisotropy, and general patterns like connectivity and star volume. Light and scanning electron micrographs published on trabecular bone indicate that the marrow spaces delineated by trabeculae (i.e. pores) range from one to several millimeters in size and are interconnected with holes ranging from approx. 0.3 to one millimeter.
When the use of the trabeculae produced of polymer forming the present invention is for physiological applications, the polymer scaffold is preferably prepared from any biocompatible polymer. The term "biocompatible" as it is used herein is meant to encompass polymers which are not toxic to cells and which allow cells to colonize thereon. Examples of suitable polymers include poly(lactide), poly(lactide-co-glycolide) (PLGA) of varying ratios, polystyrene, poly(glycolide), poly(acrylate)s, poly(methyl methacrylate), poly(hydroxyethyl methacrylate), poly(vinyl alcohol), poly(carbonate), poly(ethylene-co-vinyl acetate), poly(anhydride), poly(ethylene), poly(propylene), poly(hydroxybutyrate), poly(hydroxyvalerate), poly(urethane)s, poly(ether urethane), poly(ester urethane), poly(arylate), poly(imide), poly(anhydride-co-imide), poly(aminoacids) and poly(phosphazene). Biodegradable, aliphatic polyesters such as polylactic acid, and polymers derived therefrom, represent a particularly useful class of polymers in applications of the present scaffolds, which relate to cell transplantation due to the fact that they have already been approved for human clinical use. In this regard, a preferred polymer for use as scaffold is PLGA, particularly blends comprising more than 50% poly(DL-lactide) such as PLGA 85:15 and PLGA 75:25.
Suitable applications for the present scaffolds will vary with polymer composition and structure. For example, biodegradable polymer scaffolds are suitable for use in either, in vitro applications and/or in vivo cell transplantation. The matrices may serve then as supports or scaffolds to allow cell growth to occur in vitro prior to implantation in vivo. The scaffolds may also be used directly in vivo, without being pre-seeded with cells. In both applications (with or without prior cell seeding), biodegradable polymer matrices in accordance with the present invention are particularly useful for the growth of three-dimensional tissue and may be used in the growth of connective tissues, like bone, cartilage, paradontal tissue, as well as dental tissues and other organs, such as liver or breast tissue.
A significant characteristic of the present polymer scaffold is the presence of macropores at least 50% of which have a diameter within the range of 0.5 to 3.5 mm, a range representative of that found in the human trabecular bone. Preferably, the macropores have a diameter of at least 1.0 mm, and most preferably, the macropores have a diameter between about 1.0 mm and 3.5 mm.
In addition to its macroporous structure, the scaffold is also characterized by a high level of interconnectivity which enhances both penetration of the scaffold by cells and nutrient flow to cells. Macroporous interconnections of at least 0.35 mm provide an "open cell" environment in the polymer scaffold, which is important to encourage tissue growth throughout the scaffold, i.e. three-dimensional tissue growth.
The macropores are delineated by porous polymer walls that may or may not exhibit a lamellar structure. Total thickness of the pore walls is no greater than about 0.4 mm, and preferably no greater than about 0.3 mm. The degree of interconnectivity in the pore walls is dependent, upon other factors, of the processing temperatures.
A suprising and unexpected result is that each macropore is in flow communication with a significant number of neighboring macropores via both macro- and microporous interconnections.
Scaffolds with different pore wall structures obtained at different processing temperatures using this novel phase inversion particulate leaching process are described in the present document.
The porosity of the polymer scaffold is at least at a level of 50% for all scaffolds obtained, as estimated using Northern Eclipse image analysis software and preferably at a level of greater than 50%. The level of porosity of the present polymer scaffold also contributes to the "open cell" nature thereof, resulting in significant overlap between macropores (giving rise to the macroporous passageways) which defines the highly interconnected nature of the present scaffold and further enhances its utility as a scaffold for cell growth. In this regard, the level of porosity is preferably greater than about 75%, while the most preferred level of porosity is greater than about 85%.
The features of the present scaffold make it particularly suitable for use in tissue engineering and more notably, cell transplantation, because it provides a biocompatible scaffold that cells can colonize in a three-dimensional manner via the interconnected macroporous network of the scaffold. This is significant when considering the transplantation of any cells that yield tissues, especially those requiring neoangiogenesis such as bone tissue. Moreover, when used for cell transplantation, the scaffold is biodegradable, the degradation of which can be controlled such that cell growth may be simultaneous with the degradation of the scaffold.
It will be understood by those of skill in the art that the present polymer scaffold may be modified in order to enhance further its properties for use as a scaffold for cellular growth. Modifications typically effecting the structures used as support for cellular growth would also be suitable to modify the present polymer scaffold. Such modifications function to enhance biological response and include, for example, surface modifications with collagen, calcium phosphate, proteoglycans, proteins, peptides, carbohydrates and polysaccharides, or by acid/base treatment. Additionally, the polymer scaffold may serve as a reservoir for the delivery of active molecules, such as proteins, growth factors, etc. that enhance cellular function.
The present polymer scaffold can be made using a novel process which combines particulate leaching methodology with phase inversion methodology. In an initial step, the selected polymer scaffold is prepared as a liquid polymer. As used herein, the term a liquid polymer is meant to refer to polymer in liquid form, either alone or admixed with another liquid. This may be done by mixing the polymer in a solvent to form a polymer solution. Any solvent generally useful to prepare a polymer solution can be used for this purpose, including dimethylsulfoxide (DMSO), methylene chloride, ethyl acetate, chloroform, acetone, benzene, 2-butanone, carbon tetrachloride, chloroform, n-heptane, -hexane and n-pentane. As one of skill in the art will appreciate, non-cytotoxic solvents such as DMSO are preferably used to prepare the solution so as not to adversely affect cellular growth. The concentration of the polymer in the polymer solution will vary with the characteristics of the polymer used to make the scaffold. Alternatively, the polymer can be formed into a liquid polymer by heating to its melting point.
The liquid polymer is then admixed with particles of an appropriate size in connection with the particulate leaching phase of the process. Particles having a diameter corresponding to the desired diameter of the macropores in the polymer scaffold are suitable, specifically particles having a diameter in the range of 0.5-3.5 mm. More preferably, the particles have a diameter of greater than 1.0 mm and most preferably, the particles have a diameter of between 1.0 and 2.0 mm. Examples of suitable particles for admixture with the polymer include polysaccharides (such as glucose), organic and inorganic salts, proteins and lipids of an appropriate size which can be dissolved in a solvent other than a solvent for the polymer (i.e. a polymer non-solvent). The amount of particles admixed with the polymer solution will again vary with the characteristics of the polymer used to make the present scaffold.
Once the particles have been thoroughly mixed with the liquid polymer to form a particulate polymer mixture, the polymer is subjected to a phase inversion (precipitation) step in which it is converted from a liquid to a solid. This step is achieved by submerging the particulate polymer mixture in a polymer non-solvent, in other words a solvent in which the polymer is insoluble and in which the polymer precipitates. Such polymer non-solvent include, for example, water, alcohol, 1-4 dioxane and aniline.
The step of precipitating the polymer-particulate mixture results in the formation of elongated struts that form the interrupted pore walls defining the macroscopic pores and the macroscopic passageways connecting the macroscopic pores. The particles act as spacers separating the polymer struts. Therefore, the precipitated polymer does not form a coating that intimately envelops the particles, as is the case in previous methods, but results in an macroporous scaffold characterized by macropores interconnected by macroporous passageways.
In order to obtain a solid polymer scaffold in a particular shape, the polymer mixture can be placed in a mold during the phase inversion step. Preferably, the liquid polymer can be stabilized around the particulates by, for example, freezing the polymer-particulate slurry. Thereby, no mold is used and the phase inversion process occurs simultaneously from all outer surfaces. When the polymer solvent is DMSO, for example, the polymer mixture is cooled to a temperature less than or equal to 12� C., which is the freezing temperature of DMSO. Cooler temperatures, such as temperatures of less than 0� C. can also be used. A consequence of using low temperatures (for example, −20� C. or −80� C.) during this stage of the process is the subsequent formation of a polymer scaffold with a different morphology (cf Example 4), like a thicker skin structure, which may be removed prior to use as a scaffold for three-dimensional cell growth, as described in Example 1. In addition to cooling, other methods of stabilizing the polymer-particulate mixture may be used, for example gellation (increasing viscosity).
Following conversion of the polymer mixture from liquid to solid phase, the polymer is subjected to particulate leaching. In this step of the process, the polymer is immersed in a particulate solvent, i.e. a solvent which functions to dissolve the particles dispersed throughout the polymer but does not dissolve the polymer itself. Appropriate particulate solvents will, of course, depend on the nature of the particles and the polymer. Examples of appropriate particulate solvents include water, alcohol, 1-4 dioxane and aniline. The temperature of the particulate solvent can be varied with minimal effect on the resulting polymer scaffold. However, the temperature will generally be between the freezing point of the particulate solvent and the glass transition temperature of the polymer, so that the polymer scaffold does not melt or become viscous under the effect of the non-solvent temperature. In one example, a particulate solvent temperature of between about 0� C. and 45� C. is applied when the particulate solvent is water and the polymer is PLGA 75:25.
The polymer is submerged in the particulate solvent for an amount of time appropriate to allow complete dissolution of the particles dispersed throughout the polymer scaffold. Generally, a period of at least 24 hours is required to obtain complete particulate dissolution in the polymer scaffold, while a period of at least 48 hours is preferred. In order to expedite efficient dissolution of the particles, it is desirable to immerse the polymer in fresh solvent at frequent intervals during the dissolution period, for example at approximately 8-9 hour intervals or by the use of a circulating solvent bath.
The phase-inversion and particulate-leaching processes may occur in one step with a solvent that is simultaneously a polymer non-solvent and a particulate solvent. In one example, double distilled water (ddH2O) was used as the solvent.
The polymer scaffold is removed from the particulate solvent following an appropriate particulate dissolution period and can be either vacuum-dried prior to use or disinfected in alcohol (such as 70% ethanol), rinsed and conditioned in culture medium for subsequent use. The scaffold may also subsequently be sterilized for in vivo use, comprising the placing the scaffold in the chamber of a radio frequency glow discharge plasma instrument by applying preferably an argon gas (or other inert gas) radio-frequency glow discharge. A non-limiting process used a power of 100 W with the plasma sustained for 4 minutes. Another sterilization method that may be used is a low temperature ethylene oxide (ETO) sterilization procedure which will be known to those skilled in the art. Another sterilization process that may be used is a gamma-sterilization procedure which will also yield sterile scaffolds.
If the polymer scaffold is not required for immediate use, it is desirably stored dry in a desiccator to prevent moisture retention and possible degradation of the polymer.
The present process advantageously yields a polymer scaffold having unique characteristics, and in particular, yields a polymer scaffold having an interconnected macroporous network. Another significant advantage of the present two-stage process is that it provides amplified means for controlling the morphology of the resulting polymer scaffold. In other words, the process provides two levels, particulate leaching and phase inversion, at which to effect the morphology of the polymer scaffold. For example, macropore size and distribution can be altered during both, the particulate leaching and phase inversion stage of the process and are governed by particulate size and distribution, and, to a lesser extend by the scaffold processing temperatures. In addition, interconnection formation and size can be influenced by varying the rate of the phase inversion. The rate of the phase inversion can be altered altering a number of variables including temperature, type of polymer non-solvent and polymer concentration. Thus the final scaffold morphology can be controlled. Preferably, the resultant morphology resembles that of human trabecular bone.
In another aspect of the present invention, a method for culturing cells for three-dimensional growth is provided utilizing the polymer scaffold described herein. The novel interconnected macroporous structure of the present polymer scaffold is especially suitable for tissue engineering, and notably bone tissue engineering, an intriguing alternative to currently available bone repair therapies. In this regard, bone marrow-derived cell seeding of the polymer scaffold is performed using conventional methods, which are well known to those of skill in the art (as described in Maniatopoulos et al, in Cell Tissue Res 254, 317-330, 1988). Cells are seeded onto the polymer scaffold and cultured under suitable growth conditions. The cultures are fed with media appropriate to establish the growth thereof.
As set out above, cells of various types can be grown throughout the present polymer scaffold. More precisely, cell types includes hematopoietic or mesenchymal stem cells, and also includes cells yielding cardiovascular, muscular, or any connective tissue. Cells may be of human or other animal origin. However, the polymer scaffold of the present invention is particularly suited for the growth of osteogenic cells, especially cells that elaborate bone matrix. For tissue engineering, the cells may be of any origin. The cells are advantageously of human origin. The present method of growing cells in a three dimensional polymer scaffold according to the invention allows seeded osteogenic cells, for example, to penetrate the polymer scaffold to elaborate bone matrix, during the in vitro stage, with pervasive distribution in the structure of the polymer scaffold and particularly to a depth of at least 2.5 times the depth of the average macropore size. Osteogenic cell penetration and, as a result, bone matrix elaboration can be enhanced by mechanical, ultrasonic, electric field or electronic means. While for many human or animal applications, such as bone or tissue replacement the polymer is biocompatible and non toxic, it may preferably be biodegradable also. However, it will be appreciated that in some of these applications it may be preferred or advantageous to use a biocompatible polymer which is not biodegradable in situations where a permanent scaffold is needed to support other tissue. Further, it will be appreciated that for non biological applications the requirements for biocompatibility and biodegradable need not be invoked, for example in applications where the scaffold is used as a filter or catalyst support these requirements may not be important.
Embodiments of the present invention are described in the following specific examples which are exemplary only and not to be construed as limiting.
A PLGA 75:25 polymer scaffold in accordance with the present invention was prepared using PLGA 75:25 (obtained from Birmingham Polymer Inc), having an inherent viscosity of 0.87 dL/g. One ml of 0.1 g/ml of PLGA 75:25 in DMSO was mixed with 2 g of glucose crystals (particle size ranging from 0.8 mm to 2 mm) in an aluminum mold. The PLGA 75:25-DMSO mixture was cooled to −20� C. This temperature of the PLGA 75:25-DMSO mixture is referred to Tmix. The frozen PLGA 75:25 blocks were then immersed in an ice-water slurry of ddH2O at 0� C., which is a non-solvent for the polymer. This temperature of the water is referred to Tnonsolvent. The blocks remained in ddH2O for 48 hours during which the ddH2O was changed approximately every 8 hours. The obtained scaffolds were then removed from the water, vacuum-dried for 72 h at 0.01 mm Hg and stored at 4� C. in a desiccator under vacuum until use. Scaffolds obtained using the above mentioned conditions were then fully analyzed.
The macroporous structure of 2 mm thick polymer scaffold sections was observed at low magnification (16�) using a dissection microscope as shown in FIG. 3A. A uniform distribution of interconnected macropores ranging in size from about 0.8-1.5 mm was observed throughout the polymer scaffold. The macropores exhibited elliptic morphologies and thick porous walls (about 300 μm thick) containing micropores.
The polymer scaffold was then embedded in Tissue-Tek embedding medium (Miles #4583), and sectioned in a cryostat at −20� C. A serial set of 20 μm-thick sections (50 sections) were collected on glass slides (VWR Canlab). Sections were photographed at low magnification (16�) using a dissecting microscope and scanned. FIG. 3B is a scanned scaffold section that identifies the porous components of the scaffold, the macropores, the macroporous interconnections (passageways) and the microporous interconnections (passageways). A polymer thin film (i.e. a skin layer) was observed on the outer surface of the polymer scaffold. The images were converted to TIFF files and analyzed on a PC computer using the Northern Eclipse image analysis software. The "single measurement" menu was used to measure the pore wall sizes (area, perimeter, diameter etc.) for each scanned section. The "data measure" routine computed the area and number of pore wall struts per scanned slide.
These measurements were converted from pixel units to millimeters by calibrating the system, using the above mentioned magnification of the scanned images to determine the pixel/mm ratio. Macropore size was determined by manually drawing a line with a software tool on the digitized image of the polymer scaffold section from one pore wall to the adjacent pore wall. The characteristics of the resulting polymer scaffold as determined using the Northern Eclipse image analysis software were as follows:
The porosity of the polymer matrices was also estimated by mercury porosimetry (Quantachrome Autoscan 60). A solid penetrometer with 5 cm3 cell stem volume was used for samples in the range of 0.015 to 0.020 g. The values of void volume were calculated from the mercury intrusion volume. The porosity was calculated from the mercury intrusion volume to be 89.6%. The porosity estimated using the Northern Eclipse image analysis software (˜87%) is substantially equivalent to that of ˜90% as measured by mercury porosimetry given that the mercury porosimetry method is not accurate when analyzing polymer scaffolds with pore diameters greater than ˜75 μm.
The polymer scaffold was also prepared for analysis using a scanning electron microscope (SEM). The scaffold was cross-sectioned at a thickness of approximately 2 mm and sputter-coated with gold under argon atmosphere (Polaron Instrument Inc., Doylestown, Pa.). Scanning electron micrographs were taken on a Hitachi 2500 SEM at 15 kV acceleration voltage. The diameter of the macropores was confirmed using the SEM micrographs to be about 1 to 1.5 mm, although a clear separation between each macropore was not always observed illustrating the very open interconnected structure of these polymer scaffolds.
The microporous nature of the pore walls, as observed under the optical microscope, was confirmed by SEM, as shown in FIG. 3C.
The polymer scaffold was mechanically tested as follows. A polymer scaffold in the form of a cylinder with a diameter and a height of 1.5 cm was prepared and tested using an Instron Mechanical tester. The mechanical experiments were performed on a uniaxial servohydraulic testing machine (Instron Model 1331 load frame with Series 2150 controller). A 1 kg load cell (Sensotec, Model 31/4680) was used for all compression tests. The deflection of the actuator was measured by a DC linearly variable differential transformer (LVDT, intertechnology Model SE 374). Signals from the load cell and the LVDT were displayed during testing on a digital storage oscilloscope (Gould, Model 1425). The signals were also input into a 16-channel, 12-bit analog-to-digital (A/D) converter in an accelerated Apple lle computer. The rate of data acquisition for these experiments was 430 pairs of data points per second. Compression of the polymer scaffold occurred at a rate of 0.1 mm/s .As shown in FIG. 4A, A plot of compression strength vs. percent deformation of the polymer scaffold showed two moduli. The Young's modulus for the first elastic region (referred to Y1) was 0.76�0.12 MPa , and for the second elastic region (referred to Y2) was 0.18�0.016 MPa.
The effect of PLGA 75:25 concentration in DMSO on the structure of the resulting polymer scaffold was determined using the protocol outlined in detail in Example 1. Three different concentrations of PLGA 75:25 in DMSO (0.05 g/ml, 0.1 g/ml and 0.2 g/ml) were used to make polymer matrices while all other conditions were maintained constant as described in Example 1.
Each of the polymer scaffolds prepared were cut in half using a razor blade. A skin structure was found on each regardless of the starting concentration of PLGA 75:25 in DMSO. The mechanical properties of the 3 different polymer scaffolds were assessed and are illustrated in FIG. 4B. A significant decrease in Young's modulus was observed in the polymer scaffold prepared using the PLGA in DMSO of 0.05 mg/ml while the stiffest scaffold was obtained with a PLGA 75:25 concentration of 2 mg/ml.
These scaffolds were also observed under light microscopy and SEM. No differences in structure could be detected between the three polymer scaffolds under light microscope. However, when observed under the SEM, the scaffolds created with 0.05 g/ml PLGA in DMSO exhibited more of a lamellar wall structure with more microporous interconnections (see FIG. 5), than those created with 0.2 g/ml PLGA in DMSO, where fewer microporous porous interconnections were seen (see FIG. 6).
The effect on polymer scaffold structure of both varying the amount and size of the glucose particles admixed with the PLGA polymer was determined as follows. Differing amounts of glucose particles (0.5 g, 1 g and 2 g) were separately admixed with 1 ml polymer solution, maintaining all other conditions as described in Example 1 constant. The effect of particle size on the final scaffold morphology was also assessed by using the following sieved particles: (standard testing sieves, VWR, West Chester, Pa.): 1) NaCl crystals (<0.35 mm), 2) sucrose crystals (0.54 mm<crystal size<0.8 mm) and 3) glucose crystals (0.8 mm<crystal size<2 mm). The resulting polymer scaffolds were observed by light microscopy. When mixing the polymer solution with the particulates, it was seen that for small amounts of particulates (i.e. 0.5 g/ml), the polymer solution was not fully immersed in the particulate bed. This layer of polymer solution resulted after phase inversion in a membranous structure, similar to that seen when no particulates are used. Larger solution densities of particulates (i.e. 2.0 g/ml) completely infiltrated the polymer solution so that the resulting scaffold contained a distribution of macropores without this membranous structure.
The size of the macropores was directly proportional to the size of the particles used, e.g. macropore size were ˜0.33 mm when particles<0.35 mm were used (cf. FIG. 7A), and ˜0.75 mm when particles ranging from 0.54 to 0.8 mm were used (cf. FIG. 7B). Finally for particles bigger than 0.8 mm, the observed macropores were ˜1.4 mm (cf FIG. 7C). When no particles were mixed to the polymer-DMSO solution, the resulting polymer structure was a hollow cylinder composed of a thick skin containing micropores, as illustrated in FIG. 8. This skin closely resembled the membrane structure resulting from a normal phase-inversion process.
The effects of three different Tmix (11� C., −20� C. and −80� C.) at constant Tnonsolvent (0� C.) were studied. Two main different scaffold structures were obtained: 1) with Tmix=11� C. and 2) with Tmix=−20� C. and Tmix=−80� C. Scaffolds obtained with a Tmix=11� C. and a Tnonsolvent=0� C. were skinless and showed a very open structure. As shown in FIG. 9A, The macropores sizes seemed expanded, and were estimated by SEM at to ˜2.72 mm. The pore walls had less micropores but more macroporous interconnections, providing a generally more open structure to the scaffolds. The scaffolds obtained for Tmix=−20� C. and −80� C. both had a skin structure. For Tmix=−20� C. the macropores seemed smaller than on scaffolds obtained at higher Tmix and their sizes were estimated by SEM at ˜1.8 mm. The pore walls were lamellar, with fewer macroporous interconnections but more microporous interconnections (cf FIG. 9B). It was observed that the macropore size decreased with lower Tmix. The differences in macropore sizes were particularly important between scaffolds created at Tmix=11� C. and Tmix=−20� C., whereas minor differences in macropore size were observed between scaffolds created at Tmix=−20� C. and Tmix=−80� C. While the macropore sizes diminished with Tmix the structure of the pore wall also changed as described above. Differences in Tmix may have affected the rate of polymer precipitation, and therefore, the complexity of the pore wall structure.
Different Tnonsolvent (40� C., 20� C. and 0� C.) were also studied, with a constant Tmix of −20� C. In this case, the main difference between all scaffolds was their pore wall thickness. Lower Tnonsolvent caused thicker and more complex pore walls whereas higher Tnonsolvent created thin and compact pore walls, comparable to polymer struts delineating each macropore. FIGS. 9B and 9C show the different morphologies of the scaffold structures at Tnonsolvent=0� C. and 40� C. respectively. Most structural differences were seen between scaffolds created at Tnonsolvent=0� C. and Tnonsolvent=20� C. Fewer differences were seen between scaffolds obtained at Tnonsolvent=20� C. or 40� C. While lower Tnonsolvent (0� C.) provided lamellar pore walls (cf FIG. 9B), higher Tnonsolvent (40� C.) provided strut-like pore wall morphologies (cf FIG. 9C).
The thickness of the pore walls of scaffolds created at different Tnonsolvent was estimated by SEM. At Tnonsolvent=0� C., the pore walls were estimated at 0.29 mm, whereas at Tnonsolvent=20� C., the size of the pore walls was ˜0.10 mm; and no significant differences could be measured between Tnonsolvent=20� C. and 40� C. All scaffolds created with the various temperatures as mentioned above were sectioned, and pore size and pore wall thickness were measured. Their porosity was also estimated using the Northern Eclipse image analysis software. The following results were obtained:
The obtained scaffolds as described in Example 1 were further surface modified by acid/base treatment; plasma modification with ammonia gas; modification by ozone treatment; collagen deposition; calcium phosphate deposition and platelet rich plasma deposition. The procedures and results were as follows:
Acid/base treatment was developed to enhance surface charge and to change the surface topography. The scaffolds were maintained in several concentrations of acetic acid (0.1M, 1M, 5M) for 24 h. Scaffolds were also maintained in various concentrations of NaOH for 24 h to observe surface polymer chain hydrolysis. Under SEM, the scaffolds treated with 5M acetic acid or 0.1 M NaOH for 24 hours showed changes in surface topography with appearance of nanopores.
Plasma modification with ammonia gas was achieved by placing a scaffold in the chamber of a radio frequency glow discharge plasma instrument, evacuating the chamber and purging with ammonia. The ammonia plasma was generated at powers between 33 W and 100 W for up to 10 min. Scaffolds treated this way were found to have more nitrogen and oxygen than un-modified counterparts and were more hydrophilic. These surface properties may enhance cell-material interactions, including cell adhesion and, spreading and tissue formation.
A collagen deposition experiment was designed to enhance cell adhesion on the polymer surfaces. The scaffolds were maintained in 0.1% collagen for 1 h, 5 h, 8 h and 24 h.
A calcium phosphate deposition experiment was tested to enhance cell adhesion on the surface of the scaffolds. These were maintained for 1 week in fully supplemented medium (as described in Example 6) at 37� C. The calcium phosphate crystals on the surface of the scaffolds were visualized by Von Kossa staining.
Further CaP deposition experiments were conducted, in which the scaffolds were dipped in 1.5 mM Na2HPO4 for 2 h at room temperature, and further equilibrated in a saturated Ca2+ solution overnight. The scaffolds were then observed under SEM and crystals with leaflet morphologies were observed on the scaffold structure. (Cf FIG. 10).
A platelet-rich plasma deposition to increase in vivo responsiveness of the scaffolds in various tissues involves soaking the scaffolds in a platelet-rich plasma prior to use.
The obtained scaffolds as described in Example 1 were further bulk modified by addition of a plasticizer; incorporation of drugs/genes into the polymer; and addition of an inorganic phase into the polymer. The procedures and results were as follows:
The bulk modulus of the polymer scaffold was reduced by addition of a plasticizer, thereby yielding scaffolds for use in soft tissue applications, such as, cardiovascular and/or muscular tissues. Poly ethylene glycol (PEG) with a Mw of 5000 was added to a 1.0 g/ml solution of PLGA 75/25 in DMSO to a final weight ratio of PEG:PLGA of 0.5:1. The PEG-PLGA solution was mixed with particulates, the polymer was precipitated and the particulates and PEG polymer were leached out in H2O, as described in Example 1. This process yielded a scaffold with enhanced nanoporosity and hence, lower bulk modulus.
Addition of an inorganic phase, for example, Calcium phosphate powders to the bulk of the polymer, was investigated as a mean to enhance the osteogenic and mechanical properties of the scaffold. Dicalcium phosphate anhydrous (DCPA)_and tetracalcium phosphate (TTCP) were mixed in a 1:1 ratio, and added to the polymer DMSO solution to a ratio of particulates:polymer ranging from 1:1 to 5:1. Glucose particulates were added to the slurry and the polymer was precipitated as described above, in H2O. Calcium phosphate crystals were trapped in the resulting scaffolds.
Addition of proteins and/or DNA into the polymer slurry prior to mixing with the glucose particulates yielded scaffolds with entrapped proteins and/or DNA and/or genes and/or vectors. This is of particular interest for the use of these scaffolds as protein/DNA/gene/vectors delivery systems.
PLGA 75:25 polymer scaffolds were prepared as previously described: 2 g/ml glucose crystals were dispersed in a 0.1 g/ml PLGA 75:25 solution in dimethylsulfoxide (DMSO, BDH, Toronto, ON). The polymer slurry was frozen at 11� C. The polymer was then precipitated and the glucose crystals were extracted from the precipitated polymer in ddH2O at 40� C. Scaffolds were dried to constant mass (10 μm Hg, 72 h), disinfected in 70% EtOH for � h, rinsed 3� with a-MEM and equilibrated in sterile a-MEM at 37� C. for 6 days.
First passage primary bone marrow-derived cells were seeded on 0.25 cm3 scaffolds using protocols and media described in detail elsewhere (as described by Maniatopoulos et al, supra, and Davies et al., in Cells and Materials, 1:3-15,1991). Briefly, bone marrow-derived cells were collected from both femora of young adult male Wistar rats (approximately 150 g) into a fully supplemented medium (FSM): a-MEM supplemented with 15% fetal bovine serum, 50 mg/mL ascorbic acid, 10 mM β-glycerophosphate and antibiotics (0.1 mg/mL penicillin G, 0.05 mg/mL gentamicin and 0.3 mg/mL fungizone); 10−8 M Dexamethasone (Dex) was added to the FSM of only Dex+ cultures.
Cells were maintained in culture for 6 days, and re-fed at days 2 and 5 with FSM. At day 6, Dex− cells were trypsinized with 0.01% trypsin in PBS, whereas Dex+ cultures, in which signs of calcification were visible, were trypsinized with 0.01% trypsin and 10 μM ethylene diamine tetraacetic acid (EDTA) in PBS. Dex+ and Dex− cells were then seeded on separate pre-wefted scaffolds at a concentration of 7.5�105 cells/scaffold. The cultures were maintained for 42 days at 37� C. and 5% CO2 and refed every 2-3 days with FSM. Dex was added to the FSM of Dex+ cell cultures at a concentration of 10−8 M for each refeeding.
Tetracycline.HCl powder (Sigma, St. Louis, Mo.) was dissolved in a-MEM to prepare a stock solution of 90 mg/mL. A new tetracycline-containing fully supplemented medium (TFSM) was prepared of a-MEM containing 15% fetal bovine serum, 50 mg/mL ascorbic acid, 10 mM a-glycerophosphate and 9 mg/ml of tetracycline. The TFSM was used for the last refeeding on day 40. At day 42, cultures were washed in a-MEM (10 times, ˜3 min each), and fixed in Karnovsky's fixative (2.0% paraformaldehyde, 2.5% glutaraldehyde and 0.1 M sodium cacodylate buffer, pH 7.2-7.4) overnight. A few cultures were kept for SEM observations and were dehydrated in a series of graded alcohol solutions (70%, 100%), and freeze-dried at 0.01 mm Hg for 2 days. All other cultures were kept in 0.1 M Cacodylate buffer for histological or confocal observations.
Confocal observations were carried out as follows: samples were placed in custom-made chambers in 0.1M cacodylate buffer (obtained from BDH). The chambers were sealed with a glass coverslip. Fluorescent signals were detected by optical sectioning in a Bio-Rad MRC-600 confocal laser microscope, using the BHS filter. Scaffold seeded with Dex(+) cells showed a fluorescent label up to a depth of approx. 1 mm as seen in FIG. 11. Fluorescence could not be observed deeper within the scaffolds because the depth of field of the confocal microscope was not sufficient. Scaffolds were therefore sectioned at a thickness of approximately 2 mm and analyzed by confocal microscopy from both sides. Fluorescence was observed throughout the entire scaffold. The fluorescent label was also seen using sections of cell-seeded scaffolds seeded with Dex(+) cells (see FIG. 12). Cross sections of polymer scaffold seeded with Dex(−) and Dex(+) cells were observed under UV light. A bright fluorescent signal was only seen on the Dex(+) sections throughout the whole scaffold. Specifically, the elaborated bone matrix, as observed by the fluorescent signal, was visualized throughout the depth of a 0.5 cm polymer scaffold which was employed in culture. The limiting factor in this assay was the depth of the polymer scaffold; and thus increasing the depth of the polymer scaffold would increase the depth to which cells penetration, and thus bone matrix formation, could be achieved in this polymer scaffold.
Scaffolds were also immunolabeled for osteocalcin. Osteocalcin expression in both Dex+ and Dex− cultures were assessed by immunohistochemical methods using a goat anti-rat osteocalcin antiserum (Biomedical Technologies Inc., Stoughton Mass.) at a final dilution of 1:6000. The assay was terminated by second anti-body labeling with donkey anti-goat IgG conjugated to horseradish peroxidase antiserum, at a concentration of 1:250. A 3,3-diaminobenzidine (DAB) substrate kit for peroxidase (Vector laboratories, Burlingame Calif.) was used supplemented with nickel chloride to develop the staining. FIG. 13 shows an osteocalcin-labeled scaffold seeded with Dex+ cells and maintained in culture for 6 weeks. Histological sections of the scaffolds were obtained as following: samples were embedded in Tissue Tek and sectioned vertically at a 6 mm thickness. Cell growth within the scaffolds was also observed from the histological sections. At low magnification, the entire scaffold section could be visualized by LM. In both Dex+ and Dex − cultures, cell coverage was found throughout the entire scaffold structure. Haematoxylin and eosin staining was visible along all the macropores, on the outer surfaces as well as in the middle of the scaffolds. FIGS. 14 and 15 show low magnification of Dex+ and Dex− cultured foams The amount of matrix elaborated on Dex− cultures was far more abundant than on Dex+ cultures, as seen at higher magnification. In Dex+ cultures, only a few cell layers were found lining the pore walls and producing matrix in close apposition to the pore walls, whereas in Dex− cultures, the entire macropore volumes were filled with matrix.
PLGA 75:25 matrices were prepared as described in Example 1. These scaffolds were disinfected in 70% ethanol for 30 min prior to being seeded with human bone marrow stromal cells, from young donors, using protocols and dexamethasone (dex) containing media described in detail by Parker et al. (J. of Bone Min. Res., 12(1), S300:F298, 1997).
Three different scaffold morphologies were created: 1) scaffolds obtained by particulate leaching only, referred to as membranous scaffolds forming part of the prior art and shown in FIGS. 16A, 16B and 16C discussed briefly below, 2) scaffolds obtained by particulate leaching phase inversion using low processing temperatures, as described in Example 1, referred to Intermediate scaffolds and 3) scaffolds obtained by particulate leaching phase inversion using higher processing temperatures, as described in Example 4, referred to as bone-like scaffolds. From each of these three basic processing routes, the three scaffold structures were created with different macropore sizes, so that a total of nine different scaffold structures were obtained. These nine structures are illustrated in FIGS. 16A to 16I.
Membranous scaffolds were created using a particulate leaching technique only (as described by Mikos et al, in Biomaterials 14, 323-330, 1993), see the prior art shown in FIGS. 16A, 16B and 16C. Briefly, a PLGA 75/25 (Birmingham Polymers) solution in chloroform was cast over sieved particles, either 1) NaCl (size<0.35 mm), 2) sucrose crystals (size ranging from 0.54 to 0.8 mm) or 3) glucose crystals (size ranging from 0.8 to 2 mm). The polymer structures were left at room temperature to allow chloroform evaporation, after which the particles were dissolved in ddH2O.
Intermediate and bone-like scaffolds were produced as described in Examples 1 and 4 by extracting the same different particles as described above from the precipitated polymer. Intermediate scaffolds were created at a polymer solution temperature of −20� C. and a non-solvent at room temperature whereas Bone-like scaffolds were produced with a polymer solution temperature at 11� C. and a non-solvent at room temperature. The obtained scaffolds were disinfected in 70% ethanol for 30 min prior to being seeded with cells.
Cell colonization of the scaffolds was confirmed by confocal microscopy, and cell differentiation throughout the scaffold structure was confirmed using the osteocalcin labeling assay described in Example 6. The following results is Table 2 were observed:
Cell colonization of the scaffolds, as reported in Table 2, required a minimum interconnection size of 0.35 mm and macropore size of 0.7 mm.
In this Example, membranous scaffolds with macropore size of 1.1 mm were not colonized by cells whereas Bone-like scaffolds with macropore sizes of 0.7 mm were fully colonized by cells. In conclusion, this Example demonstrates that scaffolds obtained by particulate leaching phase inversion technique allowed cell colonization throughout the entire scaffold morphology, whereas previously published scaffold were only colonized by cells within their superficial pore layer.
The foregoing description of the preferred embodiments of the invention has been presented to illustrate the principles of the invention and not to limit the invention to the particular embodiment illustrated. It is intended that the scope of the invention be defined by all of the embodiments encompassed within the following claims and their equivalents.
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AUSTIN, Texas A grand jury indicted Texas Gov. Rick Perry on Friday for allegedly abusing the powers of his office by carrying out a threat to veto funding for state prosecutors investigating public corruption making the possible 2016 presidential hopeful his states first indicted governor in nearly a century.
A special prosecutor spent months calling witnesses and presenting evidence that Perry broke the law when he promised publicly to nix $7.5 million over two years for the public integrity unit, which is run by Travis County Democratic District Attorney Rosemary Lehmbergs office. Its the same office that indicted U.S. Rep. Tom Delay as part of a finance probe.
Several top aides to the Republican governor appeared before grand jurors in Austin, including his deputy chief of staff, legislative director and general counsel. Perry himself did not testify, though.
Grand jurors indicted Perry on abuse of official capacity, a first-degree felony with potential punishments of five to 99 years in prison, and coercion of a public servant, a third-degree felony that carries a punishment of two to 10 years.
A spokesman for the governor didnt immediately return messages seeking comment.
No one disputes that Perry is allowed to veto measures approved by the Legislature, including part or all of the state budget. But the left-leaning Texans for Public Justice government watchdog group filed an ethics complaint accusing the governor of coercion because he threatened to use his veto before actually doing so in an attempt to pressure Lehmberg to quit.
I took into account the fact that were talking about a governor of a state and a governor of the state of Texas, which we all love, said Michael McCrum, the San Antonio-based special prosecutor. Obviously that carries a lot of importance. But when it gets down to it, the law is the law.
In office since 2000 and already the longest-serving governor in Texas history, Perry isnt seeking re-election in November. But the criminal investigation could mar his political prospects as he mulls another run at the White House, after his 2012 presidential bid flamed out.
McCrum said hell meet with Perrys attorney Monday to discuss when he will come to the courthouse to be arraigned. McCrum said he doesnt know when Perry will be booked.
Asked why McCrum never spoke to Perry personally, McCrum said, Thats prosecutorial discretion that I had.
Lehmberg oversees the offices public integrity unit, which investigates statewide allegations of corruption and political wrongdoing. Perry said he wouldnt allow Texas to fund the unit while Lehmberg remained in charge.
Perry said Lehmberg, who is based in Austin, should resign after she was arrested and pleaded guilty to drunken driving in April 2013. A video recording made at the jail showed Lehmberg shouting at staffers to call the sheriff, kicking the door of her cell and sticking her tongue out.
Lehmberg faced pressure from other high-profile Republicans in addition to Perry to give up her post. Her blood-alcohol level was nearly three times the legal limit for driving.
Lehmberg served about half of her 45-day jail sentence but stayed in office, despite Perrys assertions that her behavior was inappropriate.
The jail video led to an investigation of Lehmberg by a separate grand jury, which decided she should not be removed for official misconduct.
The indictment is the first of its kind since 1917, when James Pa Ferguson was indicted on charges stemming from his veto of state funding to the University of Texas in an effort to unseat faculty and staff members he objected to. Ferguson was eventually impeached, then resigned before being convicted, allowing his wife, Miriam Ma Ferguson, to take over the governorship.
Associated Press writer Jim Vertuno contributed to this report.
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|Summer Science: Water Experiments
Event Type: Children's Program
Date: 6/19/2013
Start Time: 4:00 PM
End Time: 5:00 PM
Keep your science skills fresh this summer by joining us for simple experiments involving nature's greatest natural resource: water! Best for kids ages 7-12. Register in person, online at oppl.org/events or by calling 708.386.9032 beginning June 5.Library: Oak Park Public Library Dole Branch
Location: Meeting Room - Dole Branch
Registration Ends: 6/19/2013 at 3:30 PM
Contact: Dole Branch Library
Contact Number: 708.386.9032
Status: Closed
We're sorry. The registration deadline was 6/19/2013 at 3:30 PM.


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Come and envy the incredible talents of illustrator Eleanor Davis
Eleanor Davis has more illustrative skill than most people would know what to do with. The American creative spends her days, nights, and probably all the time in between producing a staggering volume of comics, editorial illustration and children's work, not to mention a healthy dose of personal work (what the hell else are you supposed to do with all that talent?) in all sorts of media.
We've featured some of her watercolours on the site before, but that simply doesn't do justice to Eleanor's enviable skill. Her talents extend from paint and ink to modern techniques using computers and stylus, and her subject matter isn't to be sniffed at either. At once morbidly funny and cripplingly sad, Eleanor has a gift for making you want to laugh and cry in quick succession by chopping between whimsical sketches and characters on the verge of nervous breakdown. But then life is unpredictable that way, and Eleanor is nothing if not a gifted scribe of the everyday.


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I was elected as the Member of Parliament for Rangitikei in the 2011 election. My entry into Parliament follows a nine year career in local politics as Mayor of the Manawatu District, as well as careers in agriculture and business.
My experience includes directorships in FMG and as Chair of Farmers Mutual Finance, and Special Olympics New Zealand.
I have three adult children, Diana, Cameron and Angus, and six grandchildren. I am married to Sue, and together we run the family farming business on the edge of the Rangitikei River.
I have particular interests in sustainable agriculture, including the protection of our soil and water for future generations, and sustainable economic development for rural communities.
I am a member of the Law and Order and Chair of Primary Production select committees.
Please do not hesitate to make contact with my Wellington office:
Parliament Buildings, Private Bag 18041, Wellington 6160, 8am to 5pm Monday to Friday, 04 817 8394 firstname.lastname@example.com
As the MP for Rangitikei, I am looking forward to representing and serving the people of the Rangitikei's many communities. It is my intention to spend as much time in the electorate as possible and to that end have kept the three electorate offices in Marton, Feilding and Taumarunui. So if you are local and have any issues that arise that you need help with please make contact:
47 Manchester Street, Feilding 4702, open Monday, Wednesday, Thursday & Friday 10.00am to 4.00pm - 06 323 7253
13 High Street Marton 4710, open on Tuesday's 9.30am-4.00pm – 06 327 6904
1/101 Hakiaha Street, Taumarunui 3920, open Monday 9.30am to 2.30pm and Thursday 9.30am to 12.30pm – 07 896 8008
Ian and Sue
The Team at Feilding Saleyards – Back from left: Suze, Norma & Sue. Front: Ainslie, Ian, Jacky & Kay.


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Reasons That Lead To Nail Peeling
No one likes to have chipped and peeling fingernails and the only way to get rid of all these problems is to understand the reasons behind this. Understanding the basic reasons behind flaky nails can help one to get those beautiful looking pink nails.
Some Reasons behind Nail Peeling
Some most common reasons behind nail peeling which can help one get those nice looking finger nails back are:
Apart from the above mentioned reasons, some other reasons that lead to nail peeling are improper nail treatments and improperly done acrylic nails. If one wants to get rid of the problem of nail peeling, one has to make sure that the care of finger nails is not at all compromised.


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Nectarine, Tomato, and Basil Salad With Torn Mozzarella From 'A Change of Appetite'
In this recipe from her new cookbook, A Change of Appetite, Diana Henry elevates the classic caprese combo of mozzarella, tomato, and basil with the addition of nectarine. The ripe fruit adds a juicy sweetness that I never realized was missing. Dressed with just olive oil and white balsamic, every element shows at its best. So simple, so smart. More


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Adult Super Deluxe Belly Dancer Costume
Harem or Belly Dancer Costumes
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About this product
Description:Adult Harem or Belly Dancer Costume - This Super Deluxe Edition Harem Costume features the headpiece with veil, nylon Lycra top, underpants and poly-Lurex pantaloons trimmed with metallic fringe.
All made of 100% nylon-lycra with 100% poly-Lurex.
PLEASE NOTE: The color of the costume is silver/white, not pink.
Finger cymbals sold separately.
- headpiece
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- underpants
- harem pants
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Pink Eyelashes
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These Pink Eyelashes would look great with a flapper costume.
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Costumes for Men - This Arab Sheik costume headpiece includes a headpiece with elastic band. This Sheik costume headpiece fits most sizes. This Sheik costume headpiece will compliment any Arabian costume. Sunglasses and facial hair not included.
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This Belly Dancer Jewel Kit includes a bright faceted jewel and a phial of Spirit Gum adhesive. Just the thing for any Belly Dancer, Harem or Desert Princess costume!
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Find Out What's Possible
Would you like to scout for undiscovered microorganisms and plants to remediate polluted sites or provide new medicines to treat human diseases? Are you interested in developing strategies to control insect pests in the worlds breadbasket or producing renewable alternatives to petroleum? Would you like to be part of a discovery team to find novel ways to diagnose and treat cancer or diabetes? Consider the field of biotechnology.
Biotechnology has emerged as a major contributor to the advancement of agriculture, medicine, and environmental sciences. The biotechnology industry is becoming one of the most promising sectors in the nations economy. The industrys primary goal is to invent new biologically active substances for the treatment of disease, to produce more nutritious and more plentiful sources of foods, and to innovate ways to clean up the environment and explore alternative fuel sources. Recent developments in biotechnology, particularly molecular biology, are yielding major improvements in agricultural productivity, breakthroughs in human health care, and new solutions to environmental problems.
Rutgers undergraduate biotechnology curriculum is innovative, exciting, and challenging. Many of our faculty members have received awards for excellence in teaching, academic advising, and research. All students engage in research prior to graduation, most often at Rutgers or through collaborative programs with the University of Medicine and Dentistry of New Jersey but also through placements in neighboring pharmaceutical and biotechnology companies. Opportunities for biotech internships are also available through the Student to Professional Internship Network (SPIN) program at the School of Environment al and Biological Sciences.
Biotechnology is offered as a major ONLY in the School of Environmental and Biological Sciences on Rutgers New Brunswick Campus.


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Here you can see sample works of artViper™ designstudio's customers and case studies. Additionally there's some information about the project itself.
Please click on the images to get a bigger preview of the whole website.
This website received recognition from the German Agricultural Society (DLG) for its famous Zoigl beer. Utilizing flash, AJAX and clean XHTML along with basic SEO optimization, the outcome is a satisfying website design with focus on presenting their goods and service.
Die Modellbahn claims to be one of the most thorough model train selling site, and is basically a page listing only models, spare parts and other equipment. Build with mySQL, PHP and the mootools framework, the page nicely hides the long material lists and only fades them into view as soon as its needed. Basic SEO optimization and a admin backend rounds up this website.
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The IIICS backend is completely written from scratch, utilizing functions like fulltext search, xml output for RSS and flash tag clouds, pagination, clean URL rewrites, Yahoo API integration, AJAX and flash sliders, a ultra fast engine, no use of any PHP framework which could slow down performance and a very unique design in the front end. As usual validated XHTML and CSS are used to build this website.
Build on a normal eCommerce XT shop, this webshop has turned into a powerful machine. A complete revamp of the search engine, fulltext searches, nifty PHP and AJAX addons for browsing books and finiding information, interlinking authors, keywords etc. make this shop an outstanding one. In addition the shop was de-tabled and build with CSS only.
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Comment Re:How to fix ALL the app stores... (Score 1) 249 249
A pretty good list, IMO, although perhaps a bit drastic or unrealistic as rasmusbr points out. Instead of removing all crap applications (your #1), move them to the "Junk" category then have a default hide for the category. That allows the teenagers to have their stupid apps -- and they can feel even cooler by having to go in and enable the category -- while not bothering the rest of the user base.
I suppose one way of addressing #2 (duplicating stock apps) would be to have another (hidden by default) category. That way someone who wanted a replacement music player could get one without forcing everyone to slog through garbage apps.
And while #4 (eliminating in app purchases) is important as it cuts to the core of some problems, it simply isn't realistic. Originally, Apple did not have them. They were added to appease large developers. Not going to happen, but it would help if it did.
Even though rasmusbr is correct about what will happen with #6 (time limited 100% refund policy), without in-app purchases the model would not be particularly successful.


text: Home » Photos » CHRIS BROWN MAKES A SPLASH AT DEBUT ART SHOW CHRIS BROWN MAKES A SPLASH AT DEBUT ART SHOW Comments: 0 | Leave A Comment May 3, 2012 By G-Man Chris Brown went hard in the paint at his debut art show. Following the launch of his Dum English collectibles toy line, the "Turn Up the Music" singer displayed his street art for the first time at a VIP event at the Corey HelfordGallery in Los Angeles on Wednesday. VIEW PICS


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My experience is that the better elk hunting is almost always away from the road.... As a hunter this does not scare me. As a conservative (less government) this does not scare me. Getting off the beaten path has always produced, as a berry picker this does not scare me.
What scares me is that the groups backing this (like Oregon Wild) use "saving tax dollars" as a promotional tool - yet they could give a hoot when they support letting a chronic depredating wolf pack chew threw $25,000 in taxpayer's money to pay for confirmed depredation & countless dollars in depredation management & tons of litigation dollars. All to save chronic depredating wolves in poor wolf habitat. Saving tax dollars is not what motivates a group like Oregon Wild!
My experience is that the better elk hunting is almost always away from the road.... As a hunter this does not scare me. As a conservative (less government) this does not scare me. Getting off the beaten path has always produced, as a berry picker this does not scare me.
What scares me is that the groups backing this (like Oregon Wild) use "saving tax dollars" as a promotional tool - yet they could give a hoot when they support letting a chronic depredating wolf pack chew threw $25,000 in taxpayer's money to pay for confirmed depredation & countless dollars in depredation management & tons of litigation dollars. All to save chronic depredating wolves in poor wolf habitat. Saving tax dollars is not what motivates a group like Oregon Wild!
Texaspapa, 25 years? More like 5 years. People are already getting a taste of the litigation that comes with wolves! Most will be quicker to come to the realization that wolves do not make the environmental any better around these ranches & that Oregon does not have much true "wolf habitat"
Good Comment MP, The we need them for a "healthy" environment makes most locals skin crawl. Shawano Co, in Wisconsin (99% private land) is seeing the same garbage.
What, 700 in one month is not "frivolous" for you......they know the Government can not keep up. The EAJA along with the ESA robbing the American public blind! You are either one of the few that benefit from this garbage or blind/ignorant!
facebrick ...... people can see right threw your propaganda!
wow, the wolf now is a replacement for livestock and crops.....you people are pathetic (big time).
facebrick, Wow, We both know the source of the "studies" that you speak of & the book that you tout! To say that there is no ecological benefit of these highly regulated and studied cattle lease is absurd! Plain and simple. The credibility of your type of organizations has taken huge hits of late & it's because of people like you that try to twist things to benefit things like the wolf & to destroy ranching! Yes - you cannot buy a lease tear down the fences and watering setups. These things do need to be maintained......but, my cousins have had a lease that they choose not to run cattle yet maintained the acreage. Some laws were changed because of people like you purchasing leases and destroying the capability of running cattle & making it cost prohibitive to ever run cattle again.....you and your ilk are your own worst enemy on that one! Not all leases are run this way and you know it!
Ranchers, Its not enough that you have to pay for non-confirmed depretion along with the harrassment and herding cost associated with wolves kpu7m says that you do NOT need to be repaid for livestock his pet wolves have killed. kpu7m says you can not kill that wolf that killed your cow, the government can not manage the wolf that killed your cow and undoubtedly ran and harrassed the whole herd. kpu7m don't care that it is this time of year when the cows get a little heavy with calf where they loose a few fetuses when run by preditors. kpu7m does not thank you for being a parttime land lord for her wolves and (please don't laugh)kpu7m says this animal is endangered & threatened!
Now go about your business and please make sure the safety is "on" on that 270 you have sitting by the back door, make sure the clip is sitting in the right spot in the truck!
PS, If you have kids you may want to put that 270 in a safer place!
The ranchers of Oregon are now getting a taste of the litigation that has polarized the wolf issue! It is no wonder why some have turned to SSS. True wolf lovers understand that removing depredating wolves is essential to the public's attitude & does not leave them sleepless at night know that wolves could be killing a few more livestock as they sleep. Apparently wolf advocates around the world care less about the wolves & more about curtailing hunting & ranching or lining their pockets with wolf lover donations. I'm sort of glad the anti-hunters and anti-ranchers filed the law suit in Oregon for it accomplishes two things.
* These wolves will continue to kill cattle and more articles like this will be published.
* Oregon ranchers and sportsman that were on the fence will get a taste of the senseless litigation issue those plaques the environment arena today!
GeoHydro2011, I think you have spent to much time on the anit-hunting anti-ranching hate sites that are abound! Cattle leases are managed to the livestock unit standards, the duration, the number of units & timing of the grazing. This keeps that lease in great shape for wildlife..... Sort of like having a herd of buffalo come threw to keep things in check! My cousins lease in Western MT brings in a nice chunk of change for uncle sam.... you and I have the ability to bid on that lease and others...... by all means please do so .... you can graze (or not graze) till your hearts content! Your comments show your complete ignorance to the subject!
facebrick, Environmental lawyers are robbing you blind ....... If the money spent on environmental lawyers via the EAJA dollars alone were spent on purchasing important habitat we would be tremendously better off environmentally! That does not include the cost of the government to defend these frivolous suits or the cost of the judicial system (court costs) to put the law suit on! You need to get educated!
wow, either you are ignorant to the issue, have read to many disney stories or are just our right out lying!
merwinwfinzork, have you ever seen a wolf run cattle and or depredate on livestock? Did you know that most wolves will test their prey (especially something that they have never killed before) many times before actually killing it? Do you know what effect the depredation of a calf, cow or sheep has on the rest of the herd? Have you ever helped a rancher fix fences that were overrun by cattle? Have you ever doctored up or treated an animal that ran threw a fence? How many cattle have aborted there fetus from being run by wolves? Have you ever seen cattle walking that have pulled musles from being run by wolves? Have you ever had to bury whats left of your dog after wolves were done with it? Do you know what the ratio is of confirmed depredation to non-confirmed depredation?
Keep in mind that your "miniscule" is someones lively hood. Wisconsin had almost a quarter of a million dollars in confirmed depredation by wolves in 2010 alone. What does that translate to the items listed above? Could that be why 19 Northern Wisconsin County boards have passed resolutions against the wolf? Real wolf lovers understand the limitation of this animal.... the ones that continue to push for this killer are either anti-hunting anti-ranching or just plain ignorant!
With non-confirmed depredation wolves kill at least one Domestic animal per year and countless wild animals. .... Yes, I hope a portion of the wolf population are turned into rugs and no appalogies will be forthcoming. For every one that the trapper, hunter or wildlife agent (taxpayer) kill we will save at least one domestic animal. Your the sicko to promote this killer! The true wolf lovers felt the marginal habitat (Yellowstone) was full of wolves way back in 2001! The only ones pushing for this animal now days is Anti-hunters and Anti-ranchers!
No problem with me at 24.... It's when there is a law suit put forth to stop Wildlife Serves from killing depredating wolves that I get a little hot under the collar. If the wolf is killing livestock that means he is not in very good habitat..... Oregon may not have any "Good" habitat only marginal or poor!
You're a real treat..... its always animals before people with you people. But, the thing is you don't care about animals! Wolves kill for fun.....122 animals had fun this night in Dillion Montana!
You need to be held accountable for this stuff!
The anti-hunters wolf love nothing more that to eliminate the renewable (tasty) resource that the sportsman enjoy..... They want it to go to a non-endangered non-threatened killer that eats their prey alive. They hate wildlife & run down to Lamar Valley to see wolves kill elk & rip out their fetus.....sort of like Michael Vick running down to a dog fight!
dtroutma, who are you trying to kid..... The 22,000 wolves that live remotely close to humans in north america killed two of us in the last 5 years! If the 120 million cattle kill humans at a rate of 2 per 22,000 we would see 10600 dead human every 5 years or 360000 dead people per Century. I suspect that the 2 per 22000 will go up as they put the vermin closer to humans! Quit putting forth deceptive stats....it only makes you look foolish!
Hey Susan, Do you really believe that Oregon is habitat for breeding pairs? What a joke! At three breeding pair it already takes an army of biologist to clean up after these vermin! Oregon ranchers are now getting a taste of what this wolf garbage is all about.... Now they deal with their 1st law suit. The deal in the poorly written wolf plan was that habituated depredating wolves are to be removed! Now at three breeding pair they cannot live with management already. I urge the ranchers to have the plan rewritten to three breeding pair if there are legal challenges!


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Click Here to Watch the Whole Video in HD at College Boy Physicals
Hot young college studs go for their entrance physicals! Deep anal penetration, anal probing, vacuum pumping, elector-cock therapy and MORE!
The college has now decided to introduce sports massages to the athletic department and they decided to have the medical staff do the massages on many of the athletes to prepare them for the games. Today I had scheduled Keith to be our first athlete to have his first massage. Keith is on the baseball team and we first met when he came into our clinic for his sports physical and other issues he has had so I'm very familiar with Keith. He is the boy next door type with a nice toned body and a nice cock. I had Keith undress and prepared him for his massage. He laid down on the massage table on his stomach as I applied some soothing cream and started to work and rub his tense shoulders moving my hands down his back exposing his nice round ass.
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