KR20060136475A - Preparation of a nucleated cell and/or platelet concentrate from a physiological solution - Google Patents

Abstract

The present invention relates to methods and compositions related to preparing cell concentrates, such as, for example, osteogenic cell concentrates, from physiological solutions such as bone marrow aspirate, blood, or mixtures thereof. In certain embodiments, the present invention provides methods and compositions that utilize two physiological solution-treatment techniques, particularly in terms of therapeutic environment, and do not use centrifugation.

Physiological solution, cell concentrate, bone marrow aspirate, hollow fiber filtration, leukocyte reduction filtration

Description

Preparation of nucleated cells and / or platelet concentrates from physiological solutions {PREPARATION OF A NUCLEATED CELL AND / OR PLATELET CONCENTRATE FROM A PHYSIOLOGICAL SOLUTION}

This application claims priority to US Provisional Patent Application No. 60 / 458,354 and US Provisional Patent Application No. 60 / 528,583, filed Dec. 10, 2003, both of which are incorporated herein by reference in their entirety. Is introduced.

The present invention relates generally to the fields of bone therapy, bone marrow processing, and medicine, such as orthopedic medicine. In certain embodiments, the invention relates to processing a physiological product, such as bone marrow aspirate, through a two-stage filter system with each stage lacking centrifugation.

Bone graft substitutes (BGS) are commonly used as an alternative method that is less invasive in autografts for the repair of bone defects. However, autografts still remain an absolute standard in the field of transplantation because they supply structural backbone, osteoinductive growth factors, and osteogenic cells. A promising surgical choice for autografts is to use complex implants consisting of BGS combined with bioactive (ie osteoinductive and osteogenic) elements. Examples of bioactive elements include platelets (secreting osteogenic growth factors) and stem cells (differentiated into osteogenic bone-morphogenic cells). Sources of these bioactive elements include bone marrow aspirate and blood, which are less invasive from patients than autografts. In view of this, techniques have been developed for obtaining and concentrating bioactive elements from bone marrow aspirate and blood for use in the field of tissue healing. Centrifugation is typically used to separate osteogenic / osteoinductive cells from other blood components based on differences in density. However, in operating rooms, not only the purchase and maintenance of centrifuges, but also the preparation of cell-rich suspensions can be difficult. Thus, a surgical technique at the point of treatment that can concentrate stem cells and platelets from blood or bone marrow aspirate without the need for centrifugation is desirable. This technique offers surgeons a convenient way to rapidly produce bioactive BGS, which is a viable and less invasive alternative to autografts.

In Connolly et al. (1989), bone marrow extracted from rabbits was concentrated by simple centrifugation, isopynic centrifugation, and gravity settling, all of which were average nucleated cells compared to total bone marrow. Increased number. The bone marrow bone formation effects were tested in rabbits using chambers implanted in the peritoneal cavity (ectopic site) and delayed-union model (orthotopic site). Bone formation is promoted after enrichment of bone marrow urea after centrifugation at these two sites, but not after gravity sedimentation. Although methods for increasing nucleated cell density in bone marrow aspirates have been described, centrifugation is used to concentrate stem cells in the bone marrow. In addition, concentrates were not used with osteoconductive framework material.

PCT Patent Application Publication WO 96/27397 provides a plasma coat concentrate comprising plasma, platelets, and fibrinogen. When the concentrate is combined with a fibrinogen activator at a concentration sufficient to initiate blood clot formation, a wound closure agent is obtained. Also provided is a method of processing blood to produce a plasma-smoke concentrate. This method involves centrifuging the anticoagulant blood to remove red blood cells and preparing a plasma-smoke mixture. Plasma-smoke concentrates are prepared by removing water from the mixture by hemofiltration. Fibrinogen activators can be mixed with plasma- smokescreen concentrates to prepare wound closures, which can be used in a number of clinical indications, including bone-grafting applications.

US Pat. Nos. 6,010,627 and 6,342,157 concentrate blood fractions, typically plasma, to concentrate blood coagulatory proteins such as fibrinogen, and cellular components such as platelets, leukocyte cells, or concentrates of soft tissue cells. Provided are an apparatus and a method for providing the same. Plasma- smoke concentrates are prepared by removing water from the plasma by hemofiltration. The resulting concentrate is suitable for use in making clot-based wound closure agents. This method utilizes a blood filter, a type of hollow fiber filter used in blood processing to concentrate cells and proteins in the blood fraction. The prior art defines an ultrafiltration device having a semipermeable membrane of molecular weight cut-off about 30,000 Daltons that allows for the enrichment of fibrinogen proteins useful for coagulation when fibrinogen activators are added to the cell / protein concentrate. . However, US Pat. Nos. 6,010,627 and 6,342,157 do not disclose fiber filters for enrichment of mesenchymal stem cells and platelets from whole blood or bone marrow aspirates. Thus, in the present invention, since the recovery of the protein is not important, it is possible to use a filter having a large cut-off (ie, greater than 30,000 Daltons) in concentrating the cells. Since large cut-off filters have faster flow rates and require lower operating pressures than small cut-off filters, the filters of the present invention will require less time and force to operate compared to the filters disclosed herein.

Muschler et al. (2002; US Pat. Nos. 5,824,084 and 6,049,026) provide a method for producing composite BGS in which bone marrow aspirate passes through a porous substrate. Bone progenitor cells are selectively retained within the matrix, resulting in a composite implant containing a rich (ie larger) number of progenitor cells compared to an equivalent volume of bone marrow aspirate. A method of passing bone marrow through an implantable porous backbone that serves as an affinity column for mesenchymal stem cells is provided. Stem cells are selectively retained within the matrix, resulting in a composite implant containing a rich (ie larger) number of stem cells as compared to an equivalent volume of bone marrow aspirate. However, Musler's literature does not disclose methods and compositions for platelet reinforcement.

PCT patent application publications WO 00/61256 and US Pat. No. 6,398,972 describe automated methods and devices for preparing platelet-rich plasma or platelet concentrates from physiological solutions, preferably blood. Processing is carried out by a centrifuge containing a disposable container having two chambers. Whole blood is placed in one of the two chambers and then centrifuge is operated to precipitate red blood cells at the bottom of one chamber to obtain supernatant of platelet-rich plasma. The centrifuge is stopped to allow platelet-rich plasma to be discharged into the second chamber. The centrifuge is then restarted to second centrifuge the platelet-rich plasma in the second chamber. The centrifuge was stopped so that (1) red blood cells in one chamber; (2) platelet concentrate at the bottom of the second chamber; And (3) platelet deficient plasma as a supernatant in a second chamber. A portion of platelet deficient plasma is removed from the second chamber and platelet deficient plasma and platelet concentrate are left in the second chamber. The remaining platelet concentrate is suspended in platelet-deficient plasma remaining in the second chamber to obtain platelet rich plasma.

Thus, the invention described herein meets the needs of the art in clinical therapies, in particular bone therapy, which lack the methods of the invention.

<Overview of invention>

The present invention relates to systems, methods and compositions that are advantageous for use in, for example, repairing bone defects. More specifically, the present invention provides methods for preparing cell concentrates from physiological solutions (preferably bone marrow aspirate, blood, or mixtures thereof) at the time of treatment, optionally without the need for centrifugation. This cell concentrate is preferably used in the alternative embodiment for the treatment of bone formation, but the cell concentrate is analyzed, for example for diagnostic, drug testing or prognostic purposes.

In certain embodiments of the invention, providing a physiological solution that has not been pre-centrifuged; The physiological solution is filtered to produce a filtration preserve and a permeate solution, wherein the filtration restoration comprises more than platelets, nucleated cells, or both per unit volume than in the physiological solution, wherein the permeate solution is plasma and Comprising red blood cells; And removing the filtrate retainer from the filter. In certain aspects, the filter is a leukocyte reduction filter.

In certain embodiments, the providing step may be further defined as combining a further solution comprising water or a hypotonic solution, such as sodium chloride, with a physiological solution.

In another embodiment of the invention, providing a physiological solution; Treating the physiological solution with a first filtration device to isolate nucleated cells, platelets, or both from the physiological solution, wherein the isolating step produces a first product; And treating the first product with a second filtration device to produce a second product, wherein the second product includes more than a number of nucleated cells, platelets, or both, per unit volume than in physiological solution. Provided is a method of making a cell concentrate, comprising the step. In certain embodiments the first filtration device is a nucleated cell filtration device, which may be further defined as a leukocyte reduction filtration device, and in another particular embodiment, the second filtration device is further defined as a hollow fiber filtration device.

The filtration device, such as the second filtration device, may comprise a pore size of about 0.05 μm to about 5 μm, or more preferably about 0.2 μm to about 0.5 μm.

In a preferred embodiment, the method of the present invention lacks a centrifugation step.

In certain embodiments, the physiological solution comprises bone marrow aspirate, blood, or mixtures thereof, and in some embodiments nucleated cells may comprise stem cells, connective tissue progenitor cells, bone precursor cells, cartilage precursor cells, or mixtures thereof. Include. In some embodiments, the stem cells are mesenchymal stem cells, hematopoietic stem cells, or both. In another embodiment, the methods of the invention further comprise delivering a second product to a bone defect in the individual and / or mixing the skeletal material with the second product to produce a skeletal material / second product mixture. . In some embodiments, the method may include delivering the skeletal material / second product mixture to a bone defect in the individual.

The backbone material may consist of blocks, pastes, dust, cement, powders, granules, putty, liquids, gels, solids, or mixtures thereof and / or ceramic, polymer, metal, allograft bone, autologous Graft bone, demineralized bone matrix, or mixtures thereof. The backbone material is biodegradable and is preferably osteoconductive, osteoinductive, or osteogenic. In certain embodiments, the backbone material may consist of synthetic materials, natural materials, or combinations thereof.

In certain embodiments, the methods of the present invention further comprise mixing the biological agent with the second product, the skeletal material, or a mixture thereof, wherein the biological agent mixed with the skeletal material is on, in the skeletal material, or It may be further defined as a biological agent included in both of them. In certain embodiments, the framework material itself is a biological agent.

In another embodiment of the present invention, cell concentrates produced by the methods described herein are provided.

In a further embodiment of the invention, providing a physiological solution; Treating the physiological solution with a nucleated cell filtration device to isolate nucleated cells, platelets, or both from the physiological solution, wherein the isolating step produces a first product; And treating the first product with a fiber filtration device to produce a second product, wherein the second product comprises more than a number of nucleated cells, platelets, or both per unit volume than in physiological solution. Provided is a method of increasing nucleated cell concentration and / or platelet concentration from a physiological solution.

Providing a bone marrow aspirate may be further defined as aspirating bone marrow from a subject into a first syringe to produce a bone marrow aspirate. Preferably, the first syringe comprises an anticoagulant, an isotonic solution, or both.

In a further particular embodiment, treating the bone marrow aspirate with a nucleated cell filtration device introduces the bone marrow aspirate into a first housing device, wherein the first housing device is connected in series to a leukocyte reduction filter and the leukocytes A reduction filter is connected in series to the second housing device, the leukocyte filter passes plasma and red blood cells through the filter but inhibits passage of nucleated cells, platelets, or both; Introducing a purging solution into the filter to produce a purging solution / nucleated cell / platelet mixture; And withdrawing the purging solution / nucleated cell / platelet mixture from the filter.

Treating the first product with a filtration device may include treating the first product with a hollow fiber filtration device and / or treating the first product with a device comprising a filter, wherein the filtration device is The direction of feed of the first product through is non-parallel to the flow of the first product across the membrane.

In certain embodiments, the step of providing blood can be further defined as, for example, aspirating blood from the individual into the first syringe to produce a blood aspirate. The first syringe may comprise an anticoagulant, an isotonic solution, or both. In certain embodiments, treating the blood aspirate with a nucleated cell filtration device introduces the blood aspirate into a first housing device, wherein the first housing device is connected in series to a leukocyte reduction filter, and the leukocyte reduction filter Is serially connected to the second housing device, the leukocyte filter passes plasma and red blood cells through the filter but inhibits passage of nucleated cells, platelets, or both; Introducing a purging solution into the filter to produce a purging solution / nucleated cell / platelet mixture; And withdrawing the purging solution / nucleated cell / platelet mixture from the filter.

Providing a bone marrow aspirate / blood mixture may be further defined as aspirating bone marrow and blood from a subject into a first syringe to produce a bone marrow aspirate / blood aspirate. The first syringe may comprise an anticoagulant, an isotonic solution, or both. Treatment of the bone marrow aspirate / blood aspirate with a nucleated cell filtration device introduces the bone marrow aspirate / blood aspirate to a first housing device, wherein the first housing device is connected in series to a leukocyte reduction filter, A leukocyte reduction filter is connected in series to a second housing device, the leukocyte filter passing plasma and red blood cells through the filter but inhibiting passage of nucleated cells, platelets, or both; Introducing a purging solution into the filter to produce a purging solution / nucleated cell / platelet mixture; And withdrawing the purging solution / nucleated cell / platelet mixture from the filter.

In a further embodiment of the invention, obtaining a physiological solution comprising nucleated cells, platelets, or both; Treating said physiological solution with a nucleated cell-filtration device to isolate nucleated cells, platelets, or both, wherein said isolating step produces a first product; Treating the first product with a hollow fiber filtration device to produce a second product, wherein the second product comprises more than a plurality of nucleated cells, platelets, or both per unit volume than in physiological solution; And delivering a second product to a bone defect in the subject. In certain embodiments, the method lacks a centrifugation step, but in an alternative embodiment, the centrifugation step is used, for example, to remove adipocytes and non-cellular fatty substances from platelets and nucleated cells.

Bone defects can include breaks, fractures, voids, diseased bones, bone loss, broken bones, weak bones, bone damage, or bone degeneration. The method of the present invention may further comprise mixing the backbone material with the second product. In some embodiments, the physiological solution is blood, bone marrow aspirate, or mixtures thereof.

The physiological solution is preferably obtained from an individual. In certain embodiments, the methods of the invention are carried out at the point of treatment in a hospital facility or health care providing facility. In another particular embodiment, the methods of the present invention further comprise administering an additional bone defect treatment to the individual. Additional bone defect therapies include fracture repair, surgery, bone resection, graft delivery, external stimulation, or a combination thereof. In certain embodiments, the nucleated cells comprise leukocytes, stem cells, connective tissue progenitor cells, bone precursor cells, chondrocyte cells, or mixtures thereof. Stem cells may be mesenchymal stem cells, hematopoietic stem cells, or mixtures thereof. Delivering the second product to the subject may include applying the second concentrated product directly to the bone defect. In certain embodiments, such applications use a scoop, scoopula, syringe, rod, or spatula.

In another specific embodiment, the methods of the present invention further comprise mixing the biological agent with the second product, the framework material, or a combination thereof. Biological agent agents mixed with the backbone material may be further defined as biological agents included on the backbone material, in the backbone material, or both.

In a further embodiment of the present invention, there is provided a method of producing osteogenic cell concentrate from a physiological solution comprising subjecting the physiological solution to at least one filtration step, wherein the centrifugation step is preferably performed. Lacks.

In another embodiment, a kit for preparing cell concentrate is provided, comprising a first filtration device and a second filtration device, wherein both devices are housed in a suitable container. In certain embodiments, the first filtration device and the second filtration device are housed in a suitable container. In another particular embodiment, the first filtration device is a nucleated cell filtration device and / or the second filtration device is a microfiltration device. The microfiltration device may be further defined as a hollow fiber filtration device.

The kit may further comprise a backbone material contained in a suitable container and / or may further comprise a biological agent contained in a suitable container.

In another embodiment of the invention, there is provided a kit for treating bone defects comprising a plurality of cells housed in a suitable container, wherein said cells are nucleated cells, platelets, or both. In certain embodiments, the kit further comprises a skeletal material contained in a suitable container. The kit may further comprise a biological agent contained in a suitable container.

In another embodiment of the invention, there is provided a kit comprising an instrument for the preparation of cell concentrates obtained by the methods described herein.

In certain embodiments, providing a physiological solution that has not been pre-centrifuged; The physiological solution is treated with a leukocyte reduction filter to produce a filtration preserve and a permeate solution, wherein the filtration preserve contains more than platelets, nucleated cells, or both per unit volume than the physiological solution, and the permeate solution is plasma And red blood cells; And removing the filtrate retainer from the filter.

The foregoing has outlined rather broadly the features and technical advantages of the present invention and will be better understood in accordance with the following detailed description. Further features and advantages of the present invention are described below, which form the subject matter of the present invention. It is understood that one skilled in the art can easily modify or design other structures for carrying out the purposes of the present invention based on the concepts and specific embodiments disclosed herein. Moreover, those skilled in the art will understand that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be features of the present invention, both in terms of construction and method of operation, will be better understood when considered in conjunction with the following detailed description and the accompanying drawings, together with additional objects and advantages. However, each drawing is provided only for the purpose of illustration and description, of course, is not intended to limit the scope of the invention.

For a more complete understanding of the invention, reference is made to the following description in conjunction with the accompanying drawings:

1 provides an exemplary schematic showing selective isolation of nucleated cells (including mesenchymal stem cells) and / or platelets from physiological solution by filtering bone marrow aspirate through a leukocyte reduction filter.

2 provides a schematic diagram illustrating an example of a hollow fiber filtration device operated by hand.

3 illustrates a schematic diagram illustrating increasing isolated cell concentration by filtering isolated nucleated cells and / or platelets through a hollow fiber filter.

4 provides an exemplary schematic showing the selective recovery of osteogenic cells (ie, platelets and nucleated cells) from a physiological solution by operating a first filter (leukocyte reduction-type filter).

5 illustrates an exemplary schematic showing the operation of a second filter (hollow fiber filter) to concentrate osteogenic cells (ie platelets and nucleated cells) recovered from the first filter. The syringe containing the recovered osteogenic cells is connected to a hollow-fiber filter and operated in a cross-flow manner. In cross-flow filtration, the feed stream is tangentially recycled between the feed syringe and the retentate syringe to the membrane, whereby differential pressure is generated across the membrane. This causes some particles to pass through the membrane. The remaining particles continue to flow through the membrane. Using an appropriate membrane pore size (0.2-0.5 μm), the recovery solution can pass through the filter membrane of the hollow fiber filter, but cells cannot pass through this membrane.

6 provides an exemplary schematic showing the osteogenic cell concentrate suspension obtained from the operation of the second filter. By cross-flow filtration, a portion of the recovery solution is separated from the suspended cells, thereby increasing the cell concentration in the reserve syringe.

FIG. 7 is an exemplary schematic showing the operation of a second filter (hollow fiber filter) to concentrate osteogenic cells (ie platelets and nucleated cells) recovered from the first filter. The syringe containing the recovered osteogenic cells is connected to a hollow-fiber filter, which is operated in a full amount fashion (achieved by capping the syringe syringe port). Unlike cross-flow filtration, dead-end filtration does not include recycling of the feed stream which tangentially recycles between the feed syringe and the reserve syringe to the membrane. By pressure from the forward feed syringe, differential pressure occurs across the membrane. As such, some particles pass through the membrane. Using an appropriate membrane pore size (0.2-0.5 μm), the recovery solution can pass through the filter membrane of the hollow fiber filter, but cells cannot pass through this membrane.

FIG. 8 is an exemplary schematic diagram showing osteogenic cell concentrate suspension resulting from operation of the second filter in full quantity mode. FIG. Whole filtration increases the concentration of cells remaining in the feed syringe by separating some of the recovery solution from the suspended cells.

FIG. 9 shows an exemplary schematic showing operating a fat reduction filter to reduce fat particle content in a physiological solution and selectively recovering osteogenic cells (ie, platelets and nucleated cells) from the physiological solution. to provide.

FIG. 10 illustrates certain embodiments using an aggregation filter, such as cellular and / or non-cellular fat reduction filters, in a filtration process.

11 shows a representative field of observation of nucleated cell numbers of blood, pre-treatment BMA and post-treatment BMA.

12 illustrates a schematic of an exemplary filter arrangement used to generate platelet rich concentrate from blood.

13 demonstrates platelet recovery for two exemplary starting blood volumes.

14 shows the concentration of PDGF-AB in recovered platelets and whole blood.

15 shows the concentration of TGF-β1 in recovered platelets and whole blood.

16 shows the concentration of VEGF in recovered platelets and whole blood.

I. Definition

As used herein, the indefinite article (“one” or “an”) may mean one or more. When used with the word “comprising,” the indefinite article used in the claims (s) herein may mean one or more than one. As used herein, “another” can mean at least a second or more. Also, as used herein, the terms "including", "containing" and "having" are not limiting in interpretation and are interchangeable with the term "comprising".

As used herein, the term “allograft bone material” is defined as bone tissue collected from another individual of the same species. Allograft tissue can be used in its natural state or modified to meet the needs of a wide variety of orthopedic procedures. The majority of allograft bone tissue is from deceased donors. About 70% by weight of bone is mineral. The remaining 30% are collagen and non collagen proteins (including bone morphogenic proteins, ie BMP). Allograft bone cleaned and prepared for transplantation provides a support matrix for performing bone growth, but cannot release the factors that induce the bioreaction of the patient to form bone cells and produce new bone tissue. In a preferred embodiment, allografts are cleared, sanitized and inactivated against viral transmission.

As used herein, the term “biological agent” is added to bone graft substitutes for therapeutic purposes such as facilitating bone growth, preventing disease, administering pain-reducing compounds, administering drugs, and the like. Examples of biological agents include antibiotics, growth factors, fibrins, bone morphogenic factors, angiogenic factors, bone growth agents, bone proteins, chemotherapeutic agents, pain relief agents, bisphosphonates, strontium salts, fluoride salts, magnesium salts and sodium salts. Can be mentioned.

As used herein, the term "blood" refers to circulating tissue consisting of a fluid portion (plasma) with suspended formed factors (red blood cells, white blood cells, platelets). In certain embodiments of the invention, physiological solution refers to whole blood, such as, for example, whole blood provided by a donor, whole blood obtained from the patient's circulatory system, or mixtures thereof. Blood can be obtained by any suitable means, for example in the form of a blood aspirate.

As used herein, the term "bone graft substitute (BGS)" is defined as an entity that replaces bone tissue and / or fills space within bone tissue. The term "bone defect" as used herein is defined as a bone defect such as breakage, fracture, voiding, diseased bone, bone loss, fractured or weak bone, injury, disease or degeneration. Such defects may be the result of disease, surgical adjustment, malformations or trauma. Degeneration may be the result of progressive aging. Diseased bones include osteoporosis, Paget's disease, fibroid dysplasia, dystrophic dystrophy, periodontal disease, osteopenia, osteoporosis, primary parathyroidism, hypophosphatemia, fibrous dysplasia, incomplete osteoogenesis, myeloma bone disease and bone It may be the result of bone disease such as malignancy. Bone defects may be due to a disease or condition, such as a disease that indirectly affects the bone. In addition, the bone malignancy to be treated may be a primary bone malignancy or it may be metastatic originating from another tissue or part of the body.

The term "bone marrow" as used herein refers to soft gelatinous tissue filling the bone cavity. It consists of red bone marrow, including stem cells, progenitor cells, progenitor cells, and functional blood cells, and yellow bone marrow, which primarily stores fat. Thus, the red bone marrow is actually myeloid tissue that produces blood cells. Red bone marrow produces red blood cells, white blood cells and platelets. Children have more red bone marrow throughout the body, but those skilled in the art know that it is most concentrated in flat bones, such as the sternum, in adults. Leukocytes are also produced in the bone marrow and in some embodiments are involved in immune defense. In fact, bone marrow transplantation can treat some types of immune deficiencies. Bone marrow is also referred to as bone marrow. In certain embodiments, the bone marrow includes bone marrow aspirates (bone marrow drawn through a syringe from the bones of an individual) that those skilled in the art recognize as necessarily containing some peripheral blood.

 As used herein, the term "bone marrow aspirate" refers to a substance obtained from bone upon aspiration of the bone marrow, such as by a needle.

The term "nucleated cell / platelet fraction" as used herein refers to a solution derived from bone marrow, blood or mixtures thereof comprising one or more nucleated cells and / or platelets. Nucleated cell fractions include mesenchymal stem cells, nucleated connective tissue progenitor cells, nucleated hematopoietic cells or endothelial cells, or mixtures thereof. In certain embodiments, the fraction comprises leukocytes. In certain embodiments, the nucleated cell / platelet fractions are substantially free of plasma or red blood cells. In some embodiments, the final cell concentration will contain less than approximately 30% of the original plasma and RBC volumes. The cell fraction (which may contain some plasma and red blood cells) can be coagulated by mixing it with an appropriate coagulation initiator, for example after the filtration step and before application to bone defects. Ionic calcium solutions (eg, calcium chloride solutions) and / or thrombin can initiate coagulation to form clots.

As used herein, the term "centrifugation" refers to rotation within a compartment of the device, which compartment rotates about an axis for the purpose of separating the material.

As used herein, the term "concentrate" refers to a composition comprising a greater concentration of certain components, such as particulates or particulates, as compared to the parent source.

As used herein, the term "cross-flow filtration" or "cross-flow mode" refers to deviant flow filtration associated with recycling of the retentate through the surface of the membrane filter. In certain embodiments, this refers to the direction of feed of the product through the device perpendicular to the direction of flow through the membrane. In some embodiments, the term is defined as filtration comprising multiple passes over the membrane.

As used herein, the term “whole filtration” or “whole volume mode” refers to filtration in which the feed direction of the product and the flow direction through the membrane are parallel. In some embodiments, the term is defined as filtration comprising a single pass over a membrane. In other embodiments, the endpoint filtration substantially does not comprise deviant flow filtration.

As used herein, the term "filtration retentate" refers to a composition retained by a filter upon passage of a physiological solution through the filter. In certain embodiments, it comprises nucleated cells, platelets, or both. In certain embodiments, the filtration restoration comprises very small amounts of plasma and / or red blood cells. In certain embodiments, the volume of the filtration retentate is at least about 1 mL.

As used herein, the term “filtration” is used to porous a liquid comprising a particular substance for the purpose of separating the liquid from at least some specific substance, for the purpose of separating some specific substance from another specific substance, or both. It refers to the process of passing through a material.

As used herein, the term "hollow fiber filtration device" includes a hollow tubular outer cover, wherein inside the hollow tubular outer cover there are individual tubes whose walls are filters, the individual tubes being in a direction parallel to the length of the tubes. Is a filtration device. In certain embodiments, the direction of feed of the product through the device is perpendicular to the direction of flow through the membrane. In some embodiments, this includes extraordinary flow filtration that generally involves recycling of the retentate through the surface of the membrane filter. In alternative embodiments, there is no deviant flow filtration.

The term "mesenchymal stem cell" as used herein refers to pluripotent progenitor cells located in the bone marrow that can differentiate into various non-hematopoietic tissues, including early precursors of bone, cartilage, tendons, fat, muscle, and nerve cells. Refers to.

The term "microfiltration" as used herein refers to the separation of particles from a fluid, wherein the one or more microfiltration devices comprise one or more filters having a pore size of about 0.05 μm to about 5 μm. In certain embodiments of the invention, the cells and / or cell fragments are separated from at least some fluids and the microfiltration capacity of the filter allows the cells and / or cell fragments to be retained by allowing passage of fluid through the pores.

As used herein, the term "bone conduction" refers to the ability of a material, such as a skeletal material, to allow new bone growth.

As used herein, the term "bone forming agent" refers to a substance that stimulates the growth of new bone tissue.

As used herein, the term "osteoinductive" refers to the ability to form bone at iso (ie, not bone) body parts.

As used herein, the term "permeate solution" refers to particles and liquids that are smaller than the pore size of the filter and pass through the filter membrane.

As used herein, the term "plasma" refers to blood plasma, which is a pale yellow fluid component of whole blood, including water, proteins, electrolytes, sugars, lipids, metabolic waste, amino acids, hormones and / or vitamins.

As used herein, the term “purging solution” refers to a solution that facilitates the release of bone marrow or blood derived cells from a filter. In certain embodiments, the purging solution consists at least partially of an isotonic saline solution, or a colloidal solution such as albumin or dextran, or a mixture thereof. Those skilled in the art recognize that the purging solution may consist of water, electrolytes, proteins, carbohydrates and / or gelatin and the like.

As used herein, the term "skeletal material" refers to a substance that facilitates bone growth when administered to bone defects having osteogenic concentrates such as those derived from bone marrow. In certain embodiments, the backbone material is at least partially composed of synthetic materials, natural materials, or both. In a further particular embodiment, the skeletal material consists of a biocompatible material that facilitates, permits, or enhances the dropping, bone growth and / or bone growth of the new bone matrix. In further particular embodiments, there are ceramics, polymers, metals, allograft bones, autograft bones, demineralized bone matrices, mixtures thereof, and the like, such as calcium sulfate or calcium phosphate. In more particular embodiments, the backbone material may be blocks, pastes, cements, powders, granules, putty, gels, and the like. In certain embodiments, the skeletal material is osteoconductive, osteoinductive, osteogenic, or a combination thereof. In other embodiments, the skeletal material breaks down over time when placed in the body. In further particular embodiments, the backbone material is contemplated as a matrix, carrier, solution, solid, gel, and the like. In certain embodiments, the backbone matrix comprises a viscous hydrogel, such as, for example, carboxymethylcellulose-based hydrogels. In certain embodiments, the skeletal matrix comprises JAX® Advanced Bone Void Filler and / or JAX®-tcp (tri-calcium phosphate). In a further particular embodiment, the skeletal material comprises a bone graft substitute in the form of a plurality of JAX® toy jacks. The backbone material may be dust, powder, granules, chips, putty, tablets, mixtures thereof and the like.

As used herein, the term "second filter" refers to a filter that follows the first filter in a method comprising concentrating cells. In certain embodiments, one or more additional steps can be included between the first and second filters, and in alternative embodiments, the one or more additional steps can include the use of a filter. An additional step of concentrating the cells after the second filter can be used.

The term "serum" as used herein refers to an aqueous component of an animal fluid that remains after coagulation (ie, clot formation remains after removal of fibrinogen, prothrombin and other coagulation factors from blood plasma).

As used herein, the term "stem cell" refers to an undifferentiated cell that results in a specific differentiated cell.

II. The present invention

The present invention provides novel methods and compositions relating to the production and / or enrichment of cell concentrates, such as osteogenic cell concentrates, from physiological solutions such as bone marrow aspirate, blood, or mixtures thereof. The method uses a system in which the physiological solution is processed through one or more filtration steps and preferably without centrifugation. However, in alternative embodiments, centrifugation can be used.

Thus, in some embodiments, the process of the invention comprises only one filtration step. In other embodiments, the methods of the present invention comprise two or more filtration steps. In one embodiment of the present invention, a physiological solution is provided and applied to a filter to prepare a cell concentrate by providing a filtration preservation and a permeate solution, wherein the filtration rest is platelet, nucleated per unit volume more than in a physiological solution. Cells, or both, are provided, wherein the permeate solution comprises plasma and red blood cells. After the filtration step, the filtration complement may substantially comprise most of the platelets and nucleated cells from the original physiological solution, and the permeate solution may substantially comprise most of the plasma and red blood cells from the original physiological solution. As for the filtration complement, there is an increase in the concentration of platelets and / or nucleated cells of about 4 times, 5 times, 6 times, 7 times or more relative to the physiological solution from which platelets and / or nucleated cells are filtered therefrom. In other words, about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% of platelets and / or nucleated cells are physiological solution using the method of the present invention. Filtered from. Regarding the permeate solution, about 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% of plasma and erythrocytes are filtered out of the physiological solution using the method of the present invention, which is filtered Post-permeate solution.

In one embodiment of the invention, a single filtration step of the physiological solution concentrates nucleated cells, including white blood cells and platelets, while simultaneously removing plasma and red blood cells from the physiological solution. The concentrate resulting from this single step is applied directly to the bone defect and further processed using a non-filtration step (eg, adding a biological agent), combined with the skeletal material and then combined with the bone defect. Or may be further applied to one or more filtration steps, such as concentrating the cells and / or exchanging solutions containing the cells.

Those skilled in the art recognize that the use of the present invention, based on the disclosure provided herein, obtains a concentrated composition of platelets and nucleated cells, including leukocytes, from physiological solutions. This is in contrast to many methods known in the art, for example, to remove leukocytes from the blood as desired. Filtration preservation comprising leukocytes is the preferred composition as opposed to the composition to be discarded. In other words, the present invention, as opposed to some of the waste, removes leukocytes from physiological solutions such as blood for the purpose of using them. This relates to certain embodiments of the present invention which, when administered to a bone defect, provide cells which release growth factors such as leukocytes and platelets resulting in cell migration to the wound, cell proliferation and differentiation into bone cells. Known blood processing methods of discarding leukocytes are intended to reduce or suppress an immune response when the ultimate intention of the composition is for systemic administration to an individual such as requiring blood transfusion. In contrast, the present invention relates to topical administration to wounds as opposed to systemic circulation, for example autotransfusion of cell concentrates in bone defects.

In another embodiment, the present invention is directed to a method for the treatment of nucleated cells (mesenchymal stem cells, connective tissue progenitor cells, cartilage) without first needing to centrifuge two conventional blood processing techniques (leukocyte reduction filtration and hollow fiber filtration). Precursor cells, and / or bone precursor cells) and one method for increasing platelet concentration. In addition, when the concentrated physiological solution is combined with a skeletal material such as a bone conducting material, the present invention produces a complex BGS that promotes the formation of new bone as compared to the skeletal or cell concentrate alone. In a further particular embodiment, the cell concentrate is mixed with a compound such as a powder or a liquid to form an injectable putty which may optionally include one or more coagulation initiators. Examples of coagulation initiators are solutions containing calcium ions (eg calcium chloride solution) or thrombin or both. In a further particular embodiment, the biological agent is combined with a cell concentrate, skeletal material (in or on the material or both), or a mixture thereof, prior to or upon administration to a bone defect.

Although there are methods in the art using centrifugation to concentrate stem cells for use as osteogenic bone marrow preparations, the present invention provides bone marrow concentrate using filtration to concentrate both nucleated cells (including stem cells) and platelets. Create This filtration arbitrarily eliminates the costs associated with purchasing and maintaining centrifuges in the operating room and is also a less laborious bone marrow processing method than the centrifugation methods used by others. The present invention also provides a method for concentrating bone forming (mesenchymal stem cells) and osteoinductive (platelet) elements of the bone marrow, while known methods relate to the enrichment of one of the two, It's not about.

In contrast, the invention described herein isolates and concentrates stem cells and platelets using filters rather than implantable backbones. The present invention provides a method for enriching both bone forming (mesenchymal stem cells) and osteoinductive (platelet) elements of the bone marrow, for example, Muschler (Muschler et al., 2002; US Pat. No. 5,824,084). And 6,049,026) describe methods for concentrating only mesenchymal stem cells from bone marrow. When combined with a bone conducting skeleton, the concentrated bone marrow suspension of both stem cells and platelets will heal bone better than bone marrow concentrates containing only stem cells. The present invention also results in a concentrated cell suspension that can be delivered to bone defects by syringe injection through a needle. This will allow minimally invasive delivery of cell concentrates to bone defects. The invention described by Moussler requires the use of a non-injectable bone conducting skeleton that prevents delivery of the cell concentrate / skeleton to bone defects via injection. In other embodiments, minimally invasive delivery, such as through syringe injection, is used.

Thus, the novel formulations provided herein, cell concentrates, are used to accelerate bone healing in various bone graft applications. Also provided are kits.

In one step of the invention, a physiological solution is obtained from an individual to be delivered subsequently, for example, after concentration, nucleated cells comprising osteogenic cells such as mesenchymal stem cells and leukocytes, and illustratively PDGF, Secrete osteoinductive growth factors such as TGF-β, insulin-like growth factor-I (IGF-I), insulin-like growth factor-II (IGF-II) and vascular endothelial growth factor (VEGF) or mixtures thereof One or more filtration steps may be the first filtration step in a method comprising one or more filtration steps for selectively recovering or isolating platelets. Certain non-osteogenic components, such as erythrocytes and plasma, which make up the majority of the sample volume, are not substantially recovered during the alone (or first) filtration step.

In one embodiment of the present invention, the blood or bone marrow fraction to be concentrated generally comprises an anticoagulant which is provided upon interruption using an exemplary citrate-based anticoagulant. For example, any citrate-based anticoagulant is suitable. For example, standard donor blood collection bags contain citrate-based anticoagulants. In certain embodiments, the anticoagulant heparin is added, for example when bone marrow aspirate is used.

In certain embodiments, physiological solutions, such as blood or bone marrow, include additional components such as dilutions and / or another solution that acts as a reservoir of reservoir. In embodiments where the additional component is a hypotonic solution, when it is introduced into the physiological solution, the salt concentration outside the cell consisting of the physiological solution is thereby lower than the salt concentration inside the cell. Thus, osmotic gradients are produced when water flows into the cells, and in certain embodiments they swell to better adhere them to the filter. The solution, which may be referred to as solution A in the examples, may be, for example, water or a hypotonic sodium chloride solution that is less concentrated than isotonic (normal), for example about 0.9%.

Depending on the placement of one or more filters, either alone or the first filter (leukocyte reduction filter in some embodiments) takes advantage of the tendency for certain types of cells (eg, mesenchymal stem cells) to adhere to the external substrate. When passing through the filter, nucleated cells and platelets, such as stem cells, adhere to the fiber surface in the filter, while red blood cells and plasma are not adsorbed and consequently pass through the filter. Thereafter, a purging solution, also called a recovery solution, is flushed through the filter to remove the trapped cells, thereby recovering the selected osteogenic / osteoinductive cells. The cells are then analyzed and / or applied to the wound site (eg bone defect), optionally combined with skeletal material, and then applied to the wound site or further processed. In some embodiments, the recovery solution can be hypertonic or isotonic, so they are equal to or greater than normal saline for sodium chloride concentration.

In other embodiments, the recovered osteogenic / osteoinduced cells are then subjected to a second filtration step where the concentration of platelets is increased. The filtration device of this step may be of any kind so long as it concentrates nucleated cells, platelets, or both. In a preferred embodiment, said step comprises applying the solution obtained from the first step to a hollow filtration device as known in the art. The device preferably separates cells from a portion of the suspended liquid, thereby increasing cell concentration. One skilled in the art recognizes that the method is beneficial if a larger amount of therapeutic substance can be delivered in a significantly smaller volume. In many embodiments, without this concentration of the product, it would be forbidden to apply this larger volume to the defect. The step can be performed multiple times.

In a preferred embodiment, the second filtration device comprises a tubular filter as opposed to the disk filter. In a further particular embodiment, the tubular filter is received in an outer tube comprising a hollow tube in it, and in a preferred embodiment, the flow through the tubular filtration device is parallel to the flow through the tube in the tubular filtration device. In certain embodiments, the pore diameter on the wall of the tube is from about 0.05 μm to about 5 μm, more preferably from about 0.1 μm to about 1 μm, most preferably from about 0.2 μm to about 0.5 μm.

In some embodiments of the invention, the osteogenic cell concentrate is combined with the skeletal material and delivered to the bone defect. Any method of delivery is suitable so long as the integrity of the composition is maintained and the therapy for bone defects is provided. The method of delivery may be through a syringe, scoop, spatula, scoopula, rod, tube, or the like. In other embodiments, a viscous fixative material is added to the bone forming concentrate or bone forming concentrate / skeletal material composition to facilitate retention of the concentrate or concentrate / skeleton mixture at the bone defect site. Viscous fixative materials may include coagulation factors (eg, calcium, thrombin or mixtures thereof), bone marrow aspirates, blood, platelet rich plasma, fibrinogen / thrombin, cement, slurries, pastes, combinations thereof, and the like. In certain embodiments, cements, slurries or pastes, such as those comprising one or more calcium salts, including calcium sulfate and / or calcium phosphate, are used in the present invention as for viscous fixative materials.

Any surface of the filtration device used in the present invention in contact with the physiological solution and / or the resulting concentrate is preferably inert to the components and preferably not substantially cytotoxic. In a preferred embodiment where it is desired to include cells such as, for example, nucleated cells and / or platelet concentrates, the contact surface is substantially noncytotoxic. Suitable materials include polycarbonates, polyurethanes, acrylics, ABS polymers, polysulfones, polyethersulfones, mixed cellulose esters, polyesters and the like.

In embodiments where the sterility of the osteogenic concentrate or other composition should be maintained, any filtration surface that comes into contact with any relevant composition and / or concentrate, such as in the preparation of a concentrate for bone defect therapy, may be sterilized. It is preferred that it is or that it can be easily sterilized. Commercially available filtration devices can be sterilized by performing agent treatment, for example gamma irradiation, ethylene oxide treatment, formalin treatment, hydrogen peroxide treatment, sodium hypochlorite treatment, heat treatment, steam treatment and the like. Sterile filtration devices for hematological applications are commercially available. In general, syringes and other fluid delivery systems are commercially available in sterile form, as are various valves and lockcocks and other blood processing products designed to be attached to the syringe.

In an alternative embodiment of the invention, the bone marrow concentrates are produced by the methods described herein. The bone marrow concentrate is then used for purposes other than bone defects, such as for bone marrow transplantation or for augmentation of bone marrow transplantation.

Alternatively, cell concentrates comprising platelets and nucleated cells that are not plasma or red blood cells or consisting essentially of platelets and nucleated cells are not prepared at the time of treatment, but are, for example, commercially prepared compositions or individuals prior to the time of treatment. After administration from a bone defect in the same or another individual.

In a particular embodiment of the invention, cell concentrates are prepared according to the methods described herein.

III. Specific embodiment

Discussed above relates to general embodiments of the methods described herein. The following paragraphs provide specific embodiments for this procedure, but one skilled in the art can make modifications to the following steps to optimize the product concentration method. For example, these specific embodiments may relate to the processing of whole blood, and those skilled in the art will recognize variations that are useful for optimizing this method.

In a general embodiment of the present invention, a single step filtration process concentrates these cells by isolating desired cells exemplified by nucleated cells, platelets, and the like from starting solutions such as bone marrow, whole blood or mixtures thereof and the like. This filtration step removes plasma and red blood cells from the desired cells. The resulting filtrate comprising the desired cells is optionally applied to the bone defect as a composition that also includes the skeletal material. A schematic diagram illustrating this embodiment is shown in FIG. 1.

In one particular embodiment the following steps are used in the method:

Step 1-Obtain Bone Marrow: Appropriate amount of bone marrow (approximately 5 cc or more, eg for bone graft use), for example equal or less volume of isotonic saline and a suitable anticoagulant (eg citrate-based Or heparin-based). In some embodiments, the bone marrow is obtained by another method, such as purchasing a commercial item. In another embodiment, whole blood is the primary source of said concentrate, for example with a suitable volume of anticoagulant (eg, citrate-based or heparin-based) before applying it to the next stage of nucleated cell-reducing filter. Process it by mixing.

Step 2-Bone marrow aspirate is filtered through a nucleated cell (eg, leukocyte) reduction filter to selectively recover nucleated cells such as mesenchymal stem cells and platelets, etc. (FIG. 1): Bone marrow aspirate is collected in small collection bags ( 10) inject into a housing device such as the like. A series of leukocyte reduction filters 12 is placed between the collection bag 10 and the second housing device, for example drain bag 14. Bone marrow aspirate is fed by gravity to the drain bag 14 through the nucleated cell reduction filter 12. Nucleated cell / platelet fractions (containing mesenchymal stem cells and platelets) are captured in filter 12 and the remaining blood components pass through. Subsequently, the syringe 18 is used to rinse the filter again with a suitable volume (> about 5 mL) of purging solution (eg, dextran 40 and albumin solution) and recover the smoke cells with a second sterile syringe 16. do.

Step 3-Cells suspended in the purging solution are filtered through a hollow fiber filter to increase the concentration of nucleated cells and platelets: The operation of the hollow fiber filtration device is schematically shown in FIG. A syringe containing a purging solution containing cells (labeled syringe A 20) is placed at one end of the filtration device 22, for example via Luer lock 23 at the first port 21. Attach. A second syringe (labeled syringe B 24) is attached via the second port 25 to the opposite end to collect the retentate (cells that do not pass the filter 30). A third syringe (labeled syringe C 26) is connected and attached to a third port 27 to receive the filtrate (liquid component, i.e. purging solution through the filter). The sample is passed back and forth between two opposing holding syringes (syringes A 20 and B 24) to circulate the preservation and to separate the cellular and liquid components. The liquid component passes through the semipermeable filter membrane 30 into the syringe C 26, but the cells are unable to pass through the semipermeable filter membrane 30 and are collected in the fibers 32 of the filter. In certain embodiments, the cell concentrate is discharged into a sterile syringe in filter 30 by performing the following steps, although other means may be used: (i) Syringe C 26 is removed from filtrate port 27 Close the lid of (27), (ii) withdraw the empty holding syringe (syringe A (20) or syringe B (24)) from the holding port (21 or 25, respectively), and (iii) thereby become available Connect a syringe containing a purging solution (preferably isotonic saline) to the holding port (21 or 25, respectively), and (iv) filter the purging solution in a volume approximately equal to the priming volume of the filter 30 (30) is injected to drain the cell concentrate into a second retaining syringe (syringe A 20 or syringe B 24, respectively).

Figure 3 illustrates a general embodiment where this filtration step fractionates the nucleated cell / platelet fractions and the purging solution such that the volume of the solution in which the cells are suspended is reduced. Again, the container 16 containing the smoke and purging solution from the first filtration step (which in some embodiments may be Syringe A 20 or Syringe B 24 as described for FIG. 2) is the solution. Is passed to the hollow fiber filter 22, and this solution is filtered through it. Eventually, the purging solution will remain in the container 27 illustrated by the syringe, and the desired cell concentrate will be delivered to the container 29 illustrated by the syringe and later delivered to the defect. Thus, the net effect is an increase in the number of cells per unit volume of fluid.

Step 4-Mix Cell Concentrate with Skeletal Material: The concentrated cell suspension is injected into a container containing the skeletal material, such as granular material and the like. In some embodiments various framework materials are used. The concentrate is flowed into individual skeletal granules and / or into individual skeletal granules free space. If the free volume available is greater than the volume of the cell concentrate, the carrier material (where the carrier material acts as a handling agent, increases the differentiation of mesenchymal cells into the osteoblast lineage, or these Both) can be mixed with the cell concentrate to achieve an equivalent volume to fill the intragranular and / or intergranular free space of the skeletal material. Examples of carrier materials include bio-fluids such as coagulated blood, bone marrow, platelet-rich plasma, or synthetic materials such as hydrogels, powders or granules. The cell concentrate may be combined with the carrier material by syringe mixing or by mixing in a container using a rod, spatula or other suitable instrument. The mixture is then injected into a container containing granular framework material and flow into the intragranular and / or intergranular space. The cell concentrate / skeleton mixture may be delivered to exposed defects or may be delivered transdermally via a syringe.

Whole blood (not bone marrow aspirate) can be processed in a similar manner as described in the above embodiments. The resulting concentrate will be rich in platelets (not mesenchymal stem cells), which can be mixed with the skeletal material as described in the above embodiments.

The combination of bone marrow and blood may be processed as described in the first embodiment. The resulting concentrate will be rich in both platelets and mesenchymal stem cells, which can be mixed with the skeletal material as described in the first embodiment.

In certain embodiments, after delivering the cell concentrate / skeleton material mixture to a bone defect, the defect and / or bone or bone tissue contained therein is tested. For example, bone can be tested for bone quantity and / or quality in bone defects and the like. The test may include assaying bone density, strength, bone formation rate, bone quality, and the like. Any suitable test may be used, and in some embodiments histological, radiographic and / or mechanical tests are used. Percent bone filling at bone defects after delivery of the cell concentrate / skeletal material mixture can be assayed, and in some embodiments this value is compared to the value of the surrounding bone. In some embodiments, increased deposition of calcium is tested after administration of the concentrate or concentrate / skeleton mixture.

In a particular embodiment, the bone marrow aspirate to be processed is obtained according to the methods described herein. Specific bone marrow aspiration techniques are as follows, but bone marrow aspirates may be obtained by other means: After inducing anesthesia and sterilizing the skin preparation, a small incision (less than about 1 cm) is made along the posterior iliac ridge. This incision pushes the bone marrow aspiration needle into the medullary lumen of the iliac ridge. Small amounts of bone marrow samples (less than about 4 mL) should be aspirated with a 10-mL syringe containing heparinized saline (about 1000 units / mL). After bone marrow collection, the piston of the syringe is inverted up and down several times to ensure proper mixing with the anticoagulant. In the same technique, additional aspiration can be achieved through separate cortical perforations located at least about 1 cm apart.

In a particular embodiment, the blood to be processed is obtained according to the methods described herein. Specific blood suction steps are as follows, but blood may also be obtained by other means: After sterilizing the skin preparation, a suitable large peripheral vein, typically anterior venous vein or head, using a small needle (18-21 gauge) infusion set is used. Aspirate the vein's blood. Blood is aspirated at a ratio of approximately 10: 1 (blood: anticoagulant) to a 60 cc syringe containing citrate anticoagulant. After collecting the blood, the piston of the syringe was pressed and pulled several times to ensure fusion with the anticoagulant.

IV. device

The device of the invention or the device used in the method of the invention may comprise one or more filtration components. One or more filters may be used in the instrument or part thereof. The filter may be designed to use "cross flow filtration" or "cross flow mode" in which the retentate is recycled across the membrane filter surface. In an alternative embodiment, the filter is designed to use "whole filtration" or "whole mode" that is substantially free of flow filtration elsewhere.

Microfiltration can be used in the present invention to separate particles from a fluid. The at least one microfiltration device includes at least one filter having a pore size of about 0.05 μm to about 5 μm. In certain embodiments of the invention wherein the microfiltration performance of the filter allows fluid to pass through the pores so that cells and / or cell fragments remain, the cells and / or cell fragments are at least somewhat separated from the fluid.

The filtration device may be of any type as long as it concentrates nucleated cells, platelets or both. In a preferred embodiment, the device preferably separates the cells from the portion of the liquid in which they are suspended so that the cell concentration is increased.

In a preferred embodiment, the filtration device may comprise a tubular filter of the opposite concept as the disk filter. In a further particular embodiment, the tubular filter is received in an outer tube in which the hollow tube is contained, and in a preferred embodiment the flow through the tubular filtration device is parallel with the flow through the tube in the tubular filtration device. In certain embodiments, the pore size on the tube wall is about 0.05 μm to about 5 μm, more preferably about 0.1 μm to about 1 μm, most preferably about 0.2 μm to 0.5 μm.

In some embodiments of the present invention a hollow fiber filtration device is used which can be purchased commercially. In a particular embodiment, the device comprises a hollow tubular outer cover containing individual tubes whose walls are filters, which individual tubes are arranged in a direction parallel to the tube length. In certain embodiments, the direction of feed of the product through the device is perpendicular to the direction of flow across the membrane. In some embodiments, this generally includes the recirculation of the preservatives by flow filtration across the surface of the membrane filter and elsewhere. In alternative embodiments there is no flow filtration elsewhere.

One skilled in the art knows that any filtration surface that comes into contact with any relevant composition and / or concentrate is sterile or can be easily sterilized. Commercial filtration devices can be sterilized by performing agent treatment, for example gamma irradiation, ethylene oxide treatment, formalin treatment, hydrogen peroxide treatment, sodium hypochlorite treatment and the like. Sterile filtration devices for hematological applications are commercially available. In general, syringes and other fluid delivery systems are commercially available in sterile form, as are various valves and lockcocks and other blood processing products designed to be attached to the syringe.

Disposable may be considered as a device used in the method described herein, but may be reused after sterilizing them by gamma-ray sterilization or the like. Filters and syringes may be purchased commercially. In certain embodiments, the device is plastic. In a further particular embodiment, the size of the leukocyte reduction filter is approximately 12 cm in diameter, 25 mm thick and / or the size of the hollow fiber filter is approximately 120 mm long × 20 mm.

V. Addition of Biological Agents to the System

In a preferred embodiment of the present invention, a biological agent is included in a substance (cell concentrate, skeletal substance or both) delivered to a bone defect. Examples thereof include antibiotics, growth factors, fibrin, bone morphogenic factors, bone growth agents, chemotherapeutic agents, pain relief agents, bisphosphonates, strontium salts, fluoride salts, magnesium salts and sodium salts.

Unlike oral administration of high doses of antibiotics to an organism, the present invention allows topical administration of antibiotics to be included in the materials of the composition. This reduces the amount of antibiotics necessary for the treatment or prevention of infection. Administering antibiotics with a substance in the composition also results in less diffusion of the antibiotic, especially when the antibiotic is contained in the fibrin matrix. Alternatively, the skeletal material of the present invention may be coated with antibiotics and / or contained in the skeletal material or concentrate or combination thereof. Examples of antibiotics include tetracycline hydrochloride, vancomycin, cephalosporin, and aminoglycosides such as tobramycin and gentamycin, and quinolone antibiotics such as ciprofloxacin.

Growth factors may be included in the topical administration to aid bone growth. Examples of growth factors that may be included include platelet derived growth factor (PDGF), transgenic growth factor β (TGF-β), insulin-like growth factor-I (IGF-I), insulin-like growth factor-11 (IGF- 11), fibroblast growth factor (FGF), beta-2-microglobulin (BDGF II), bone morphogenic protein (BMP), growth and differentiation factor-5 (GDF-5), vascular endothelial growth factor (VEGF) or Mixtures thereof and the like. The skeletal material of the present invention may be coated with growth factors and / or contained in the skeletal material or concentrate or combinations thereof.

Proteins or agents that are attached to and / or bind to growth factors can be used in the present invention. Examples of growth factor binding / accessory proteins include follistatin, osteonectin, sog, cordin, dan, cyr61, thrombospondine, type IIa collagen, endoglin, cp12, nell, cream, acid-1 glycoprotein and alpha -2HS glycoproteins.

In some embodiments, the compositions of the present invention comprise cells such as osteoblasts, endothelial cells, fibroblasts, adipocytes, myoblasts, mesenchymal stem cells, chondrocytes, pluripotent stem cells, pluripotent stem cells and pluripotent stems. Cells or musculoskeletal precursor cells.

Bone morphogenesis factors include proteins from demineralized bone or DBM (demineralized bone matrix), in particular BP (bone protein) or BMP (bone morphology) which actually contains a plurality of components such as osteonectin, osteocalcin and osteogenin, etc. Growth factors that have specific activity in bone tissue, including proteins called forming proteins). The factor may coat the backbone material of the present invention and / or be contained in the backbone material, concentrate or combinations thereof.

Angiogenic factors may be included on the skeletal material or may be included in the skeletal material or concentrate or combinations thereof. Some examples of angiogenic factors include monobutyrin, dibutyrin, tributyrin, butyric acid, vascular endothelial growth factor (VEGF), erumid, thymosin beta 4 (TB4), synthetic peptide analogues for heparin binding proteins, Nicotine, nicotinamide, spermine, angiogenic lipids, ascorbic acid and derivatives thereof and thrombin and analogs and peptide fragments thereof.

In certain embodiments, bone growth agents may be included in the framework materials, concentrates or both in the compositions of the present invention. For example, a nucleic acid sequence encoding an amino acid sequence that facilitates bone growth or bone healing or the amino acid sequence itself may be included in the concentrate and / or framework material of the present invention. For example, leptin is known to inhibit bone formation (Ducy et al., 2000). Leptin, leptin ortholog, or any nucleic acid or amino acid sequence that negatively affects the leptin receptor can be included in the composition. As a specific example, since the antisense leptin nucleic acid in the composition of the present invention can be moved to a bone defect site to inhibit leptin amino acid formation, any inhibitory effect that leptin may have on bone regeneration or bone growth can be avoided. Another example is leptin antagonists or leptin receptor antagonists.

Nucleic acid sequences can be delivered in a nucleic acid vector in which the vector is contained within a delivery vehicle. Examples of such delivery vehicles are liposomes, lipids or cells. In certain embodiments, the nucleic acid is transferred to a carrier-assisted lipofection method [Subramanian et al., 1999] for ease of delivery. In this method, the cationic peptide is attached to the M9 amino acid sequence, which binds to the negatively charged nucleic acid. M9 then binds to a nuclear transport protein such as transportin, and the entire DNA / protein complex can cross the cell membrane.

The amino acid sequence can be delivered in a delivery vehicle. An example of such a delivery vehicle is liposomes. Delivery of amino acid sequences can utilize protein transduction domains, such as the HIV virus TAT protein [Schwarze et al., 1999].

In a preferred embodiment, the biological agent of the invention has a high affinity for the fibrin matrix.

In certain embodiments, the particles of the present invention may contain a biological agent in or on the agent, which agent elutes from the particle as the particle degrades or through diffusion.

The biological agent may be a pain reliever. Examples of such pain relievers include lidocaine hydrochloride, bipivacaine hydrochloride, and non-steroidal anti-inflammatory drugs such as ketorolac trometamine.

Other biological agents that may be included in the suspension material or contained on or in concentrates and / or backbone materials of the invention are chemotherapeutic agents, such as cis-platinum, ifosfamide, methotrexate and doxorubicin Hydrochloride. It is known to those skilled in the art that chemotherapeutic agents are suitable for bone malignancies.

Another biological agent that may be included in the concentrate and / or framework material is bisphosphonate. Examples of bisphosphonates include alendronate, clathronate, ethidronate, ibandronate, (3-amino-1-hydroxypropylidene) -1,1-bisphosphonate (APD), zoleronate, dichloromethylene bisphosphonate, Aminobisphosphonatezolendronate and pamidronate.

The biological agent may be in purified form, partially purified form or commercially available, and in preferred embodiments is recombinant in form. This agent is preferably free of impurities or contaminants.

VI. Combination therapy

Those skilled in the art will appreciate that in some embodiments the invention provided herein is combined with another therapy of the individual. For example, in embodiments in which bone defects are treated with the methods of the invention and / or the compositions of the invention, the patient may also be prescribed standard bone therapy treatment. Standard bone therapy treatments for bone defect repair include surgery, additional bone grafts, antibiotic administration, implant use, external stimuli such as low intensity ultrasound or electromagnetic pulses.

VII. Pharmaceutical Compositions and Routes of Administration

The composition of the present invention will contain a therapeutically effective amount of a osteogenic substance. In some embodiments, the composition is used in combination with an effective amount of a second compound (second agent) which is a bone therapeutic agent as exemplified above. Such compositions are generally dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. As used herein, the term "effective" refers to providing bone growth properties, preventing bone breakdown, strengthening bones, preventing fractures, healing fractures, and the like.

The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular substances and compositions that do not result in side reactions, allergic reactions or other inappropriate reactions when administered to an animal or, if appropriate, a human. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. It is known in the art to use such media and agents for pharmaceutically active substances. Any conventional medium or agent is contemplated for use in the therapeutic composition except where they are incompatible with the active ingredient. Supplementary active ingredients, such as other bone therapies, may also be incorporated into the compositions.

The composition of the present invention is advantageously administered in a form suitable for administration of a bone defect. The composition form may be an injectable composition as a liquid solution or suspension, but may also be prepared in liquid prior to injection using solid forms suitable for the preparation of solutions or suspensions. In other embodiments, the composition is applied in a non-injectable manner. Examples thereof may be by applying the cell concentrate alone or using a device such as a scoop, scoopula, spatula, rod, spoon, syringe, pipette, forceps, metering spoon, or any medically approved delivery vehicle, etc. Mixed with and applied.

In a particular embodiment of the invention, the solution comprising the desired cell concentrate comprises phosphate buffer, dextrose, salt, dextran 40, dextran 70 or mixtures or combinations thereof. Other pharmaceutically acceptable carriers include aqueous solutions, nontoxic excipients such as salts, preservatives, buffers and the like. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters such as tailoleate and the like. Aqueous carriers include water, alcohols / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, and the like. Preservatives include antimicrobials, antioxidants, chelating agents and inert gases, but in certain embodiments the concentrate is used immediately after concentration at the time of treatment. The pH and exact concentration of the various ingredients in the medicament are adjusted according to known parameters.

All essential materials and reagents required for bone therapy can be put together in a kit. The components of the kit are provided in at least one liquid solution, which liquid solution is preferably an aqueous solution, particularly preferably a sterile aqueous solution. The kit may contain a device and / or platform for combining physiological solution collection devices, one or more filters and / or filter devices, framework materials, cell concentrates and framework materials and / or combinations thereof. .

Typically, the kits of the present invention will also include means for containing vials tied together for commercial use, such as injection containers or blow molded plastic containers with the desired vials. Regardless of the number or type of containers, the kits of the present invention may also include or be packaged with devices for assisting injection / administration or for placing the final composite composition into the animal body. Such an instrument may be a scope, scoopula, spatula, rod, spoon, syringe, pipette, forceps, metering spoon, or any medically approved delivery vehicle.

The concentrate or concentrate / skeletal material may also be included with a solvent or dispersion medium comprising, for example, blood, plasma, water, sugars, salts or suitable mixtures thereof. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like.

VIII. Kit

In some embodiments of the invention, kits are provided, for example, to perform the methods described herein and / or to prepare the compositions described herein. In particular embodiments, these kits are used for the treatment of bone defects. In some particular embodiments, the present invention provides a kit for preparing cell concentrate, comprising a first filtration device housed in a suitable container and a second filtration device housed in a suitable container. The first filtration device may be a nucleated cell filtration device and / or the second filtration device is a microfiltration device, for example a hollow fiber filtration device.

The kit may further comprise a framework material contained in a suitable container. The kit may further comprise a biological agent contained in a suitable container. Examples of biological agents include growth factors, antibiotics, strontium salts, fluoride salts, magnesium salts, sodium salts, bone morphogenic factors, angiogenesis factors, chemotherapeutic agents, pain relief agents, bisphosphonates, growth factor binding / accessory proteins, cells, Bone growth agents or mixtures thereof. In certain embodiments, the growth factor is platelet derived growth factor (PDGF), transgenic growth factor β (TGF-β), insulin-related growth factor-I (IGF-I), insulin-related growth factor-II (IGF- II), fibroblast growth factor (FGF), beta-2-microglobulin (BDGF II), nerve growth factor (NGF), epidermal growth factor (EGF), keratinocyte growth factor (KGF), bone morphogenic protein (BMP) ) And mixtures thereof. In a further particular embodiment, the antibiotic is selected from the group consisting of tetracycline hydrochloride, vancomycin, cephalosporin, quinolone, aminoglycosides and mixtures thereof. In certain embodiments, the quinolone is ciprofloxacin. In certain embodiments, the aminoglycoside is tobramycin or gentamycin.

In another embodiment, the present invention provides a kit for treating bone defects comprising a plurality of cells contained in a suitable container, wherein the cells are nucleated cells, platelets or both. Such kits may comprise a backbone material contained in a suitable container and / or a biological agent contained in a suitable container.

The kit may comprise an instrument for preparing the cell concentrate obtainable by the method (s) described herein. Kits of the present invention may include at least any device suitable for carrying out any of the methods described herein.

Kits provided herein can be considered as disposable kits for single use.

The following examples are included to demonstrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques which have been found to be useful for the practice of the invention by the inventors, and thus can be regarded as constituting a preferred manner for the practice of the invention. . However, one of ordinary skill in the art appreciates that various specific embodiments disclosed in the context of the present disclosure can be varied without departing from the spirit and scope of the present invention, and that such changes result in the same or similar results.

Example 1

Double filtration of physiological solutions

Although some embodiments may use blood or a mixture of blood and bone marrow, in this example an in vitro study to determine the efficacy of a filter device that concentrates nucleated cells (eg, mesenchymal stem cells) and platelets in bone marrow aspirate. Was performed. Volumes of exemplary bone marrow aspirates, such as 25 mL, were obtained from animal donor (s) (eg sheep). Each sample was divided into two such that half of the sample was not processed and the other half was processed by the filtration device (s) described herein. The volume (V0) of the untreated sample and the volume (V1) of the sample after being treated were measured. For each of the untreated and treated samples, nucleated cell numbers, mesenchymal stem cell numbers and platelet counts were determined using hemocytometer and fluid cytometry. In addition, to quantify the concentration of mesenchymal stem cells that differentiate into osteoblast lineage, cells and cell concentrates obtained from untreated bone marrow are cultured in osteogenic medium and osteoblast markers such as, for example, alkaline phosphatase Analyzed using. Nucleated cell number, platelet count and alkaline phosphatase activity were normalized to sample volume (V0 or V1) to provide a measure of nucleated cells, mesenchymal stem cells and platelet concentrations. Bone marrow aspirate will retain significant amounts of mesenchymal stem cells and platelets relative to untreated bone marrow (preferably greater than about 60%) and significantly reduce volume (preferably greater than about 80%) . Filtered bone marrow aspirate will increase cell concentration approximately 3 times or more compared to untreated bone marrow aspirate.

To demonstrate the efficacy of a filter device to concentrate bone marrow in vitro, subcutaneous injection studies were performed to determine the acceleration of bone healing by concentrated bone marrow suspensions in animal models (eg sheep). Autologous bone marrow aspirates were processed through a filter instrument to increase the concentration of mesenchymal stem cells and platelets. The concentrated bone marrow suspension was combined with bone conducting skeletal material, and if necessary carrier material, and injected subcutaneously into a risky sized defect (eg, a defect that does not spontaneously heal if empty). Concentrated bone marrow aspirate + skeletal group was compared for untreated bone marrow aspirate + skeletal group for bone healing. Bone healing was measured mechanically and / or histologically by roentgen imaging, by standard means in the art. The concentrated bone marrow aspirate group preferably exhibited an excellent bone healing effect compared to the untreated bone marrow aspirate group.

Example 2

Description of Example Filter Configurations

Exemplary filter configurations in the present invention may be as follows:

1. Leukofilter alone (see FIG. 4);

2. Leucco filter + hollow fiber filter (cross-flow mode) (see FIGS. 4, 5, 6);

3. leuco filter + hollow fiber filter (whole volume method) (see Figs. 4, 7, 8); And / or

4. Use of any of the three configurations and a fat reduction filter (FIG. 9).

4 shows the operation of a single (or, in some embodiments, first) filter (eg, leukocyte reduction filter) to selectively recover osteogenic cells (ie, platelets and nucleated cells) from physiological solution. Provide a schematic. In the filling step, a physiological solution containing both osteogenic and non-osteoblastic cells, optionally comprising an anticoagulant, was injected into the collection bag 10. In the filtration step, the physiological solution was passed from the collection bag 10 through the leukocyte reduction filter 12, such as by gravity feeding. After passing through a filter holding cells (eg, nucleated cells), platelets, or mixtures thereof, the remaining physiological solution and its components (eg, red blood cells) were flowed into drain bag 14. In the back-flush step, the valves 11 and 13 of the collection bag 10 and the drain bag 14 were closed, respectively. Valves 15 and 17 of the syringes 9 and 19 were opened, respectively. In the recovery step, osteogenic cells were back-flushed from the filter 12 when the recovery solution flowed from the syringe 19 to the valve 17 to force the osteogenic cells into the syringe 9. Collections in the syringe 9 include recovered cells, which in some embodiments are not further processed, but are optionally applied to bone damage after combining the cells with the skeletal material. In other embodiments, the cells of the syringe 9 are referred to as the feed used for later steps in this process, which syringe may be referred to as the feed syringe 9.

FIG. 5 shows a schematic diagram showing the operation of a second filter, such as a hollow fiber filter, for example to concentrate osteogenic cells (ie, platelets and nucleated cells) recovered from the first filter 12 of FIG. 4. Doing. A syringe containing osteogenic cells, such as the syringe 9 illustrated in FIG. 4, was connected to the hollow fiber filter 30 and operated in a cross-flow manner. In cross-flow filtration, the feed stream is recycled to the membrane of the hollow fiber filter 30 tangentially, for example, between the feed syringe 9 and the retentate syringe 36, thereby generating a differential pressure across the membrane. I was. This causes some particles to pass through the membrane. The remaining particles continue to flow through the membrane. Using an appropriate membrane pore size (eg, about 0.2-0.5 μm), the recovery solution can pass through the filter membrane of the hollow fiber filter 30, but cells cannot pass through this membrane. 6 shows a schematic showing the osteogenic cell concentrate suspension resulting from the operation of the second filter. Cross-flow filtration fractionated a portion of the recovery solution from the suspended cells, thereby increasing the cell concentration in the preserve syringe 36.

7 provides a schematic showing the operation of a second filter (eg, hollow fiber filter 30) to concentrate osteogenic cells (ie, platelets and nucleated cells) recovered from the first filter. An exemplary syringe containing osteogenic cells, such as the syringe 9 of FIG. 4, is connected to the hollow fiber filter 30 and operated in a full amount fashion (this is achieved by capping the preserve syringe port 34). being). Unlike cross-flow filtration, full filtration does not include the step of recycling the feed stream into the membrane tangentially between the typical feed syringe 9 and the condensate syringe. The pressure from the forward feed syringe generated differential pressure across the membrane. This causes some particles to pass through the membrane. Using an appropriate membrane pore size (eg, about 0.2-0.5 μm), the recovery solution can pass through the filter membrane of the hollow fiber filter 30, but cells cannot pass through this membrane. FIG. 8 is an exemplary schematic diagram showing osteogenic cell concentrate suspensions resulting from operation of the second filter in full volume mode. FIG. Whole filtration fractionated a portion of the recovery solution from the suspended cells, thereby increasing the concentration of cells remaining in the feed syringe 9.

Example 3

Description of Cell Concentrates

In a particular embodiment of the invention, the final cell concentrate should have one or more of the following properties: 1) more nucleated cells per unit volume in the starting physiological solution; 2) more platelets per unit volume in the starting physiological solution; And / or 3) upon platelet activation, the concentration of growth factor is higher than the growth factor concentration of an equivalent volume of starting physiological solution. Examples of growth factors include platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF), endothelial growth factor (EGF) and insulin-like growth factor (IGF). have.

In a particular embodiment, the method of obtaining the cell concentrate and / or the cell concentrate itself may have the following advantages: 1) the leukocyte concentration is at least about 6 times the reference blood value; 2) substantially free of exogenous animal-derived or human-derived components in the reverse-flushing solution (in other embodiments exogenous animal-derived or human-derived components such as exemplary human serum albumin May be added); 3) substantially no exogenous animal-derived or human-derived components in the coagulation initiator; And / or 4) the filter must activate less than about 25% of platelets (Babbush et al., 2003).

In other embodiments, the method for obtaining the cell concentrate and / or the cell concentrate itself more preferably has the following advantages: 1) a single (or first) filter having at least about 60 mL and at most about 120 mL Processing blood; 2) mixing the blood with one or more anticoagulants; 3) the recovery solution comprises biologically compatible and / or non-pyrogenic components; 4) the filtration step (s) takes less than about 10 minutes; 5) the filter recovers at least about 70% platelets from the blood; 6) the filter concentrates platelets about 6 times or more relative to the reference physiological solution (eg blood or bone marrow concentrate) value; 7) Platelet concentrates contain sufficient coagulation factors to initiate coagulation (examples of coagulation factors are fibrinogen, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, prekallikrein (prekallikrein), prothrombin, tissue factor, von Willebrand Factor (vWF) (while performing this method, the fibrigen concentration can be measured by ELISA to monitor the protein Can be)); 8) platelet concentrate coagulates within about 5 minutes after addition of the coagulation initiator; 9) final coagulant volume comprises about 5 to 10 mL; And / or 10) once solidified, the concentration of growth factors (eg PDGF, TGF-β, VEGF, EGF and IGF) is at least about 6 times the concentration of growth factor in the coagulated blood with equal or substantially equivalent volume Including.

In certain embodiments of the invention, the materials used in the present invention meet state regulatory standards, such as those approved by the FDA.

Example 4

How to Measure Cell Concentrate Characteristics

Nucleus  Cells (white blood cells) and Platelet count  [ Wintrobe's  Clinical Hematology, 10 th  ed.]

By exemplary means, white blood cells and platelets can be counted using manual and / or automated blood analyzers, both methods being known in the art. Manual counting was performed after proper dilution of the sample in a blood cell counter, a specially designed counting chamber containing a specific volume. The cells may also be counted under a microscope, for example.

Automated hematology analyzers may be preferable to manual counting because they increase the accuracy and speed of the assay. Two exemplary main types of automatic counters, aperture-impedance counters and optical method counters, can be used to count white blood cells and platelets. Aperture-impedance counters include, for example, Coulter (Hialeah, FL), Sysmex (Baxter Diagnostics, Waukengan, Illinois) and some cell- Cell-Dyne instruments (Abbott Diagnostics, Santa Clara, Calif.). Optical method counters include, for example, Technicon (Bayer Diagnostics, Kend, WA) and some cell-dyne instruments.

Cell number was determined for the original physiological solution and cell concentrate after final treatment.

Growth factor quantification

Enzyme-linked immunosorbent assay (ELISA) was used to quantify the concentration of growth factors in exemplary embodiments. Growth factor concentrations were determined for the original physiological solution after activation of platelets and for cell concentrates after final treatment. Appropriate platelet activators (eg, thrombin, calcium chloride solution, adenosine diphosphate (ADP), or a combination thereof) may be used to activate platelets. After sufficient time (eg, less than about 1 hour) of activating platelets and releasing growth factors, the concentration of growth factors was quantified, such as using commercially available ELISA kits.

Example 5

How to remove fat from physiological solution

In some embodiments of the invention, additional steps or means, such as filters, are included in the design to remove fat, such as particles (which may include fat cells), from physiological solutions. Fat can be in cellular or non-cellular form. Thus, one of ordinary skill in the art recognizes that fat refers to fat cells, coagulated fats that are in non-cellular form in nature, or both. The physiological solution was passed through the above steps or means, eg, an exemplary filter referred to herein as a fat reduction filter, to substantially reduce the amount of fat (or, in some embodiments, to remove it). In this process, the physiological fluid then passes through the filter (s), so removing fat leads to faster flow and better capture of osteogenic cells. In a preferred embodiment, the fat is sufficiently removed to prevent blockage, blockage, coagulation, and blockage, blockage, coagulation of the filter (s). Those skilled in the art recognize that it is desirable to completely remove fat or fat cells, but it is unnecessary as long as filtration can be achieved.

Commercially available filters that have been used clinically to remove microaggregates from blood may be suitable for the present invention. Examples include, but are not limited to, standard blood transfusion filters such as SQ40SJKL (Pall Corp., Port Washington, NY) and LipiGuard (Paul Corporation, Port Washington, NY).

9 shows a schematic of the introduction of a fat reduction filter 8 into a device used to prepare osteogenic cell concentrates. The physiological solution was passed through the syringe (6) to the collection bag (10) via the exemplary fat reduction filter (8) component. The reduced flow in the fat particle contents was then processed through a leucofilter 12 as shown in FIGS. 4 and 9. If desired, the cell suspension recovered from the leucofilter 12 was then treated via a hollow fiber filter 30 in a cross-flow mode (FIGS. 5 and 6) or a full quantity mode (FIGS. 7 and 8).

Example 6

Immature New Zealand White Rabbit  marrow Aspirate  ( BMA Filtration

BMA was harvested from New Zealand white rabbits whose two skeletons were not mature and processed through the filters exemplified in the present invention to give nucleated cells (in some embodiments, a cell population comprising undifferentiated cells such as bone precursors). It was proved that the concentration of was increased. This study shows that the filter can concentrate BMA obtained from immature New Zealand white rabbits within the desired range (about 6 to 10 times) in the specified time frame (about 15 minutes).

Materials and methods

Blood and bone marrow aspirates (BMA) were taken from three New Zealand white rabbits that were not mature. At the time of this procedure each rabbit was two months old and weighed approximately 1.4 kg. Rabbits were not treated with any medications or steroids before harvesting BMA. Anesthetics were administered intramuscularly with 50 mg / kg ketamine and 10 mg / kg xylazine. Approximately 2 mL of blood was drawn from each rabbit's aorta and mixed with 0.3 mL of heparinized saline. The volume of blood taken from each rabbit is shown in Table I. Bone marrow was aspirated from both the left and right sides of the adjacent tibia of each rabbit. The medial side of the metaphyseal adjacent to the anterior of the tibia was incised 0.5-1 cm. The 18-g needle was gently rotated to puncture the cortical bone and put it into the bone marrow cavity. If the first needle was clogged during this drilling, the needle was replaced with a new one. After pulling the plunger back to form a negative pressure until the bone marrow began to flow into the syringe, the pressure was reduced and the bone marrow was collected for about 10 seconds. In each tibia, 2 to 3.5 mL of BMA was aspirated with a 10 cc disposable syringe filled with 0.5 mL of heparinized saline (1000 units / mL) using an 18-g needle. After the syringe was removed, inverted up and down several times to mix thoroughly, the BMA was transferred to graduated sterile plastic test tubes. The volume of BMA collected from each rabbit is shown in Table II.

The initial nucleated cell number in each sample was determined by lysing red blood cells in 2% acetic acid solution. 100 μl of aspirate was mixed with 900 μl of acetic acid. A portion of the resulting cell suspension was injected into a hemocytometer grid and cells were counted at 200-fold magnification using compound microscopy (BX60, Olympus Optical Corporation, Olympus Optical Co., Ltd., Japan). The concentration of nucleated cells was calculated as follows:

N = C × SF × D _{acetic acid}

Wherein N = nucleated cell concentration; SF = scale factor of the blood cell counter grid = 104; D _{acetic acid} = dilution factor in _acetic acid = 10. The counts were repeated two or more times for each sample.

After the initial nucleated cell number was determined, 4 mL of BMA was collected from each rabbit to bring the combined volume to 12 mL. The collected BMA samples were then passed through an exemplary filtration device as schematically shown in FIGS. 5, 6 and 10. In FIG. 10, the cohesive filter 7 can remove large particles of material in certain embodiments comprising fat particles.

This particular embodiment includes a system having a one-step process using at least a leukocyte reduction filter to capture osteogenic cells, and optionally a hollow fiber filter to concentrate the captured cells. Leukocyte Reduction Filter (P / N B-1462G; L / N 312700) Recovery Solution (L / N SLS051303) was made by Pall Medical, Inc., Port Washington, NY, USA. Hollow fiber filters (P / NX12E-100-20N; L / N DH157 / N) were manufactured by Spectrum Laboratories, Inc. (Rancho Dominguez, Calif.). Nucleated cell concentrations of ACS-treated cell suspensions were determined as described in the previous paragraph.

result

The mean nucleated cell numbers of blood, pre-treatment BMA and post-treatment BMA were 6.0 × 10 ⁶ , 20.1 × 10 ⁶ and 131 × 10 ¹¹ cells / ml (Tables I, II and III, respectively). A representative cell clock of the hemocytometer is shown in FIG. 11. The nucleated cell number of BMA before treatment was approximately 3.5 times the nucleated cell number of blood [Holdrinet et al., 1980]. This indicates that bone marrow cells are collected in the aspirate and the BMA consists entirely of peripheral blood with nucleated cell numbers similar to blood. 12 mL of BMA collected from three donor rabbits were treated via ACS rounds in approximately 5 minutes. The final volume was 0.6 mL, 20 times less volume. The cell concentration of the post-filtered sample was approximately 6.5 times the BMA cell concentration before treatment. The cell concentration increased 6.5-fold with a 20-fold decrease in volume, and the cell recovery efficiency of the filtration prototype was 33%.

This study shows that circular filters can concentrate BMAs obtained from New Zealand white rabbits with a skeletal maturation within the desired range (about 6 to 10 times) in the specified time frame (about 15 minutes). Since age-related changes in the bone marrow cytoplasm and fat composition of rabbits are known (Kita et al., 1987; Bigelow and Tavassoli, 1984), in other embodiments the present invention is directed to nucleated cells in BMA obtained from rabbits with mature skeletal bone. A concentrated filtration is provided.

Volume and nucleated cell numbers of blood samples obtained from each rabbit Sample Blood volume (ml) Repetition of cell counts Nucleated cell concentration (10 ⁶ / ml) Rabbit 1 Rabbit 2  2.8 1.4 1.8  88; 119; 91 32; 48; 40 43; 49; 36  9.7 4.0 4.2 Mean = 6.0 ± 3.2

Volume and nucleated cell number of untreated bone marrow aspirate (BMA) samples obtained from each rabbit Sample BMA Volume (ml) Repetition of cell counts Nucleated cell concentration (10 ⁶ / ml) Rabbit 1 Rabbit 2   8 7 5  214; 229 203; 180 172; 213; 183  22.2 19.2 18.9 Mean = 20.1 ± 1.8

Volume and nucleated cell number of filtration-treated bone marrow aspirate (4 ml of bone marrow was taken from each of the three rabbits and processed through a filtration system) Sample Starting volume (ml) Final volume (ml) Repetition of cell counts Nucleated cell concentration (10 ⁶ / ml) Filtration-treated BMA   12  0.6 989; 1590; 1335  131 ± 30

Example  7

Platelet activation and cell concentrate coagulation method

Platelet activators (eg calcium chloride solution or thrombin, or a combination thereof) can be added to the cell concentrate to activate platelets and induce coagulation. The addition of platelet activators to cell concentrates will in turn exhibit higher growth factor concentrations (due to increased platelet activation) and good handling properties (due to coagulation) in preferred embodiments.

Platelet activators should be added to the cell concentrate at an appropriate rate to induce rapid platelet activation (less than 30 minutes) and coagulation. For example, if the activator is a CaCl ₂ / thrombin (100 unit / ml) solution, an activator: cell concentrate ratio of 1:10 is used.

A portion of the cell concentrate prepared for each blood donor was added to a glass beaker and the appropriate platelet activator was added to form a coagulum. This mixture was allowed to undergo coagulation shrinkage for an appropriate time (at least about 30 minutes). The contents of the beaker will then be transferred to a centrifuge tube and centrifuged at about 1,000 g for about 30 minutes. Growth factor concentrations in supernatants were quantified using a commercially available enzyme-linked immunosorbent assay (ELISA) kit in a representative embodiment. For reference, this experiment was repeated for (i) untreated blood samples obtained from each donor, and / or (ii) a portion of cell concentrate without platelet activator added.

Example 8

Method for preparing platelet rich concentrate (PRC) from human blood using a single filter

Whole blood was taken from healthy human donors and processed through the filters exemplified in the present invention to demonstrate an increase in the concentration of at least platelets and platelet-derived growth factors. This study shows that by treating human blood through a single filter (eg, leukocyte reduction filter), platelets and growth factors can be concentrated above the baseline observed in untreated blood.

A schematic of the evaluated filter is shown in FIG. 12. Each round filter includes two standard cc cc blood collection bags, a white blood cell reduction filter, and two syringe attachment inlets. To evaluate the filter, whole blood units (each unit of 450 mL) were taken from healthy human volunteers and collected in blood bags containing 50 mL of the anticoagulant citrate dextrose (ACD-A) of Formula A. An aliquot of 60 ml was transferred to a 60 mL syringe. The contents of the syringe were then injected into a blood bag containing 10 mL of platelet capture solution A (injectable water). In some cases, 120 mL of anticoagulated blood was injected into a bag containing 20 mL of Solution A to determine if the filter could effectively process a double dose of blood. Blood bags containing diluted blood samples were attached to platelet recovery filters (Purecell PL, Paul Medical, Inc., Port Washington, NY). The filtration height (vertical distance between the upper blood line of the collecting bag and the point of entry into the drainage bag) was adjusted to 12.5 inches. The diluted blood sample was then filtered at room temperature. The filtration time for each sample was recorded. After filtration was complete, the filter was back-flushed using a syringe filled with 7 mL of platelet recovery solution B (5% saline solution) and 13 cc of air. The contents of the filter were back-flushed with a 20 cc syringe. The recovered cell suspension was transferred from a syringe into a 15 mL centrifuge tube. The total volume of platelets recovered was measured using a grid on a centrifuge tube.

For each donor, platelet concentrations of recovered cell suspensions and whole blood samples were measured using a Cell Dyne blood analyzer (Abbott Labs, Ill.). ELISA for PDGF-AB, TGF-β1 and VEGF-A (Quantikine, R & D Systems, Minneapolis, Minneapolis), recovered suspension, recovered suspension + 10% CaCl ₂ / Thrombin (5000 units / ml) and whole blood. ANOVA determined that the mean showed a significant difference at the 95% confidence level.

Filtration times were 10-15 minutes for 60 mL blood samples (n = 20) and 45-90 minutes for 120 mL samples (n = 9). The flow rate of the 120 mL sample was reduced by back pressure from the drain bag filled near the maximum volume. The final volume of recovered cell suspension was 9-10 mL (for both starting blood volume), average 9.5 mL.

Average platelet recovery in both 60 mL and 120 mL blood samples was approximately 65%, with no significant difference between the two groups (FIG. 13). Mean platelet concentration factors for 60 mL and 120 ml samples were 4.1 ± 0.4 and 8.3 ± 1.2 times, respectively, compared to whole blood (p <0.05).

Average concentrations of PDGF-AB, TGF-β1 and VEGF-A in whole blood and platelet concentrates are shown in FIGS. 14-16 (n = 15), respectively. Cell concentrates prepared by the filter showed 16, 25, and 14-fold increases in PDGF, TGF-β, and VEGF concentrations compared to whole blood (p <0.05) in all cases. When cell concentrates were activated with calcium chloride / thrombin, the concentration factor increased 26-fold and 34-fold for PDGF and TGF-β, respectively (p <0.05). When activated with calcium chloride / thrombin, the VEGF concentration factor decreased by 14 to 3 times. In certain embodiments, this relates to the apparent ability of fibrin to bind VEGF, which can mask ELISA epitopes.

This study shows that the filters exemplified in the present invention are useful for conveniently preparing platelet rich concentrates from whole blood. The filtration process took 10-15 minutes, resulting in 9.5 mL of a final product more than four times rich in platelets. In addition, the concentrations of PDGF-AB, TGF-β1 and VEGF-A were significantly higher in platelet concentrates compared to whole blood.

references

All patents and publications mentioned in this specification are indicative of the level of skill in the art to which this invention pertains. All patents and publications are incorporated herein in their entirety to the same extent as by designation such that each individual publication is specifically and individually incorporated by reference.

Patent

U.S. Patent 5,824,084

U.S. Patent 6,010,627

U.S. Patent 6,049,026

U.S. Patent 6,342,157

U.S. Patent 6,398,972

WO96 / 27397 PCT application

Publication

Babbush CA, Kevy SV, Jacobson MS. An in vitro and in vivo evaluation of autologous platelet concentrate in oral reconstruction. Implant Dent. 2003; 12 (1): 24-34.

Bigelow CL and Tavassoli M, "Fatty involution of bone marrow in rabbits", Acta Anat (Basel), 118 (1): 60-4 (1984).

Connolly, J., Guse, R., Lippiello, L., Dehne, R. Development of an Osteogenic Bone-Marrow Preparation, J. Bone and Joint Surgery, vol. 71-A, no. 5, pp. 684-691, 1989.

Holdrinet RSG, Egmond JV, Wessels JMC, and Haanen C, "A method for quantification of peripheral blood admixture in bone marrow aspirates", Exp. Hemat., 8 (1): 103-7 (1980).

Kita K, Kawai K, and Hirohata K, "Changes in bone marrow flow with aging", J Ortho Res, 5 (4): 569-75 (1987).

Takigami, H., Muschler, GF, et al. Spine Fusion using Allograft bone matrix enriched in bone marrow cells and connective tissue progenitors, Trans. of 48 ^th Orthopaedic Res. Soc., P. 807, 2002.

Although described in detail in the invention and its advantages, it should be understood that various changes, substitutions and alterations can be made without departing from the spirit and scope of the invention as defined by the appended claims. In addition, the scope of the present application is not limited to the specific embodiments of the processes, machines, manufacture, compositions, means, methods and steps described in the specification. As those skilled in the art will readily appreciate from the disclosure of the present invention, a process, machine, presently or later developed, which performs substantially the same function or achieves substantially the same results as the corresponding embodiments described herein, Preparations, corresponding compositions, means, methods or steps can be used according to the invention. Accordingly, the appended claims are intended to be within the scope of the above procedures, machines, manufacture, compositions, means, methods or steps.

Claims (102)

Providing a physiological solution that has not been pre-centrifuged; The physiological solution is filtered to produce a filtration preserve and a permeate solution, wherein the filtration restoration comprises more than platelets, nucleated cells, or both per unit volume than the physiological solution, and the permeate solution is plasma and red blood cells. To include; And Removing the filtration retainer from the filter Comprising a method for producing a cell concentrate. The method of claim 1, wherein the physiological solution comprises bone marrow aspirate, blood, or mixtures thereof. The method of claim 1, wherein the nucleated cells comprise leukocytes, stem cells, connective tissue progenitor cells, bone precursor cells, chondrocyte cells, or mixtures thereof. The method of claim 1, wherein the stem cells are mesenchymal stem cells, hematopoietic stem cells, or both. The method of claim 1, wherein said providing step comprises combining the additional solution with a physiological solution. The method of claim 5, wherein the additional solution is water. The method of claim 5, wherein the additional solution is a hypotonic solution. The method of claim 5 wherein the additional solution is a hypotonic solution comprising sodium chloride. The method of claim 1, further comprising delivering the filtration restoration removed from the filter to a bone defect in the subject. 2. The method of claim 1, further comprising mixing the skeletal material into the filtered retainer removed from the filter to produce a skeletal material / filtration retainer mixture. The method of claim 10, further comprising delivering the skeletal material / filtration restoration mixture to a bone defect in the subject. The method of claim 10, wherein the backbone material consists of blocks, pastes, dust, cement, powders, granules, putty, liquids, gels, solids, or mixtures thereof. The method of claim 10, wherein the backbone material consists of a ceramic, polymer, metal, allograft bone, autograft bone, demineralized bone matrix, or mixtures thereof. The method of claim 10, wherein the backbone material is biodegradable. The method of claim 10, wherein the backbone material is osteoconductive, osteogenic, osteoinductive, or a combination thereof. The method of claim 10, wherein the backbone material consists of synthetic materials, natural materials, or combinations thereof. The method of claim 10, further comprising mixing the biological agent with a filtration retentate, a skeletal material, or a combination thereof removed from the filter. The method of claim 17, wherein the biological agent admixed with the backbone material is further defined as a biological agent included on the backbone material, in the backbone material, or both. The method of claim 10 further comprising mixing the coagulation initiator with the second product, the framework material, or both. The method of claim 1 wherein the filtration retentate removed from the filter is subjected to one or more further processing steps. Providing a physiological solution; Treating the physiological solution with a first filtration device to isolate nucleated cells, platelets, or both from the physiological solution, wherein the isolating step produces a first product; And Treating the first product with a second filtration device to produce a second product Comprising more than a number of nucleated cells, platelets, or both, per unit volume, than in physiological solution. The method of claim 21, wherein the first filtration device is a nucleated cell filtration device. The method of claim 22, wherein the nucleated cell filtration device is further defined as a leukocyte reduction filtration device. The method of claim 21, wherein the second filtration device is further defined as a hollow fiber filtration device. The method of claim 24, wherein the device comprises a pore size of about 0.05 μm to about 5 μm. The method of claim 25, wherein the device comprises a pore size of about 0.2 μm to about 0.5 μm. The method of claim 21, lacking a centrifugation step. The method of claim 21, wherein the physiological solution comprises bone marrow aspirate, blood, or mixtures thereof. The method of claim 1, wherein the nucleated cells comprise leukocytes, stem cells, connective tissue progenitor cells, bone precursor cells, chondrocyte cells, or mixtures thereof. The method of claim 29, wherein the stem cells are mesenchymal stem cells, hematopoietic stem cells, or both. The method of claim 21, wherein said providing step comprises combining the additional solution with a physiological solution. 32. The method of claim 31, wherein the additional solution is water. The method of claim 31, wherein the additional solution is a hypotonic solution. 32. The method of claim 31, wherein the additional solution is a hypotonic solution comprising sodium chloride. The method of claim 21, further comprising delivering the second product to a bone defect in the subject. 22. The method of claim 21, further comprising mixing a skeletal material into the second product to produce a skeletal material / second product mixture. The method of claim 36, further comprising delivering the skeletal material / second product mixture to a bone defect in the subject. 37. The method of claim 36, wherein the backbone material consists of blocks, pastes, dusts, cements, powders, granules, putty, liquids, gels, solids, or mixtures thereof. The method of claim 36, wherein the backbone material consists of a ceramic, polymer, metal, allograft bone, autograft bone, demineralized bone matrix, or mixtures thereof. The method of claim 36, wherein the backbone material is biodegradable. The method of claim 36, wherein the backbone material is osteoconductive, osteogenic, osteoinductive, or a combination thereof. 37. The method of claim 36, wherein the backbone material consists of synthetic materials, natural materials, or combinations thereof. The method of claim 36, further comprising mixing the biological agent with a second product, framework material, or combinations thereof. The method of claim 43, wherein the biological agent admixed with the backbone material is further defined as a biological agent included on the backbone material, in the backbone material, or both. The method of claim 21, further comprising mixing the coagulation initiator with the second product, the framework material, or both. The method of claim 21 further comprising applying the physiological solution to the fat reduction step. 47. The method of claim 46, wherein the form of fat is cellular or non-cellular. 47. The method of claim 46, wherein said fat reduction step comprises treating said physiological solution with a fat cell reduction filter. A second product made by the method of claim 21. Providing a physiological solution; Treating said physiological solution with a nucleated cell filtration device to isolate nucleated cells, platelets, or both from said physiological solution, wherein said isolating step produces a first product; And Treating the first product with another filtration device to produce a second product, wherein the second product comprises more than a number of nucleated cells, platelets, or both per unit volume than in physiological solution. Comprising, nucleated cell concentration and / or platelet concentration from a physiological solution. 51. The method of claim 50, lacking a centrifugation step. 51. The method of claim 50, wherein said physiological solution comprises bone marrow aspirate, blood, or mixtures thereof. 51. The method of claim 50, wherein said providing step comprises combining the additional solution with a physiological solution. The method of claim 53, wherein the additional solution is water. The method of claim 53, wherein the additional solution is a hypotonic solution. 54. The method of claim 53, wherein the additional solution is a hypotonic solution comprising sodium chloride. 51. The method of claim 50, wherein providing the bone marrow aspirate is further defined as aspirating the bone marrow from a subject into a first syringe to produce a bone marrow aspirate. The method of claim 57, wherein the first syringe comprises an anticoagulant, an isotonic solution, or both. 58. The method of claim 57, wherein treating the bone marrow aspirate with a nucleated cell filtration device Bone marrow aspirate is introduced into a first housing device, wherein the first housing device is connected in series to a leukocyte reduction filter, the leukocyte reduction filter is connected in series to a second housing device, and a leukocyte filter is passed through the filter Passing plasma and red blood cells but inhibiting passage of nucleated cells, platelets, or both; Introducing a purging solution into the filter to produce a purging solution / nucleated cell / platelet mixture; And Recovering the purging solution / nucleated cell / platelet mixture from the filter As further defined. 51. The method of claim 50, wherein treating the first product with a filtration device comprises treating the first product with a hollow fiber filtration device. 51. The method of claim 50, wherein treating the first product with a filtration device comprises treating the first product with a filtration device comprising a filter, wherein the direction of supply of the first product through the filtration device crosses the membrane. 1 non-parallel to the flow of product. 51. The method of claim 50, wherein treating the first product with a filtration device comprises treating the first product with a filtration device comprising a filter, wherein the direction of supply of the first product through the filtration device crosses the filter. Method parallel to the flow of the first product. 51. The method of claim 50, wherein substantially no recycle of the first product is present. 51. The method of claim 50, wherein the nucleated cell filtration device comprises a filter and most of the first product passes substantially through the filter once. 62. The method of claim 61, wherein the device comprises a filter and most of the first product passes substantially through the filter at least once. The method of claim 52, wherein providing blood is further defined as aspirating blood from the subject into the first syringe to produce a blood aspirate. The method of claim 66, wherein the first syringe comprises an anticoagulant, an isotonic solution, or both. 67. The method of claim 66, wherein treating the blood aspirate with a nucleated cell filtration device Introduce a blood aspirate into the first housing device, wherein the first housing device is connected in series to the leukocyte reduction filter, the leukocyte reduction filter is connected in series to the second housing device, and the leukocyte filter is passed through the filter Passing plasma and red blood cells but inhibiting passage of nucleated cells, platelets, or both; Introducing a purging solution into the filter to produce a purging solution / nucleated cell / platelet mixture; And Recovering the purging solution / nucleated cell / platelet mixture from the filter As further defined. The method of claim 52, wherein providing the bone marrow aspirate / blood mixture is further defined as aspirating bone marrow and blood from a subject into a first syringe to produce a bone marrow aspirate / blood aspirate. The method of claim 69, wherein the first syringe comprises an anticoagulant, an isotonic solution, or both. 53. The method of claim 52, wherein treating the bone marrow aspirate / blood aspirate with a nucleated cell filtration device. Bone marrow aspirate / blood aspirate is introduced into the first housing device, wherein the first housing device is connected in series to a leukocyte reduction filter, the leukocyte reduction filter is connected in series to a second housing device, and the leukocyte filter is Passing plasma and red blood cells through the filter but inhibiting passage of nucleated cells, platelets, or both; Introducing a purging solution into the filter to produce a purging solution / nucleated cell / platelet mixture; And Recovering the purging solution / nucleated cell / platelet mixture from the filter As further defined. 51. The method of claim 50, further comprising applying the physiological solution to the fat reduction step. 73. The method of claim 72, wherein said fat reduction step comprises treating said physiological solution with a fat reduction filter. Obtaining a physiological solution comprising nucleated cells, platelets, or both; Treating the physiological solution with a nucleated cell filtration device to isolate nucleated cells, platelets, or both, wherein said isolating comprises producing a first product; Treating the first product with a hollow fiber filtration device to produce a second product, wherein the second product comprises more than a plurality of nucleated cells, platelets, or both per unit volume than in physiological solution; And Delivering the second product to a bone defect in the subject Including a method of treating bone defects in a subject. 75. The method of claim 74, lacking a centrifugation step. 75. The method of claim 74, wherein the bone defect comprises breakage, fracture, voiding, diseased bone, bone loss, broken bone, weak bone, bone damage, or bone degeneration. 75. The method of claim 74, further comprising mixing a backbone material with the second product. 75. The method of claim 74, wherein said physiological solution is blood, bone marrow aspirate, or mixtures thereof. 75. The method of claim 74, wherein said physiological solution is obtained from said subject. 75. The method of claim 74, performed at a medical facility or a healthcare provider facility. 75. The method of claim 74, further comprising performing additional bone defect therapy on the subject. The method of claim 81, wherein the additional bone defect therapy comprises fracture repair, surgery, bone resection, graft delivery, external stimulation, or a combination thereof. The method of claim 74, wherein the nucleated cells comprise stem cells, connective tissue progenitor cells, bone precursor cells, chondrocyte cells, or mixtures thereof. 84. The method of claim 83, wherein said stem cells are mesenchymal stem cells, hematopoietic stem cells, or mixtures thereof. 75. The method of claim 74, wherein delivering the second product to the subject comprises adding the second concentrated product directly to the bone defect. 86. The method of claim 85, wherein the method is applied with a scoop, scoopula, syringe, rod, tube, or spatula. 75. The method of claim 74, further comprising mixing the biological agent with a second product, framework material, or a combination thereof. 88. The method of claim 87, wherein the biological agent mixed with the skeletal material is further defined as a biological agent that is included on, in, or both. 75. The method of claim 74, further comprising applying the physiological solution to the fat reduction step. 75. The method of claim 74, wherein said fat reduction step comprises treating said physiological solution with a fat reduction filter. A method of making osteogenic cell concentrate from a physiological solution, comprising applying the physiological solution to two filtration steps. 92. The method of claim 91, lacking centrifugation. And a first filtration device and a second filtration device, wherein both of the devices are housed in a suitable container. 95. The kit of claim 93, wherein the first filtration device is a nucleated cell filtration device. 95. The kit of claim 93, wherein the second filtration device is a microfiltration device. 97. The method of claim 95, wherein the microfiltration device is further defined as a hollow fiber filtration device. 95. The kit of claim 93, further comprising a framework material contained in a suitable container. 95. The kit of claim 93, further comprising a biological agent contained in a suitable container. A kit for treating a bone defect comprising a plurality of cells housed in a suitable container, wherein the cells are nucleated cells, platelets, or both. 103. The kit of claim 99, further comprising a framework material contained in a suitable container. 105. The kit of claim 99, further comprising a biological agent contained in a suitable container. Providing a physiological solution that has not been pre-centrifuged; Treating the physiological solution with a leukocyte reduction filter to produce a filtration preserve and a permeate solution, wherein the filtration preserve comprises more than platelets, nucleated cells, or both per unit volume than the physiological solution, and the permeate solution Comprising plasma and red blood cells; And Removing the filtration retainer from the filter Comprising a method for producing a cell concentrate.

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