Patent Application
20210154369
This invention relates to a cutting device for cutting a substrate, processes for the production of substrate fibers, and the substrate fibers produced. Suitable substrates include but are not limited to bone tissue, including allogenic and xenogenic cortical bone. The fibers are cut from a substrate using the device, such that an individual fiber produced has a length that is typically greater than 10 to 200 times its width and thickness. The invention further relates to compositions including bone fibers and other agents, including, for example, bioactive agents, including stem cells, which bind to the bone fibers and are induced to form new bone.
This invention also relates to the formation of a tissue-engineered material using in vitro cell culture, in a bioreactor system(s), in the presence of biomaterials suitable for the induction of new bone formation. This invention further relates to the use of specific forms of reactors to cause the formation of a shaped material suitable to specific clinical applications. For example, the formation of a mandible-shaped reactor for in vitro growth of a shaped bone graft substitute for the use in repair of fractured jaws is within the scope of the present invention. This invention further relates to a bone forming tissue that will remodel into load-bearing bone when implanted in the surgical repair of bone defects.
Ground demineralized cortical and cancellous bone have been widely used in the induction of new bone formation for the treatment of a variety of clinical pathologies. Typically, the bone materials are obtained from human or animal sources, ground and demineralized. Such bone has been demonstrated over the past two decades to induce new bone formation when implanted in animal models, to stimulate elevated levels of the enzyme alkaline phosphatase, and to contain extractable amounts of bioactive molecules, such as bone morphogenetic proteins (BMPs).
The ground demineralized bone matrix (DBM) has also been called demineralized bone (DMB), and demineralized freeze-dried bone allograft (DFDBA). DFDBA materials are provided for clinical use in a freeze-dried state. DBM (or DMB) can be provided for clinical use in either a freeze-dried state or as a hydrated state—usually in some form of an aqueous carrier, for example, glycerol in GRAFTON™ (GRAFTON™ is a registered trademark of Osteotech, Inc., Shrewsbury, N.J.), pluronic polymer in DYNAGRAFT™ (DYNAGRAFT™ is a registered trademark of GenSci Regeneration Technologies, Inc., Irvine, Calif.), and collagen in OPTIFORM™ (OPTIFORM™ is a registered trademark of Regeneration Technologies, Inc., Alachua, Fla.). These various commercially available demineralized bone products primarily contain demineralized cortical ground bone distributed for clinical applications. The use of carriers with demineralized bone particles are more acceptable to clinicians because such particles acquire a static charge in the dry state making them difficult to dispense into containers and following rehydration, the clinician typically has difficulties in getting the bone particles to remain at the implant site and in a compacted state wherein they are presumed to be most osteoinductive. DBM is considered to be osteoinductive if it induces the formation of new bone, for example, at the site of clinical application. By adding carriers to the DBM, the biomaterials become easier to aliquot into containers and tend to remain tightly aggregated at the implant site making them easier to handle.
The osteoinductive nature of DBM arises from the interaction between bone-forming cells and the DBM. Such interaction takes place at both a molecular and physical level. At the molecular level, attachment of the bone-forming cells to the DBM involves the presence of “receptors” on the surface of the plasma membrane of mammalian cells that bind to “ligands” present on the surface of the biomaterial. An example of this type of attachment or binding is illustrated in the role of RGD-containing amino acid sequences in the attachment of mammalian cells to a wide variety of molecules present within matrices of tissues. The RGD amino acid sequence refers to the amino acids arginine (R), glycine (G), and aspartic acid (D). Holland, et al. (Biomaterials. 1996. 17(22):2147-56) described the research on a synthetic peptide, gly-arg-gly-asp-ser-pro-lys (GRGDSPK) (which includes the cell-adhesive region of fibronectin, and arg-gly-asp (RGD) peptide sequence covalently bound to a dialdehyde starch (DAS) coating on a polymer surface. The authors concluded that the GRGDSPK/DAS-coated surface could be substituted for an adhesive-protein coated surface in the culture of anchorage-dependent cells.
On the other hand, binding at the physical level in the context of surface patterning has been described, for example, in Goodman, et al. (Biomaterials. 1996. 17(21):2087-95). Goodman et al. described clinical and experimental investigations on manufactured surface topographies that have significant effects on cell adhesion and tissue integration stating that micro- and nano-scale mechanical stresses generated by cell-matrix adhesion have significant effects on cellular phenotypic behavior. Details of surface patterning effects on cell attachment and proliferation were described by Schmidt and Recum (Biomaterials. 1992. 13(15):1059-69) measuring macrophage responses to microtextured silicone. Schmidt and Recum measured the effects of seven different silicone surface textures on macrophage spreading and metabolic activity in vitro. Variables of the textured arrays important to cell spreading and metabolic activity included size, spacing between, depth, density, and orientation of the individual surface events and the roughness of the surfaces. It was found that pattern dimensions of about 5 microns textures were associated with small cells, whereas a smooth (untextured) surface was associated with large cells. The authors put forth a hypothesis that included a possible mechanism of how a micrometer-sized surface texture could modify cell function.
There are thus several issues pertinent to the ability of implanted bone compositions to induce the formation of bone. These issues include providing an environment suitable for the infiltration of cells, a confined environment that restricts the diffusion of synthesized matrix-forming molecules (for example, collagens, proteoglycans, and hyaluronins), promotes cell attachment to DMBs, and includes the presence of bioactive molecules (for example BMPs). Additionally, the method for making bone fibers for these bone implanted compositions in an efficient and consistent manner is addressed by the present invention.
Demineralized bone matrix (DBM) is widely used in the repair of pathologies associated with skeletal defects and periodontal diseases. This material is typically produced from cortical bone of long-bones (chiefly those bones found in the legs and arms of human cadaveric donors) by cutting the shafts of these long-bones into small chunks (1-4 mm) using methods well-known in the field. The resulting pieces and chunks of bone are subsequently cleaned and grinded into a finer bone powder. The resulting bone powder is typically in the about 125 to 1000 micron particle size ranges. The bone powder may be demineralized by exposure to dilute (normally 0.4 to 0.6 N) hydrochloric acid, organic acids, calcium chelating agents, etc. as is known in the art. For example, U.S. Pat. Nos. 5,275,954; 5,531,791; 5,556,379; 5,797,871; 5,820,581; 6,189,537; and 6,305,379 describe methods of demineralizing bone material and are hereby incorporated by reference in their entirety. This ground demineralized bone matrix material has been called demineralized freeze-dried bone allograft (DFDBA), demineralized bone allograft (DBA), demineralized bone matrix (DBM), and demineralized bone (DMB) and is currently produced by a number of for profit and not-for-profit companies for use in orthopaedic, spinal fusion, and periodontal applications.
The use of DBM in the formation of new bone has been assessed using in vivo (usually a mouse or rat implant system), in vitro (cell culture or extraction and quantitation of bone forming molecules reportedly present in bone), and in situ (where the formation of new bone in patients has been assessed during clinical applications) applications. Methods of assessing this new bone formation and the effects of the demineralization process on new bone formation by DBM are described in Zhang et al., “A quantitative assessment of osteoinductivity of human demineralized bone matrix,” J. Periodontol. 68:1076-1084 (1997) and Zhang et al., “Effects of the demineralization process on the osteoinductivity of demineralized bone matrix,” J. Periodontol. 68:1085-1092 (1997). An in vitro assessment of the ability of DBM to induce cells towards an osteoblastic phenotype has also been described (Wolfinbarger and Zheng, “An in vitro bioassay to assess biological activity in demineralized bone,” In Vitro Cell Bio. Anim. 29A:914-916 (1993)).
DBM is assumed to form new bone when implanted in animal models via an endochondral pathway. The implanted DBM is presumed to cause mesenchymal stem cells (typically undifferentiated fibroblasts) to migrate towards the implanted biomaterial(s). This induced chemotaxis results in cells infiltrating the implanted DBM biomaterial(s) where they are induced to undergo phenotypic changes from a fibroblastic cell phenotype to a chondrocyte phenotype and eventually to an osteoblast cell phenotype. These induced phenotypic changes have been reported to be due to the action(s) of one or more small molecular weight proteins falling in the TGF-β family commonly referred to as bone morphogenetic proteins (BMPs). As the change in cell phenotypes occurs, the proliferative potential of the cells declines. For example, the population doubling times increases from approximately 12 hours to approximately 40 hours. As a result, the cells synthesize and secrete collagens and other matrix-forming proteins/glycoproteins laying down a cartilagenous matrix and finally an osteoid-like matrix, which if left implanted in the animal long enough, can be shown to mineralize. This process is analogous to the formation of new bone. If the implanted materials lack the cell-inducing protein factors, only providing an environment suitable for cellular infiltration and cellular proliferation and differentiation, the implanted materials are deemed to be osteoconductive. If the implanted materials possess the cell inducing protein factors and provide an environment suitable for cellular infiltration and cellular proliferation and differentiation, the implanted materials are deemed to be osteoinductive. If the implanted materials already contain cells suitable for new bone formation, such as autogenously transplanted bone, the materials are deemed to be osteogenic.
The present invention is directed to a fiber, preferably bone fiber, having a textured surface, which acts as an effective binding substrate for bone-forming cells and for the induction or promotion of new bone growth by bone-forming cells, which bind to the fiber. The bone fiber of the present invention may be demineralized or mineralized, or may be used in a composition comprising a combination of demineralized and mineralized bone fibers and bone particles. The bone fibers of the invention may be made from any type of bone, such as allogenic or xenogenic bone. Preferably, the bone fiber is made from cortical bone or cancellous bone, more preferably cortical bone. The bone fiber may be of any length. Preferably, the bone fiber has an average length of from about 1 mm to about 100 mm, an average width of from about 0.5 mm to about 2.5 mm, and an average thickness of from about 0.2 mm to about 1.4 mm. The fiber may then be processed according to known processes. In a preferred embodiment, the bone is freeze-dried.
The present invention further is directed to bone material compositions comprising the bone fibers of the present invention. In a preferred embodiment of this aspect of the invention, the bone material composition comprises a bone fiber and bone-forming cells, wherein the bone fiber has a textured surface, which acts as an effective binding substrate for bone-forming cells, and wherein the composition induces or promotes new bone formation from the bone-forming cells bound to the bone fiber. Preferably, the bone-forming cells are selected from stem cells, connective tissue progenitor cells, fibroblast cells, periosteal cells, chondrocytes, osteocytes, pre-osteoblasts, and osteoblasts. Most preferably, the bone-forming cells are stem cells. The bone fibers used in the bone material composition may be any type of bone, including allogenic or xenogenic bone. Preferably, the bone fibers are comprised of cortical or cancellous bone, more preferably comprised of cortical bone. In addition, the composition may further comprise cancellous bone. The composition may further comprise both demineralized and non-demineralized bone fibers or bone particles. The bone material composition may further comprise an agent effective to initiate or promote the induction of bone growth.
Yet another aspect of the invention is a method for inducing or promoting bone growth. This method comprises providing a bone fiber according to the present invention, contacting the bone fiber to bone-forming cells, which adhere to the textured surface of the bone fiber, and wherein the binding induces or promotes new bone growth from the bone-forming cells. The method may further comprise contacting the bone fibers and bone-forming cells with an agent effective to initiate or promote the induction of the new bone growth. Suitable agents to induce or promote bone growth include bone morphogenic proteins, angiogenic factors, growth and differentiation factors, mitogenic factors, and osteogenic/chondrogenic factors. Preferably, the bone fiber used in the method is demineralized. Preferred bone-forming cells include stem cells, connective tissue progenitor cells, fibroblast cells, periosteal cells, chondrocytes, osteocytes, pre-osteoblasts, and osteoblasts. Preferably, the bone-forming cells are stem cells. Moreover, the bone-forming cells may be contacted to the bone fibers via a biological fluid. Preferably biological fluids include plasma, bone marrow, blood, or blood products.
According to another aspect of the present invention a cutter is provided for producing substrate fibers. The cutter preferably includes a leading edge designed to make initial contact with the substrate and a trailing edge. The trailing edge preferably is configured such that it is raised above the leading edge by a prescribed height. The cutter includes a cutting surface upon which a blade section is disposed. The blade section is used to cut the substrate. At least one substrate channel may be provided near the blade section in order to direct the substrate fibers away from the substrate.
According to one exemplary embodiment of the present invention, the blade section can include at least one row of teeth designed specifically for cutting the substrate. Furthermore, each tooth can be configured with at least one predetermined cutting angle to reduce stress and achieve desired substrate properties. For example, one specific implementation of the invention provides a preferred primary cutting angle ranging from 3-6. Preferably the primary cutting angle can be selected to be approximately 4′ A secondary cutting angle can also be provided. The secondary cutting angle can vary between 10-18′ but is preferably selected to be approximately 14′
According to another aspect of the invention, a substrate cutting device is provided. The substrate cutting device includes a base and a tower. The base further includes a cutter that can be moved along a predetermined cutting path. A substrate chute extends through the base in order to position the substrate in a location where it will be in contact with the cutter. The tower includes a lower surface, which contains a recess. The recess can be aligned with the substrate chute. A clamping mechanism is provided to keep the substrate in contact with the cutter during the cutting process. The substrate cutting device can further include a fiber receptacle to receive the substrate fibers after they have been cut.
According to one exemplary embodiment of the present invention, the base is mounted on a slide mechanism, which moves along the predetermined cutting path. An actuation unit, such as a pneumatic actuator, can be used to supply the force necessary for moving the slide mechanism. According to one specific implementation of the present invention, the first actuation generates a force ranging between 600 lbs-900 lbs, and preferably about 750 lbs. A second actuation unit can also be provided to control the clamping mechanism. The second actuation unit can be configured to generate a force ranging from 150 lbs-250 lbs, and preferably about 200 lbs. The present invention can also include a computer controller for controlling operation of the substrate cutting device, including the first and second actuation units. For example, the computer controller can be used to adjust the force applied by the first actuation unit and/or adjust the speed at which the slide mechanism is moving. The computer controller can also be used to adjust the force applied on the substrate during the cutting process.
According to another aspect of the present invention, a method for cutting a substrate comprises the steps: placing the substrate into a substrate cutting device; applying a predetermined force on the substrate; moving a substrate cutter along a grain direction of the substrate; cutting substrate fibers from the substrate; detecting when the substrate has reached a predetermined minimum thickness; and terminating the process if the substrate has reached the predetermined minimum thickness.
The present invention is further directed to the substrate fibers produced using the substrate cutting device of the present invention.
The present invention is also directed to a method of growing new bone or bone-like tissue under in vitro cell culture conditions comprising providing ground demineralized bone and bone-forming cells in a bioreactor under conditions sufficient to form bone or bone-like tissue suitable for transplantation by causing a flow of nutrient solutions into, through, and out of the bioreactor. The bone or bone-like tissue is formed by proliferation and/or differentiation of the bone-forming cells in the presence of the ground demineralized bone and under suitable bioreactor conditions.
The bone-forming cells are preferably selected from the group consisting of stem cells, fibroblast cells, periosteal cells, chondrocytes, osteocytes, pre-osteoblasts, and osteoblasts. The most preferred bone-forming cells are fibroblast cells and pre-osteoblasts. The bone-forming cells can be autogenic, allogenic or xenogenic with respect to the intended recipient.
In accordance with the invention, the ground demineralized bone may be in the form of particles or fibers. The particles are about 50 microns to about 4 mm, preferably about 250 microns to about 710 microns. The fibers have a width of about 0.1 mm to about 0.5 mm, a thickness of about 0.05 mm to about 0.5 mm, and a length of about 1 mm to about 500 mm. If the ground demineralized bone is freeze-dried, it should be rehydrated. The invention provides that rehydration may occur either prior to or after being added in the bioreactor.
The invention further provides that additional components may be added to the bioreactor, such as collagen or hyaluronin, which may create a viscous bone-like matrix. Additionally, growth factors, such as vascular endothelial growth factor or differentiation factors such as bone morphogenetic proteins may be added.
The nutrient solution may comprise at least one of Dulbecco's modified Eagle's medium, fetal bovine serum, L-ascorbic acid-2-phosphate, antibiotics, dexamethasone, beta-glycerolphosphate, glucose, glutamine, amino acid supplements, glutathione-ethyl ester, antioxidants, caspase inhibitors, and inorganic ions suitable for mineralization-related metabolic events.
The nutrients solution may be delivered to the ground demineralized bone and bone-forming cells by resorbable hollow fibers. The hollow fibers are also sufficient to remove metabolic waste products from the bioreactor.
In another aspect of the invention, nondemineralized bone may be added along with the demineralized ground bone. The ratio of demineralized ground bone to nondemineralized bone may be about 1:1 to about 20:1 or as necessary to control availability of biologically active agents and available volume for cell growth.
The present invention is further directed to the bone or bone-like tissue formed according to the process disclosed herein. Moreover, implants comprising the bone or bone-like tissue are within the scope of the invention.
Furthermore, a method for growing an extracellular matrix capable of forming bone when transplanted into a patient is described. The method comprises providing bone-forming cells in a bioreactor under conditions sufficient to promote the growth and differentiation of cells resulting in the formation of an extracellular matrix, wherein said conditions include the flow of nutrient solutions through the bioreactor. Preferably, ground demineralized bone is added to the bioreactor. The present invention further encompasses the extracellular matrix made by this process and a method of implanting bone into a patient in need thereof comprising transplanting the formed extracellular matrix into the patient under conditions sufficient to form bone.
In yet another aspect of the invention, a device for the growth of new bone or bone-like tissue under in vitro cell culture conditions is provided. The device comprises a bioreactor, wherein the bioreactor comprises inlet and outlet ports for the flow of nutrient solutions, sample injection ports, and an inlet port and outlet port for the bioreactor to cyclically receive negative pressure and positive pressure. The bioreactor may optionally include hollow fibers for the delivery of nutrients and removal of wastes. The bioreactor is capable of applying mechanical/electrical stimuli to the formed or forming bone.
The bioreactor may further comprise an outer nondeformable chamber and inner deformable chamber. Either of these chambers may receive or remove the nutrient solutions via the inlet and outlet ports. In addition, the sample injection port may contact either chamber in which the bioreactor will receive biomaterials. Additional ports may be available to allow the bioreactor to receive cyclical negative and positive pressure in the volume between the outer nondeformable chamber and the inner deformable chamber through the inlet and outlet ports. Endplates may be used to secure the bioreactor and provide apertures to receive the ports.
Preferably, the device comprises hollow fibers, which can be in any shape. The hollow fibers can be round and tubular, or in the form of concentric rings. The hollow fibers may be made of a resorbable or non-resorbable membrane comprising polydioxanone, polylactide, polyglactin, polyglycolic acid, polylactic acid, polyglycolic acid/trimethylene carbonate, cellulose, methylcellulose, cellulosic polymers, cellulose ester, regenerated cellulose, pluronic, collagen, elastin, or combinations thereof. The pores of hollow fibers are of a specified diameter that extend from the inside to the outside of the wall of the hollow fiber. For example, the pores may have a diameter of about 2 kiloDaltons to about 50 kiloDaltons, preferably about 5 kiloDaltons to about 25 kiloDaltons, or alternatively, about 2 kiloDaltons to about 15 kiloDaltons.
In accordance with the present invention, the device may include an inner deformable chamber comprising a deformable wall. The deformable comprising a flexible permeable barrier. The flexible permeable barrier may comprise a resorbable or non-resorbable membrane made up of polydioxanone, polylactide, polyglactin, polyglycolic acid, polylactic acid, polyglycolic acid/trimethylene carbonate, cellulose, methylcellulose, cellulosic polymers, cellulose ester, regenerated cellulose, pluronic, collagen, elastin, or a combination thereof. In addition, the inner deformable chamber may further comprise a fine mesh. Preferably, the fine mesh comprises sterilizable materials and is made up of stainless steel, titanium, plastic polymer, nylon polymer, braided collagen, silk polymer, or a combination thereof. The fine mesh may have any suitable pore size range such as, for example, between about 0.1 to about 10 mm, about 1 mm and about 5 mm. The fine mesh may be on the inner surface of the flexible permeable barrier, outer surface or both.
The invention provides a bone fiber having surface properties that offer a suitable environment for the attachment of infiltrating cells, such that they can attach (normal mammalian cells are “attachment dependent” meaning they do not typically proliferate or maintain synthetic functions unless attached to a solid matrix) and synthesize bone matrix-forming molecules. Appropriate attachment surfaces can also contribute to the stimulation of cells to proliferate, differentiate, and to synthesize appropriate bone matrix-forming molecules.
The present invention is also directed to a method of making the bone fibers of the present invention involving the use of an apparatus suitable for cutting bone to produce fibers having the enhanced cell-binding surface to increase the bone-forming induction properties of demineralized bone material and to facilitate formation of a matrix suitable for perfusion, percolation, and infusion of viscous cell materials into the matrix.
Finally, the present invention is directed to an apparatus suitable for cutting a substrate. The apparatus includes a unique arrangement that allows the substrate to be cut into fibers having consistent properties for a particular application. A special cutter is used to cut the fibers along a grain direction of the substrate in order to produce substrate fibers. The apparatus includes various safety features, such as sensors to detect whether all access doors are shut prior to commencing operation. If a sensor is triggered during operation, the apparatus is immediately powered down in order to prevent an operator from being harmed. The present apparatus can also include a computer controller to control various operations.
The terms used herein are given their plain, ordinary meaning as understood by those having ordinary skill in the art, unless otherwise defined herein.
The “bioactive agents” of the present invention refer to the agents capable of initiating and inducing the differentiation and/or proliferation of bone cells and/or the induction of bone cell growth. The bioactive agents may include, for example, bone morphogenic proteins, stem cells, blood, blood elements, bone marrow and bone marrow extracts, platelets and platelet extracts, homogenates of skin and skin homogenate extracts, growth factors, selenium and transferrin, calcium salts, and CYMETRA™ (CYMETRA™ is a registered trademark of LifeCell Inc., New Jersey).
“Bone formation,” as used in the present invention, refers to the act of the bone-forming cells taking the form of bone cells, bone, cartilage, osteoids, and bone matrices.
The term, “bone material composition,” means a composition comprising the bone fibers or bone fibers plus anorganic or inorganic components mixed with the bone fibers of the present invention and bone-forming cells. Typically, this combination has physical characteristics that allow infusion of visous materials such as bone marrow and osteoinductive effect so as to allow the bone-forming cells to form into new bone cells under appropriate conditions.
The “cutting cycle” is a single forward plus backward stroke of the cutter across the substrate as disclosed herein.
A “cutting event” is the complete cutting run of a load chute of a substrate.
“Demineralization” refers to the act of removing minerals from tissues containing minerals. The demineralization may be conducted by processes known in the art.
The “fiber bone” or “bone fiber” of the present invention is the fiber made from bone by shaving or cutting along the length of the bone to provide the bone fiber its textured surface to which bone-forming cells may bind and the induction of bone growth may be initiated under appropriate conditions.
“Osteoinductive” shall mean the ability to induce or promote the formation of new bone either in vivo or in vitro. For example, the bone fibers of the present invention have been found to induce or promote the formation of new bone by bone-forming cells attached to its surface. The induction of new bone may be fostered by the presence of bioactive agents that assist in the initiation of this induction process.
The “substrate” of the present invention may be any material, i.e., non-biological or biological materials, which may be cut using the cutting device of the present invention. Where the substrate is bone, for example, the bone fibers act as a material upon which an organism such as bone-forming cells may grow or attach.
The term “bioreactor” is intended to mean a contained or enclosed system or vessel for the culture of cells, such as mammalian or vertebrate cells, by which sterility or the freedom from microbial contamination can be achieved. Nutrient solutions can be aseptically delivered into the bioreactor and waste solutions can be aseptically removed from the bioreactor.
The term “newly formed bone” is intended to mean a matrix secreted by bone-forming cells. This newly formed bone is best illustrated by histological evidence of newly formed bone when demineralized bone is implanted intermuscularly in a nude mouse (or rat) bioassay system. For example,
The term “bone tissue” is intended to include the organic phase or organic and inorganic phases of that tissue comprising a bone. Within the context of this invention, bone tissue can include newly formed bone, implant bone, and associated cells, bone marrow, bone marrow-like tissue, and cartilage (and cartilage-like tissues).
The term “bone-like tissue” is intended to include a matrix similar to cartilage and/or osteoid similar to that tissue found in articular cartilage, mineralized adult bone, nonmineralized fetal bone, or tissues consisting primarily of type 1, type 2 collagens, hyaluronic acid (hyluronans), proteoglycans, and non-collagenous proteins similar to those proteins found in bone and/or cartilagenous tissues. This matrix will be suitable for the growth and differentiation of chondrocytes, chondrocyte-like cells, osteocytes, osteoblasts, and/or osteoblast-like cells.
The term “transplantable bone” is intended to include a nonmineralized, partially mineralized, or fully mineralized viable construct produced, using a bioreactor, that is nonload-bearing, partially load-bearing, or fully load-bearing at the time of transplantation.
The term “implantable bone” is intended to include a nonmineralized, partially mineralized, or fully mineralized nonviable acellularized construct produced, using a bioreactor, that is nonload-bearing, partially load-bearing, or fully load-bearing at the time of implantation.
The term “strain” is intended to include forces applied to the cells and matrix contained in a bioreactor that contribute to manipulation of phenotype of the cells contained therein. As used in the present invention, strain is expected to be applied to the cells and matrix in the bioreactor through forces applied to and within the bioreactor.
The term “stress” is intended to include forces applied to the cells and matrix contained in a bioreactor that contribute to manipulation of phenotype of the cells contained therein. As used in the present invention, stress is expected to be applied to the cells and matrix in the bioreactor through forces applied to and within the bioreactor.
The term “hollow fiber” is intended to include tubular structures containing pores of defined size, shape and density for use in delivering nutrients (in solution) to cells contained within a bioreactor and for removal of waste materials (in solution) from cells contained within a bioreactor. For purposes of the present invention, hollow fibers may be constructed of a resorbable or nonresorbable material.
The term “nutrient solution” is intended to include solutions entering a bioreactor and containing those nutrient materials essential to the culture of mammalian or vertebrate cells. Nutrient solutions may also contain additives that affect specific changes in phenotype of cells under culture or to contribute to changes in the matrix structure of the forming newly formed bone, such as, mineralization.
The term “waste solution” is intended to include solutions exiting a bioreactor and containing waste byproducts of cellular metabolism. The concentrations of waste byproducts, for example ammonia, lactic acid, etc. and residual levels of nutrients such as glucose, in the waste solution can be used to assess the levels of metabolic activity of cells being cultured in a bioreactor.
It must be noted that as used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a nutrient solution” includes a plurality of such solutions and reference to “the vessel” includes reference to one or more vessels and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices, or constructs similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices, or constructs are now described.
The bone fibers of the present invention have the ability to induce or promote bone formation and have properties particularly suitable as a component in bone implants. The bone fibers can be made from cortical or cancellous bone, and from any source, i.e., allograft or xenograft, by the essentially linear cutting from a bone-cutting device. The essentially linear cuttings, i.e., cuttings along the grain direction of the bone, result in bone fibers that optionally curl with the cutting process to form ribbon-like structures such as shown in
Fibers can be cut from any substrate that is capable of being cut using the device. Suitable substrates include non-biological materials, and biological materials. For example, suitable substrates include bone, bone tissue, plasticized bone, plasticized soft tissue, freeze-dried bone, freeze-dried soft tissue, frozen bone, frozen soft tissue, newly formed bone, implant bone, and associated cells, bone marrow, bone marrow-like tissue, cartilage, and cartilage-like tissues. Preferably, the substrate is bone tissue. Any type of bone may be used, such as allogenic and xenogenic bone. The bone tissue may be derived from any mammalian source, but is preferably human.
Production of bone fibers begins with the procurement of bone suitable to the preparation of fiber bone and includes any bone in an animal, such as bone diaphyseal shafts of long bones, for example the femur, tibia, humerus, ribs, radius, fibula. In humans, such bones are composed primarily of cortical bone tissue, but may also include cancellous bone.
The bone used to make the bone fibers may be processed in known manners prior to forming the fibers of the present invention. For example, the bone may be treated with enzymes to partially digest the organic components of the bone, such as collagenase, papain, protease, hyaluronidase, endonuclease, lipase, and/or phosphatase, or organic acids, such as acetic or citric acid. Alternatively, the bone may be partially digested by breaking or fragmenting the covalent bonds in the individual collagen molecules contained in the demineralized bone. Once the bone is cleaned of associated soft tissue, it can then be optionally cut into lengths and shapes appropriate for use in the cutting device. Alternatively the fiber bone can be cut directly from the shaft portions where the cutting blade can be attached to a manual (hand-held) cutting blade holder.
The bone tissue is used to form the bone fibers by contacting the bone tissue with an instrument capable of cutting along the length or along the grain direction of the bone tissue. The cutting instrument should be capable of cutting to provide serrated edges and grooves on the resulting bone fibers, which act as a surface-enhanced binding substrate for bone-forming cells. It has been found that bone-forming cells have an increased ability to attach to these bone fibers. While not intending to be bound by particular theory, it is believed that the edges and grooves formed on the bone fibers of the present invention provide more attachment sites to which the bone-forming cells may bind.
Cell binding to the fiber bone may be easily observed and quantitated using any number of assay methods known in the art. For example, cell populations present in any number of suspension formats, for example, bone marrow, concentrated platelets, blood, liver homogenates, etc., can be incubated with the fiber bone. The fiber bone can then be separated from the cell solution(s), gently washed to remove loosely adherent cells and other biomaterials present in the cell suspensions. The cells retained on the fiber bone can be quantitated using the traditional methyltetrazolium (MTT) assay where an insoluble chromogenic compound is formed due to the presence of metabolically viable cells (active mitochondrial enzymes) where fiber bone incubated with the suspension format lacking cells is used as a control. Alternatively, the fiber bone can be fixed with any number of fixatives, for example, formalin, and the DNA in adherent cells stained for visualization using light microscopy. The phenotypic identity, for example, fibroblasts, chondroblasts, osteoblasts, etc. can be verified using traditional enzyme assays such as alkaline phosphatase activity stains. Fibroblasts (less differentiated cells) stain only minimally for this enzyme, whereas chondrogenic and osteogenic cells stain heavily for this enzyme.
In the method of inducing new bone formation, the bone fibers of the present invention may be used in either a mineralized or demineralized state or a combination thereof. Whether mineralized or demineralized, the bone fibers have the textured surface to which the bone-forming cells may efficiently attach. In the case of demineralized bone fibers, the ribbon-like structures typically unwind into essentially linear strips of bone.
The bone fibers may be of any length, width, and thickness as deemed necessary or useful for its intended use. For example, the fibers may be the length of bone tissue from which they are being made. Alternatively, the fibers may be designed to be cut at shorter lengths to accommodate their use in particular bone implants. Bone fibers preferably have average length of from about 1 mm to about 100 mm, an average width of from about 0.5 mm to about 2.5 mm, and an average thickness of from about 0.2 mm to about 1.4 mm, more preferably having an average length of from about 20 mm to about 30 mm, an average width of from about 1.0 mm to about 2.0 mm, and an average thickness of from about 0.4 mm to about 0.8 mm. Furthermore, it is noted that the length of the fibers produced according to the invention may be substantially greater than the width and thickness of the fibers. For example, the bone fibers may have a length that is greater than about 10 to about 200 times its width and thickness, preferably about 40 to about 100 times its width and thickness. As will be described further herein, the cutting apparatus of the present invention may be modified to accommodate any desired length, width or thickness of the fibers.
For demineralization, the mineral content of the bone fibers may be removed using any known process for demineralization causing the bone fibers to be demineralized. Preferably, the bone fibers are demineralized to contain calcium at a level of from about 0.5 wt % to about 4.5 wt %, more preferably from about 1.0 wt % to about 4.0 wt %, and most preferably from about 1.5 wt % to about 3.5 wt %, for example, as disclosed in U.S. Pat. Nos. 6,189,537 and 6,305,379; and co-pending U.S. patent application Ser. Nos. 09/655,711 and 10/180,989, the disclosures of which are herein incorporated by reference in their entireties. Once demineralized, the bone fibers may optionally be combined with agents including for example, biological carriers, bioactive agents, or other agents including for example, surface active agents, preservatives including for example glycerol, and inorganic mineral compositions, either before or after further processing, such further processing including but not limited to, freeze-drying, terminal sterilization processes, and/or retaining as a hydrated fiber bone in the presence or absence of preserving agents, or combined immediately prior to implantation in a patient. Moreover, the bone fibers of the present invention may be further combined with other carriers and agents as one having ordinary skill in the art would appreciate for the use DMBs. For example, suitable biological carriers include collagen, gelatin, saccharides, fibrin, fibrinogen, alginates, hyaluronins, methylcelluloses, and biologically compatible thixotropic agents. Suitable bioactive agents include but are not limited to, bone morphogenic proteins, stem cells, blood, blood elements, bone marrow and bone marrow extracts, platelets and platelet extracts, homogenates of skin and skin homogenate extracts, growth factors, selenium and transferrin, calcium salts, and CYMETRA™.
Production of demineralized bone biomaterials and the induction of new bone by these biomaterials are described in U.S. Pat. Nos. 5,275,954, 6,189,537 and 6,305,379, of which are herein incorporated by reference in their entireties. The bone fibers of the present invention may induce or promote new bone formation by serving as a source of one or more chemoattractants that diffuse from the bone biomaterials to cause cells to migrate to the implanted bone fibers wherein cells adhere to the bone particles (normal mammalian cells are “attachment dependent,” meaning they typically require attachment to some surface in order to function metabolically) and differentiate towards a chondrocytic (cartilage forming) or osteocytic (bone forming) phenotype. In accordance with the present invention, it is believed that surface characteristics of the bone fibers of the present invention render the fibers more accessible and are a more accepting substrate to receive and bind bone-forming cells. Thus, the surface characteristics of the bone fibers may result in improved cell attachments, and consequently, act as a means for selectively attaching cartilage or bone-forming cells from a mixed population of cells, such as are present in platelet-rich plasma, blood, blood products, or bone marrow.
In accordance with the present invention, the surface patterning present on the bone fibers preferably contains parallel striations, cracks, and serrated edges and grooves to which cells may attach. This surface pattern of the bone fibers of the present invention permits a multitude of cells to bind to the bone fibers allowing less specific cells to bind and grow based on the functional properties of the fibers.
In a preferred embodiment of the present invention, the surface patterning of the bone fibers is created by the bone-cutting device of the invention as described herein. As illustrated in
The “bone-forming cells” or “bone-matrix forming cells” of the present invention are those cells suitable for the induction of new bone formation when infiltrated with the bone fibers of the present invention and include those cell types suitable for differentiating into bone cells or suitable for forming a matrix similar to osteoid of forming new bone. Suitable cell types may include differentiated, partially differentiated, or undifferentiated cells. For example, cell types include, but are not limited to stem cells, connective tissue progenitor cells, fibroblast cells, periosteal cells, chondrocytes, osteocytes, pre-osteoblasts, and osteoblasts. Preferably, the stem cells are multipotent, the fibroblast cells are undifferentiated, the periosteal cells are partially differentiated, and the chondrocytes or osteocytes are differentiated.
Preferably, the bone-forming cells are stem cells. Stem cells represent a population of cells present throughout the body of mammals that are undifferentiated possessing the potential for differentiating into virtually any other, more differentiated, cell in the body. For this reason, stem cells represent a unique opportunity to repair and/or remodel damaged tissues such as broken bones, abraded cartilage, skin, etc.
Moreover, the bone-forming cells may be tissue progenitor cells. For example, U.S. Pat. Nos. 5,824,084 and 6,049,026 (and U.S. patent application 2002/0161449) describe kits and composite bone grafts contained in the kits, wherein the composite bone graft(s) are designed to contain an enriched population of connective tissue progenitor cells and a greater number of connective tissue progenitor cells per unit volume than found in the original bone marrow aspirate.
In one aspect of the invention, the bone fibers of the present invention allow for the formulation of “bone material compositions” comprising the bone fibers for use in bone implants. These bone material compositions provide increased accessibility of the bone fibers to bone-forming cells by permitting suitable voids through which viscous solutions of platelet rich plasma, bone marrow, blood or blood products may flow. For example, the bone fibers may be demineralized and compacted to form a bone material composition suitable for implantation. Because the bone fibers of the present invention are easily handled without breaking apart, the bone fibers may be molded to create an implantable composition, which retains its shape in the implant and further has appropriate spacing through which such solutions comprising bone-forming cells may pass. These bone material compositions may further have integrated therein other components, such as inorganic particles, organic particles, or more specifically non-demineralized cancellous or cortical bone chunks, which may increase the ability of such solutions to flow through the composition by providing structural spacing of the fiber bone. Under such conditions, the surface of the fiber bone fibers would be presented to the infiltrating bone marrow/platelet rich plasma preparations to promote cellular attachment, selectively concentrating the cells most appropriate to the formation of bone or cartilage when the bone material composition is then implanted into some clinical site in the body. Such ex-vivo exposure of the bone fiber biomaterials to osteogenic or chondrogenic cells would serve to concentrate cells that would normally be expected to migrate into the implanted materials through the normal chemoattractive properties of demineralized bone. Thus, this pre-implantation exposure of cells to the bone fibers should reduce the time required for the initiation of new bone formation and lessen the clinical times needed to affect a repair of the damaged site in the body, i.e. a broken bone or fusion site in an intervertebral fusion procedure for repair of cervical or lumbar complications in the spine. Other suitable components for integration into the bone material include, but are not be limited to, inorganics such as particulate calcium salts, such as calcium phosphates, calcium sulfates, and/or calcium carbonates, organics such as particulate skin, particulate cartilage, particulate tendons and ligaments, particulate dextrans, particulate alginates, and particulate resorbable and non-resorbable synthetic polymeric materials.
The bone material compositions may be formed in manners known in the art. In one embodiment of the present invention, the bone fibers of the present invention and bone-forming cells are preferably placed in a bioreactor capable of simulating the nutrient flow and waste removal present within an implant site. The flow of nutrient solutions into, through, and out of the bioreactor permit the associated ground demineralized bone and bone-forming cells to form into bone or bone-like biomaterial suitable for transplantation. In the present instance, the bioreactor use aspect of the present invention would simulate the actions of the fibers and fiber bone compositions when used clinically. The process of making bone in a bioreactor is described in Application No. 60/466,772, for example, which is herein incorporated by reference.
In another aspect of the invention, the bone fibers of the present invention have exhibited superior properties for the formation of bone implants. Bone implants may be formed using the bone fibers of the present invention based on their ability to be easily handled for molding, retaining its shape, and allowing appropriate spacing for biological solutions to pass therethrough even upon compaction. For example, the fibers may be hydrated, which renders then pliable and malleable, but capable of retaining its shape without losing durability. In fact, the fibers have been shown to retain its integrity even upon hydration, molding, and subjection to other bone implant-forming treatments. Therefore, the bone fibers of the present invention have superior properties making them ideal for the formation of bone implants.
In another aspect of the invention, the bone fibers can be used alone or in conjunction with a bone material composition and placed in a suitable container through which blood, blood products, bone marrow, or platelet rich plasma can be induced to flow through such that the cells capable of adhering to the bone material composition, specifically the fiber bone, are suitably concentrated for implantation into a site in the body wherein the formation of new bone is desired.
Referring to
A first actuation unit 122 generates to force necessary to move the slide mechanism 116. According to the disclosed embodiment of the invention, the first actuation unit 122 is pneumatically operated. It should be noted, however, that the first actuation unit 122 can also be operated hydraulically, electrically, and/or mechanically depending on the specific requirements. As illustrated in
According to the disclosed embodiment of the invention, the first actuation unit 122 is configured to generate a force ranging from 600 lbs to 900 lbs. Preferably, the first actuation unit 122 generate a force ranging from 700 lbs to 800 lbs. Most preferably, the force is approximately 750 lbs. Additionally, the force can be varied during operation of the substrate cutting device 100, or it can be maintained at a constant level. For example, according to one embodiment of the invention, the computer controller 188 can be used vary the force applied by the first actuation unit 122 by reducing the amount of force applied during a return stroke and increasing the force applied during a cutting stroke.
As best illustrated in
Turning to
According to the disclosed embodiment of the present invention, when the cutter 114 is mounted on the slide mechanism 116, the cutter surface is substantially flush with the surface of the slide mechanism 116. Such a configuration advantageously minimizes movement of the substrate 162 during operation. Furthermore, as shown in
Referring to
The secondary cutting angle 144 can be selected in the range of 10 to 18. The secondary cutting angle 144 can also range from 12 to 16. Preferably, however, the secondary cutting angle 144 is selected to be approximately 14.
Turning now to
Turning again to
Referring to
A second actuation unit 172 is used to generate the force necessary to operate the second actuation unit 172. As illustrated in the embodiment of the invention shown in
As shown in
According to the disclosed embodiment of the invention, the tower 112 includes three sensor devices 190(e-g). Sensor device 190e detects when the clamping mechanism 178 is in the “up” (or home) position. Sensor device 190f detects when the clamping mechanism 178 is in the vicinity of the clamp stopper 192. Accordingly, sensor device 190f and the clamp stopper 192 both function to prevent accidental contact with the cutter 114. Sensor device 190g detects the presence of the door 184. If an error signal is obtained from sensor device 190g, then operation of the substrate cutting device 100 is immediately halted.
Once all access doors are determined to be closed, control passes to step S318. The clamp is then activated. As previously discussed, this can be accomplished by second actuation unit applying pressure on the substrate. At step S320, the cutter is activated. At step S322, the sensor devices are checked to see if the substrate size has been reduced to a thickness, which is less than a minimum value. If the substrate thickness is greater than the minimum value, then control returns to step S322 and the cuter remains active, i.e., continues to cut the substrate. If the substrate thickness is less than or equal to the minimum value, then the system is stopped as step S326.
As illustrated by the dashed lines, the system continuously monitors the state of the sensor devices throughout the process. Thus, if any of the access doors are opened during operation of the substrate cutting device, control will pass to step S316 and the system will be immediately halted. As previously discussed, this is done, in part, to prevent injury to an operator. The system continues to operate until the either the substrate thickness reaches the minimum size, or one of the access doors is opened.
The present invention provides a method of growing bone in vitro involving providing a biomaterial, such as ground demineralized bone, suitable for inducing cells to form an extracellular matrix and cells capable of forming bone or bone-like biomaterials, and placing the biomaterial and bone-forming cells in close association under conditions suitable for forming bone or bone-like biomaterial. In particular, the ground demineralized bone and bone-forming cells are preferably placed in a bioreactor capable of simulating the nutrient flow and waste removal present within an implant site. The flow of nutrient solutions into, through, and out of the bioreactor permit the associated ground demineralized bone and bone-forming cells to form into bone or bone-like biomaterial suitable for transplantation.
The biomaterial, ground demineralized bone, is capable of inducing selected cell types to form an extracellular matrix consistent with the osteoid materials comprising the organic phase of bone tissue when implanted in heterotopic or orthotopic sites in a living organism. Ground demineralized bone is obtained in manners known in the art and may be available in any form, including as particles or fibers. Ground demineralized freeze-dried bone particles may be used in any particle size suitable for inducing the growth of bone in a bioreactor, such as from about 50 microns to 4 mm, preferably, about 125 microns to 850 microns, and most preferably, about 250 microns to 710 microns. Ground demineralized bone fibers may be produced in known manners, such as by skiving or shaving the surface of the cortical bone to produce short fibers that easily entangle. The fibers are suitable for growing bone in a bioreactor and preferably have physical dimensions of about 0.1 mm to 0.5 mm in width, 0.05 mm to 0.5 mm in thickness, and 1 mm to 500 mm in length. The bone used to make the ground demineralized bone may be processed in known manners prior to forming the ground demineralized bone used in connection with the present invention. For example, the bone may be treated with enzymes to partially digest the organic components of the bone, such as collagenase, papain, protease, hyaluronidase, endonuclease, lipase, and/or phosphatase, or organic acids, such as acetic or citric acid. Alternatively, the bone may be partially digested by fragmenting the covalent bonds in the individual collagen molecules contained in the demineralized bone. The covalent bond breakage of the formed fragments of a collagen molecule may be in the range of about 2 to about 50, and should be sufficient to modify the resorption rate of the demineralized bone. Subsequent to forming the fibers or particles, the fibers and particles are demineralized by exposure to dilute (about 0.4 to 0.6 N) hydrochloric acid or organic acids, calcium chelating agents, etc., as one skilled in the art would appreciate. Alternatively, non-acid chelators of calcium, such as ethylene diamine tetraacetic acid (EDTA), may be used to demineralize the bone.
In addition, the weight percent residual calcium in ground demineralized bone is a factor in defining the bioavailability of bioactive molecules, such as, for example, bone morphogenetic proteins (BMPs), to the cellular population contained within the bioreactor. In fact, it has been found that the ability to extract BMPs from ground bone particles has been shown to be approximately a linear function of the extent of demineralization of the ground bone. Thus, a suitable amount of residual calcium is that amount sufficient to optimize the bioavailability of bioactive molecules, such as BMPs, to the bone-forming cells in the bioreactor. Preferably, the residual calcium is present in the range of about 0-8 weight percent, more preferably about 1-4 weight percent, and most preferably about 2 weight percent.
In accordance with the present invention, the ground demineralized freeze-dried bone particles are added aseptically to the bioreactor. They may be directly added to the bioreactor in a freeze-dried state and rehydrated in the bioreactor or rehydrated in culture medium prior to addition to the culture chamber of the bioreactor. The ground demineralized bone may be added alone or in combination with other components. Preferably, the other components do not inhibit the effect of the ground demineralized bone to induce bone formation. For example, ground nondemineralized bone may be added with ground demineralized bone. In such cases, the ground demineralized bone to nondemineralized bone may be added in any ratio, but preferably is added in a ratio of about 1:1 to about 20:1, more preferably about 8:1, and most preferably about 3:1. The ground nondemineralized bone may take any form, e.g., particles or fibers, and typically will have similar physical dimensions as the ground demineralized bone.
Particle size ranges of the ground demineralized bone particles in the bioreactor determine the “void volume” or available volume outside of the ground demineralized bone particles in which the bone-forming cells and other components may be added. It has been found that the bone particle spacing or availability of space around the ground demineralized bone particles within the bioreactor relates to the void volume and has an impact on the ability of bone-forming cells in the bioreactor to differentiate and/or proliferate. It is desired that bone-forming cells have sufficient contact to allow those cells to infiltrate the voids or space between the ground demineralized bone particles, which permits the in vitro growth of bone or bone-like tissue. Therefore, the void volume or spacing around the ground demineralized bone particles should be that which is effective in allowing for the optimal contacting and infiltration of voids by bone-forming cells between the ground demineralized bone particles.
In accordance with the present invention, the ground demineralized bone particles may be rehydrated in the bioreactor or prior to being added to the bioreactor. Preferably, the particles are rehydrated and mixed with bone-forming cells prior to addition to the bioreactor. The ground demineralized bone particle spacing will differ depending on whether or not the bone particles are rehydrated prior to addition to the bioreactor growth chamber. First, the ground demineralized bone particles may be added to the bioreactor growth chamber and subsequently rehydrated prior to adding bone-forming cells. In this approach, the ground demineralized bone particles may be added to the bioreactor growth chamber in a freeze-died state, which provides a relatively simple step and allows the particles to pack tightly filling the available space. Subsequent rehydration of these freeze-dried ground demineralized bone particles in the bioreactor will cause the bone particles to swell to a tighter state of packing due to rehydration. The bone-forming cells may then be added to the rehydrated bone matrix void volume (that volume outside of the bone particles) in the bioreactor. It has been found this tighter state of packing ground demineralized bone particles in the bioreactor is effective in more tightly packing the added bone-forming cells. While the tight packing may hinder some infiltration of the void volume present throughout the bioreactor, it has been found that the more tightly packed added cells promotes better retention of synthesized matrix molecules during the differentiation process and may be best utilized when seeding more differentiated cells into the bioreactor system.
Alternatively, the ground demineralized bone particles may be rehydrated prior to the addition to the growth chamber of the bioreactor. The bone-forming cells may then be added to the packed ground demineralized bone particles in the bioreactor or directly to the rehydrated bone particle suspension prior to its addition to the bioreactor. While rehydrating freeze-dried ground demineralized bone particles prior to addition to the growth chamber of the bioreactor has been found to increase the difficulty in adding the bone particles to the bioreactor, it has been found that directly adding the bone-forming cells to the rehydrated ground demineralized bone particle suspension results in fully dispersed bone-forming cells and ground demineralized bone particles. More uniform distribution within the growth chamber is thereby achieved and is less likely to contribute to damage to the hollow fibers present within the growth chamber.
In either case, centrifugal forces can be used to cause the rehydrated bone particles and cells to pack throughout the growth chamber with excess fluids removed from the packing port.
The “bone-forming cells” of the present invention are those cells suitable for the induction of new bone formation when infiltrated with ground demineralized bone in a bioreactor and include those cell types suitable for differentiating into bone cells or suitable for forming a matrix similar to osteoid of forming new bone. Suitable cell types include, but are not limited to stem cells, fibroblast cells, periosteal cells, chondrocytes, osteocytes, pre-osteoblasts, and osteoblasts. Preferably, the stem cells are multipotent, the fibroblast cells are undifferentiated, the periosteal cells are partially differentiated, and the chondrocytes or osteocytes are differentiated. In the case of differentiating cell types, such as fibroblasts or stem cells, these cell types may be placed in close proximity to the ground demineralized bone, which, in the bioreactor and under appropriate conditions, will cause the cells to differentiate into bone cells. In the case of cell types suitable for forming an osteoid-like matrix, such as osteoblasts or chondroblasts, such cell types may be placed in close proximity to the ground demineralized bone in the bioreactor and under appropriate conditions, will cause the cells to synthesize matrix similar to osteoid of forming new bone. The type of cells selected for in vitro bone growth is dependent upon the desired time frame for new bone formation, seeding cell densities, and nutrient medium provided.
The source of the bone-forming cells may be autogenic, allogenic, or xenogenic. The use of a potential recipient's own cells in the formation of the bone or bone-like biomaterial will result in a tissue unlikely to be rejected for some immunological reason, rendering the transplantable newly formed bone autogenous in nature. The use of allogenic cells in the formation of new bone with subsequent implantation can be achieved by decellularizing any newly formed bone or bone-like structure prior to implantation using any decellularizing technology known in the art depending on the desired characteristics of the acellular bone or bone-like structure desired for a given clinical application.
The bone-forming cells are added either to the void volume space of the packed ground demineralized bone particles or directly to the rehydrated ground demineralized bone particles prior to addition to the growth chamber of the bioreactor. The cell density of the bone-forming cells may be in the range of from about 102-108 cells per ml, preferably 103-106 cells per ml, and more preferably about 104-105 cells per ml. The density of bone-forming cells added depends on several factors. For example, previous cell culture work in development and validation of in vitro bioassays for assessing the osteoinductive potential of demineralized bone demonstrated the importance of cell density difference depending on the phenotypic status of the cells. (Wolfinbarger, L and Y. Zheng. 1993. An in vitro bioassay to assess biological activity of demineralized bone. In Vitro Cell Dev. Biol. Anim. 29:914.) Less differentiated cells (e.g., dermal fibroblasts), where proliferation constituted a component of the differentiation process, involved a lower seeding density in in vitro bioassays than more differentiated (periosteum derived cells, for example) cells. Presumably, cells more differentiated along the pathway leading from a “stem-like” cell to a differentiated cell phenotype proliferated less well (longer population doubling times of approximately 40 hours) than less differentiated cells (shorter population doubling times of approximately 12 hours) and could be seeded at higher cell densities when used in an in vitro bioassay. Consequently, seeding densities of cells in the bioreactor depends in part on the phenotype of the cells added to the bioreactor, the availability of biologically active materials, and the culture medium used. In addition, seeding cell density in the bioreactor depends on the ability to deliver nutrients to the cells and remove waste byproducts from the bioreactor culture chamber. For example, greater cell densities in the bioreactor require more nutrient delivery and greater waste product removal than lower cell densities.
The bioreactor can be in virtually any shape based on the shape of the bioimplant desired as a newly formed bone or structure that will form load-bearing bone when implanted clinically. The wall of the bioreactor can be deformable and contained within a nondeformable chamber such that positive and negative pressure environments can be applied between the inner wall of the nondeformable chamber and the outer wall of the deformable chamber such that the volume of the bioreactor containing the demineralized bone, cells, and matrix can be decreased or increased over time to simulate stress and strain application to the bone matrix being formed.
The demineralized bone and bone-forming cells can be preloaded into the bioreactor in the presence, or lack thereof, of a viscous matrix designed to provide attachment sites for the cells and/or to restrict diffusion of synthesized osteoid forming molecules. The viscous nature of the matrix may be obtained by the incorporation of polymers, for example, collagenous, hyaluronin, or similar resorbable or nonresorbable polymers.
Nutrients are delivered to the ground demineralized bone and bone-forming cells in the bioreactor and may impact the growth and differentiation of cells contained in the bioreactor. The nutrient solutions are selected to provide sufficient nutrition to the bone-forming cells to maintain viability, growth, and/or differentiation in the bioreactor. Those skilled in the art are capable of selecting an appropriate nutrient solution for the present invention. For example, media such as Dulbecco's modified Eagle's medium may be used and may be further supplemented with other suitable nutrients. Other suitable nutrients include fetal bovine serum, L-ascorbic acid-2-phosphate, antibiotics, cell modulators such as dexamethasone, beta-glycerolphosphate, glucose, glutamine, amino acid supplements, inhibitors (or activators) of apoptosis such as glutathione-ethyl ester, antioxidants, caspase inhibitors, and cations and anions, e.g., magnesium, manganese, calcium, phosphate, chloride, sodium, potassium, zinc, and sulfate ions, and nitrates and nitrites. The concentration of fetal calf serum must not inhibit induced cell differentiations due to diffusible agents from the demineralized bone. The remaining concentration of components in the nutrient solution should be sufficient to promote growth and/or differentiation in the bioreactor and maintain viability of the bone-forming cells and the resulting bone or bone tissue.
In accordance with the present invention, the nutrient solutions may be modified during different phases of the process. For example, during initial culture, seeded cell densities may be minimal, especially for fibroblast cell seeding cultures, and thus nutrient solutions may contain low concentrations of fetal calf serum (such as <2% vol:vol) to facilitate the role of growth and differentiation factors diffusing from the ground demineralized bone particles in modulating phenotypic changes in the added cells. Monitoring the concentration of the nutrients, such as glucose, glutamine, and amino acid supplements, via the eluent flow of medium allows for the determination of nutrient consumption permitting control of flow (delivery) of nutrients into the cell population. Moreover, waste products of metabolism, for example, ammonia and lactic acid, can be monitored via the eluent flow of medium from the bioreactor to determine the metabolic state/function of the resident cell population. Changes in cell phenotype during the culture phase can be monitored by sampling the eluent flow of medium from the bioreactor for proteins associated with specific cell phenotypes, for example, osteopontin and osteocalcin. Should it be desired, for example, other components may be added to the medium during culture to promote a desired function. For example, to induce mineralization during a specific phase of the culture period, chemical components such as β-glycerolphosphate may be added to the medium as a substrate for alkaline phosphatase and to serve as a source of phosphate to be complexed with calcium in the formation of crystalizable calcium salts such as hydroxyapatite. Alternatively, hormonal stimulation of cells can be accomplished via the addition of certain compounds such as, for example, vitamin D. The levels of oxygen tension can be controlled by oxygenation of the nutrient medium being added to the cells being cultured in the bioreactor to manipulate the metabolic state of the cells during the culture phase such that mildly hypoxic conditions can be used to manipulate chondrogenesis and/or osteogenesis. Manipulation of the ionic composition of the medium can be used to control hydrolytic enzyme degradation of demineralized bone matrix, enzyme mediated cross-linking of the formed extracellular matrix being synthesized by the resident cell population, and the osmotic balance of the nutrient solution. Induction and/or inhibition of cellular apoptosis can be controlled by the addition of inhibitors (or activators) of apoptosis such as glutathione-ethyl ester, antioxidants, and caspase inhibitors or activators. For example, use of allogenic cells may require induction of apoptosis to produce a cellular formed bone tissue. In addition, gamma irradiation treatment of the bone particles, either before or after demineralization, can be used to promote cell-mediated resorption of the demineralization bone particles facilitating new bone formation within the areas where the bone particles are resorbed.
The nutrients may be delivered in any manner suitable for the formation of bone in the bioreactor. For example, resorbable hollow fibers can be used to deliver nutrients and remove metabolic waste products during the cellular proliferations and/or differentiation process. The nutrient solutions used can be sequentially introduced into the bioreactor growth chamber as needed to induce cellular morphogenesis, growth, secretion of osteoid biomaterials, and/or to cause mineralization of the formed matrix as desired depending on the type of implantable bone material desired. The resorbable hollow-fibers used to deliver nutrients and remove wastes from the bone forming part of the bioreactor provide an opportunity to leave a series of hollow tube-like openings within the formed bone tissue through which the formed bone tissue can be vascularized. Growth factors such as vascular endothelial growth factor (VEGF) can be final delivered through these hollow fibers once the bone tissue has been formed to promote angiogenesis within the hollow structures following transplantation.
Delivery of nutrients and removal of waste products depends primarily on two factors: numbers of hollow fibers per unit volume of the culture chamber of the bioreactor and flow rates of nutrient solutions through the hollow fibers.
The hollow fibers of the present invention are those suitable for the delivery of nutrients and removal of waste in the bioreactor. The hollow fibers may be any shape, for example, they may be round and tubular or in the form of concentric rings. The hollow fibers may be made up of a resorbable or non-resorbable membrane. For example, suitable components of the hollow fibers include polydioxanone, polylactide, polyglactin, polyglycolic acid, polylactic acid, polyglycolic acid/trimethylene carbonate, cellulose, methylcellulose, cellulosic polymers, cellulose ester, regenerated cellulose, pluronic, collagen, elastin, and mixtures thereof. Moreover, the hollow fibers of the present invention include pores to allow the nutrients and waste to pass in and out of it. The pores of the hollow fibers are a sufficient diameter to allow the diffusion of a molecule from one side of the hollow fiber to the other side of the hollow fiber. Preferably, the molecules that may pass through the hollow fiber pores are about 0.002 to about 50 kDa, more preferably about 5-25 kDa, or most preferably 2-15 kDa.
The number of hollow fibers per unit volume of the culture chamber of the bioreactor is determined based on the cross-section of the hollow fibers, the bioreactor per se, and the distance the bone-forming cells can live from the hollow fibers for nutrient delivery and waste removal. As an example of determining the number of hollow fibers per unit volume,
Bioreactor ID(A)=2 cm 1)
Hollow Fiber ID(B)=1 mm 2)
Distance of Cells Can Live From Any Conduit for Nutrients Delivery and Waste Removal approximates 20˜30 μm depending on the diffusion rates of the nutrient molecules. According to human physiology, it is rare that any single functional cell of the body is more than 20-30 μm away from a capillary.
Calculation:Total Area of Cross-section of Bioreactor=(A/2)2*π=(2 cm/2)2*π=(10 mm)2*π=100 mm2*π 3)
Total Area of Cross-section of One Hollow Fiber=(B/2)2*π=(1 mm/2)2*π=0.25 mm2*π
Total Area of Nutrients Delivery and Waste Removal of One Hollow Fiber=(C/2)2*π=(1 mm/2+0.02 mm)2*π=(0.5 mm+0.02 mm)2*π=0.2704 mm2*π
Number of Hollow Fibers for Bioreactor with Cross-Section ID of 2 cm=100 mm2*π/0.2704 mm2*π=369.82 370
Percentage of Total Area Covered by Hollow Fibers=(0.25 mm2*π)*370/100 mm2*π*100=92.6% Percentage of Total Area Covered by Nutrients Delivery and Waste Removal=(0.2704 mm2*π)*370/100 mm2*π*100=100.48%
Although the flow of nutrient solutions through the hollow fibers will generate some minimal turbulent flow of solutions through the bulk volume of the growth chamber of the bioreactor, the primary mechanism for nutrient dispersal through the growth chamber and to the cells in culture will be diffusion and/or the alternating positive and negative pressure applications applied to the deformable bioreactor wall used to apply stress/strain to the demineralized bone, cells, and extracellular formed/forming matrix mixture during the culture process. Diffusion of nutrients from capillary beds in tissue typically limits the provision of nutrients (for example oxygen, glucose, etc.) to 20-30 μm from an individual capillary. Thus, if diffusion were the sole determinant of nutrient delivery and waste removal, it should be expected that cells located more than 20-30 μm from a hollow fiber will receive less nutrients and exist in a greater concentration of waste byproducts than cells close to a hollow fiber. With application of stress/strain to the demineralized bone, cells, and extracellular formed/forming matrix mixture via alternating applications of positive and negative pressure, it becomes possible to affect greater nutrient solution delivery and waste removal permitting cultivation of cells at greater distances from the hollow fibers than would be allowed by simple diffusion.
Shear stress to cells present in the bioreactor due to flow of nutrient solution will be minimal. Thus, optional addition of mechanical stress and strain to the forming bone matrix will occur primarily via manipulation of the inner vessel in the bioreactor used to contain the demineralized bone, cells, and extracellular formed/forming matrix. This component of the bioreactor includes the option of placing an inner vessel constructed of a deformable material within an outer vessel to which cyclic positive and negative pressure can be applied via a port in the outer vessel wall. It is to be expected that such positive and negative pressures will be minimal and designed to gently compress and expand the forming extracellular matrix in order to provide cyclic mechanical stimulation to the cells contained within the inner vessel of the bioreactor and to promote nutrient solution flow into, through, and out of the bioreactor containing the cells and matrix mixture.
In addition to the cyclic mechanical stimulation to cells contained within the inner vessel of the bioreactor, the inclusion of a series of micro-electrodes within the inner wall of the inner vessel in liquid contact with the forming, or formed, extracellular matrix will allow cyclic, low-level, electrical stimulation of cells and/or the creation of a small electrical gradient from one end to the other end, or side to side, of the bioreactor for use in electrical stimulation of cellular metabolism during induced new bone formation. This cyclic electrical stimulation can occur concurrent with, or not concurrent with, other mechanical or media changes to the forming, or formed, extracellular matrix containing the cells being manipulated to form new bone or bone-like tissue(s).
One aspect of the present invention is practiced by sterilizing all aspects of the bioreactor (tubing, fittings, valves, reagent (solution) containers, filters, sampling ports, bioreactor components, etc.).
The bioreactor 100 as shown in
Referring to
The deformable wall of the inner chamber of the bioreactor may be constructed out of a flexible permeable barrier and a fine deformable mesh that can be molded to a specific shape as needed. The flexible permeable barrier is mechanically supported by a fine mesh, which is present either on the inside or the outside of the flexible permeable barrier. The flexible permeable barrier is made of any suitable resorbable or non-resorbable membrane, such as those comprising polydioxanone, polylactide, polyglactin, polyglycolic acid, polylactic acid, polyglycolic acid/trimethylene carbonate, cellulose, methylcellulose, cellulosic polymers, cellulose ester, regenerated cellulose, pluronic, collagen, elastin, or mixtures thereof. The fine mesh is suitably made up of sterilizable materials, such as stainless steel, titanium, plastic polymer, nylon polymer, braided collagen, and silk polymer, but must be capable of deforming to any desired shape. The fine mesh may have any suitable pore size dictated by the desired bone plug properties. For example, suitable pore sizes for the mesh is between about 0.1 to 10 mm and, preferably, 1-5 mm. The deformable wall may be made to be permeable for some metabolites and not others. For example, the deformable wall may be made to not be permeable to small or large molecular weight metabolites. In particular, a small molecular weight metabolite would fall within the range of 0.001-25 kDa, preferably 0.1-2.5 kDa. A larger molecular weight metabolite would fall within the range of 25-200 kDa, preferably 25-50 kDa. The deformable wall may further be constructed to allow for its use in the bioreactor of the present invention. For example, the tensile properties of the deformable wall should make it capable of deforming under the cyclic negative and positive pressure, such as between 10-30 mmHg. The mesh used to construct the deformable wall preferably will conduct an electrical current. The resorbable or non-resorbable hollow fibers can be used to deliver nutrients and remove waste for the inner chamber. The deformable inner chamber can be contained within a nondeformable outer chamber. The cyclic application of positive and negative pressures to the deformable wall of the inner chamber of the bioreactor to be used in the in vitro growth of bone or bone-like tissue serve to transform this bone or bone-like tissue into bone following transplantation into a recipient.
Inlet and outlet ports of the outer chamber can deliver nutrients and remove waste for this deformable chamber (
The nutrient medium provided and the flow rate of this nutrient medium will vary depending on cell type added to the bioreactor, the packing density of the demineralized bone, presence/absence of a pre-added “extracellular matrix”, and numbers and kinds of hollow fibers contained within the inner vessel of the bioreactor. Nutrient flow will continue until such time as it has been previously determined that the appropriate matrix (structure) has been obtained. At this time, the bioreactor is aseptically dismantled and the bone or bone-like structure aseptically removed for further use.
The formed new bone can consist of a nonmineralized and nonload-bearing osteoid-like material that will mineralize when transplanted into a heterotopic or orthotopic site in a patient or a partially mineralized and partially load-bearing osteoid material that will further mineralize when transplanted into a patient. Given time, it is also possible to produce an almost completely mineralized bone-like tissue that will be load-bearing when implanted clinically.
In another aspect of the invention, the demineralized ground bone and bone-forming cells may form an extracellular matrix that is capable of forming bone when implanted in a patient. In this manner, the demineralized bone and cells may be gelled in a viscous material and have non-loading bearing implantable material that will form in vivo similar to the in vitro bone-forming process described above.
The bone, bone-like tissue, and extracellular matrix made according to the present invention is suitable for transplantation into a patient in need thereof. As one having ordinary skill in the art would appreciate, the bone, bone-like material or tissue, and extracellular matrix can be made into a desired shape that the body will remodel into the appropriate bone when implanted into a patient in some clinical application. For example, as shown in
Moreover, the bone, bone-like tissue, and extracellular matrix may be further treated prior to implantation in manners known in the art. For example, these materials may be acellularized using known methods prior to implantation. Preferred methods of acellularization include, but are not limited to, methods described in U.S. patent application Ser. Nos. 09/528,371 and 09/660,422, which are hereby incorporated in their entirety. The acellularized bone, bone-like tissue and extracellular matrix is within the scope of the present invention. In addition, these acellularized may be recellularized by known methods either in vitro or in vivo. Alternatively, any residual resorbable hollow fibers present in the bone, bone-like tissue, or extracellular matrix may be removed using hydrolytic enzymes, such as cellulase, chitinase, collagenase, elastase, proteases such as chymotrypsis, trypsin, ficin, papain and/or specific enzymes that are capable of degrading the polymers comprising the resorbable and non-resorbable hollow fibers and dialysis films. Other known methods of processing bone prior to implantation are further within the scope of the present invention.
The following examples are for purposes of illustration only and are not intended to limit the scope of the appended claims.
Diaphysyl shafts (total of approximate 520 grams wet weight of bone material) from the long bones and ribs of a given donor (human donor information is confidential) were mechanically debrided (as disclosed in co-pending U.S. patent application Ser. No. 10/108,104, incorporated by reference herein) to remove associated periosteal tissue and bone marrow in the intramedulary canal. The shafts and ribs were then cut into linear pieces with widths, thickness, and lengths approximating <45 mm x<45 mm x<6 cm using a bone saw. A cut piece of cortical bone (wet weight 48 grams) was then loaded individually into the load chute of the cutting device and the clamping cylinder was locked into the closed position. The cutting slide having the cutting blade disposed therein was activated and cut fiber bone was collected into the receiving bin. A total of 42 grams of fiber bone were accumulated during the 60 cutting cycles (cutting cycle equals one back/forthpass of the cutter/cutter slide across the bone surface) for approximately 70 seconds with additional bone materials being added to the feeder chute at each cutting event. After each cutting event, another cortical shaft and/or cortical pieces were added and another cutting event was initiated. The amount of the bone materials loaded into the chute for each cutting event varied. However, the number of cutting events performed were sufficient to accumulate a bulk fiber mass of approximately 490 grams (wet weight).
The cut fiber bone was stored in a sterile container in the freezer (minus 80 C.) for three days. Prior to demineralization, the cut fiber bone was cleaned with LifeNet's patented ALLOWASH technology. For demineralization, a total of 463 grams of bone materials were added to the Pulsatile Acid Demineralization (PAD) chamber (as disclosed in co-pending U.S. patent application Ser. No. 09/655,371 herein incorporated by reference) and demineralized to 2.5% residual calcium using 2 cycles of 0.5 N HCl and acid volumes of 4.0 liters/cycle and 3.0 liters/cycle, 1 cycle of ultrapure water of 3.0 liters/cycle, and 2 cycles ultrapure water plus buffer of 3.0 liters/cycle to terminate the demineralization process. The bone fibers were finally washed in 3.0 liters of ultrapure water and stored frozen at minus 80 C in a sterile container.
Aliquots of the demineralized fiber bone were removed from a sterile container and transferred to the animal implantation laboratory. Aliquots of fiber bone (20 and 40 mg wet weight) were manually compacted and implanted intramuscularly into the hindquarters of athymic (nude) mice as compressed fiber bone materials using established Institutional Animal Care and Use Committee approved protocols (Old Dominion University). After 28 days of implantation, the implanted materials were explanted and the explants fixed in formaldehyde. The fixed explants were embedded in paraffin and sectioned for use in preparation of histology slides. The prepared histology slides were stained using Hematoxylin and Eosin (H&E staining) and viewed under the microscope for induced new bone formation. The induced new bone formation is illustrated in
The attachment of fibroblast cells to fiber bone may be quantitated using the methyltetrazolium dye assay method (MTT) where metabolic activity reduces the methyltetrazolium dye to an insoluble (chromogenic) substrate that can be quantitated using the spectrophotometer. In this particular assessment, cell attachment is compared with cell attachment to particle bone (cortical bone ground, using impact fragmentation) ground to a particle size range of 250 to 710 microns, demineralized and used in equal gram equivalents.
Fibroblast cells (NIH 3T3) were chosen for the study in that these cells represent relatively undifferentiated cells present in the body and are presumed to represent those cells that primarily migrate to the site of implantation of demineralized bone such as used in nude (athymic) mouse implant studies to assess the osteoinductivity of demineralized bone.
Fibroblast cells (1-5×105 cells/ml) grown in RPMI 1640 tissue culture medium (supplemented with 10%, by volume, fetal calf serum (FCS) and glutamine) were harvested from the T-75 culture flasks using trypsinization. The residual trypsin associated with the cells put into suspension was neutralized by resuspending the cells in fresh RPMI 1640 tissue culture medium (supplemented with 10% FCS). Demineralized fiber bone (100 mg, wet weight) was aliquoted into replicate (20) 15 ml sterile centrifuge tubes and demineralized particle bone (100 mg, wet weight) was aliquoted into replicate (20) 15 ml sterile centrifuge tubes. The twenty tubes of fiber bone and 20 tubes of particle bone were divided into two groups each of 10 replicates such that one group of 10 would be incubated with tissue culture medium without cells and the remaining group of 10 would be incubated with tissue culture medium with cells. Each tube received 5 mls of medium (medium containing or not containing cells) such that tubes receiving medium with cells received approximately 5×105 to 1×106 cells/100 mg of demineralized bone (fiber or particle). The tubes were statically incubated at 37 C for one (1) hour, at which time the medium was decanted off of the bone and fresh medium (5 ml) added and decanted to affect a “washing” of the demineralized bone. This “washing” process was repeated a total of three times. All steps were conducted using aseptic techniques such that the demineralized bone could be incubated overnight at 37 C to permit the attached cells to proliferate.
Following the overnight incubation, the demineralized bone/medium/“cells” (if added in the centrifuge tube) were vigorously vortexed to dislodge cells and the medium decanted to a fresh centrifuge tube. The dislodged cells were concentrated by low speed (1,500 to 2,000 rpm in a clinical table top centrifuge) centrifugation and the medium decanted. The cell pellets were assayed using the standard MTT assay and the numbers of cells “quantitated” by comparison to a standard curve where known numbers of cells were aliquoted into centrifuge tubes, centrifuged to concentration and assayed.
Background absorbance values were obtained using the demineralized bone (fiber and particle) incubated in the absence of cells. On average, the fiber bone presented 1-5×103 cells/100 mg of bone whereas the particle bone presented approximately 2-4×102 cells/100 mg of bone, i.e., an approximate 10-fold greater numbers of cells per unit wet weight of fiber bone to particle bone.
Implantation of biomaterials into muscle pouches of athymic (nude) mice (two implants/mouse, implanted bilaterally in the gluteal region of the mouse) represents the current “gold-standard’ method of assessing the osteoinductivity of such biomaterials. Between 10 and 20 mg (dry weight) of biomaterials (demineralized bone in this example) are rehydrated with isotonic saline and implanted just under the fascia using a dental amalgum tool (such as typically used by a dentist to add the filing materials to a cavity formed in teeth).
In this study, human “shaved” (fiber) bone and human “DMB Positive Control” (particle) bone were implanted into muscle pouches of athymic mice (two implants/mouse and three mice per implant group). The implanted materials were explanted after 28 days, and the explants (explanted as “hard” nodules) were fixed in buffered formalin. The samples were decalcified and embedded in paraffin prior to preparation of histological sections for staining (hematoxalin/eosin; H&E). As illustrated in
Dialysis tubes (Spectrum, Spectra/Por) made with different membrane pore sizes (MWCO 10,000-25,000) and different material (regenerated cellulose or cellulose ester) were used for musculoskeletal bone tissue regeneration. The hydrogen peroxide in sterile dialysis tubes was removed and the tubes were soaked in tissue culture media for 1-2 hours in order to remove all remnants of hydrogen peroxide. Demineralized bone matrices were weighed aseptically and hydrated with cell suspension (human dermal fibroblasts or human periosteal cells) in RPMI 1640 tissue culture medium. The DBM and cell mixtures were introduced into dialysis tubes and the tubes were incubated in culture media containing 2% FBS, 50 μg/ml L-ascorbic acid, 1 μM dexamethasone, and 50 mM beta-glycerolphosphate. The dialysis system was incubated either under static (that means the dialysis tubes are incubated in a media container), stirred dynamic (that means the dialysis tubes are incubated in a media container which stays on stir plate to give constant mixing speed), or fluid-flow dynamic (that means the dialysis tubes are incubated in a media flow chamber which controls the media flow rate for dialysis tubes by peristaltic pump) conditions. The culture media were replaced by fresh media once a week to keep sufficient nutrients for cell growth and differentiation.
During various time of incubation (1-7 weeks), the culture media were taken out from the containers for osteocalcin quantitation by ELISA, the tissues from the dialysis tubes were taken out for histology analysis, alkaline phosphatase quantitation, percentage of calcium quantitation, and double strand DNA quantitation. The samples of culture media were taken out from bioreactor each week for osteocalcin quantification by ELISA.
Various bone plugs produced according to this example were further examined. Specifically, some of the bone plugs formed according to this example are depicted in
The bioreactor was constructed from glass tubing (inner diameter, 5 mm; length, 50 mm) and contained forty porous regenerated cellulose hollow fibers (outer diameter, 216 μm; inner diameter, 200 μm; MWCO of 18,000; Spectra/Por®; Spectrum Laboratories, Inc.; Laguna Hill, Calif.). The hollow fibers were embedded in biomedical grade silicon rubber (Nusil Silicone Technology, Carpenteria, Calif.).
To determine the optimal cell seeding density in the bioreactor system, human periosteal (HPO) cells were inoculated into the bioreactor at various cell density of 0.5×106, 1×106, 5×106, and 1×107 cells with DBM (1.5 cc or 500 mg). The culture medium used comprises Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics, ascorbic acid, beta-glycerophosphate, dexamethasone, and 2% fetal bovine serum (FBS). Two hundred and fifty ml of cell culture medium was recirculated with a medium flow rate of approximately 5 ml/min. After inoculation, the bioreactors were perfused using a peristaltic pump and maintained in a 5% CO2/95% air incubator. After 5 days, the samples of cells with DBM were removed and in vitro alkaline phosphatase assay was performed.
To study the growth of new bone or bone-like tissue using hollow-fiber bioreactor system, the bioreactor was inoculated with 1×107 cells and DBM (1.5 cc or 500 mg) through either end into the extracapillary space of the bioreactor. Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics, ascorbic acid, beta-glycerophosphate, dexamethasone, and 2% fetal bovine serum (FBS) was used as culture medium throughout the experiments. Culture medium was changed weekly. Two hundred and fifty ml of cell culture medium was recirculated with a medium flow rate of approximately 5 ml/min. Diffusive nutrient supply and removal of metabolic waste products across the membrane of hollow fiber was advanced by constantly recirculating culture medium through the system using a peristaltic pump maintained in a 5% CO2 incubator. After 3 weeks, samples were taken from the bioreactors, fixed in neutral buffered formalin, embedded in paraffin and sectioned. Sections were stained with Haematoxylin & Eosin. The results were illustrated in
Each of the patents and publications cited herein are incorporated by reference herein in their entirety. It will be apparent to one skilled in the art that various modifications can be made to the invention without departing from the spirit or scope of the appended claims.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general the principles of the invention and including such departures from the present disclosure as within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims. Any references including patents and published patent applications cited herein are incorporated herein in their entirety.
This application is a Continuation-in-Part of U.S. application Ser. No. 16/179,173, filed Nov. 2, 2018; which is a Continuation of U.S. application Ser. No. 15/494,001, filed Apr. 21, 2017, allowed; which is a Continuation of U.S. application Ser. No. 12/692,879, filed on Jan. 25, 2010, abandoned; which is a Divisional of U.S. application Ser. No. 10/606,208, filed on Jun. 26, 2003, now U.S. Pat. No. 7,744,597, issued Jun. 29, 2010; this application is also a Continuation-in-Part of U.S. application Ser. No. 16/059,430, filed Aug. 9, 2018; which is a Continuation of U.S. application Ser. No. 14/730,458, filed Jun. 4, 2015, abandoned; which is a Continuation of U.S. application Ser. No. 11/518,566, filed Sep. 11, 2006, now U.S. Pat. No. 9,080,141, issued Jul. 14, 2015; which is a Divisional of U.S. application Ser. No. 10/835,529, filed Apr. 30, 2004, now U.S. Pat. No. 7,494,811, issued Feb. 24, 2009, which claims benefit of U.S. Provisional Application No. 60/466,772, filed May 1, 2003, the contents of each of which are all hereby incorporated herein by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| 60466772 | May 2003 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10606208 | Jun 2003 | US |
| Child | 12692879 | US | |
| Parent | 10835529 | Apr 2004 | US |
| Child | 11518566 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15494001 | Apr 2017 | US |
| Child | 16179173 | US | |
| Parent | 12692879 | Jan 2010 | US |
| Child | 15494001 | US | |
| Parent | 14730458 | Jun 2015 | US |
| Child | 16059430 | US | |
| Parent | 11518566 | Sep 2006 | US |
| Child | 14730458 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16179173 | Nov 2018 | US |
| Child | 17164616 | US | |
| Parent | 16059430 | Aug 2018 | US |
| Child | 10606208 | US |
