US 9,808,558 B2Grant

Allografts combined with tissue derived stem cells for bone healing

Issue Date:2017-11-07

•24 Claims

•6 Drawing Sheets

Abstract

There is disclosed a method of combining mesenchymal stem cells (MSCs) with a bone substrate. In an embodiment, the method includes obtaining tissue having MSCs together with unwanted cells. The tissue is digested to form a cell suspension having MSCs and unwanted cells. The cell suspension is added to the substrate. The substrate is cultured to allow the MSCs to adhere. The substrate is rinsed to remove unwanted cells. In various embodiments, the tissue is adipose tissue, muscle tissue, or bone marrow tissue. In an embodiment, there is disclosed an allograft product including a combination of MSCs with a bone substrate in which the combination is manufactured by culturing MSCs disposed on the substrate for a period of time to allow the MSCs to adhere to the substrate, and then rinsing the substrate to remove unwanted cells from the substrate. Other embodiments are also disclosed.

Metadata

Assignee

AlloSource

Inventor

Yaling Shi

Application Information

Application Number:US 14/880,675

Filing Date:2015-10-12

Priority Date:2008-11-20

Art Unit:1651

Classifications

IPC:

A61K35/38A61L27/38A61L27/36A61L27/54C12N5/0775A01N1/02A61K35/12

Patent Drawings (6 sheets)

Description

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No. 12/612,583, filed Nov. 4, 2009, which claims the benefit under 35 U.S.C. §119 (e) of U.S. Provisional Patent Application No. 61/116,484, filed Nov. 20, 2008, both of which are hereby incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

[0002] Regenerative medicine requires an abundant source of human adult stem cells that can be readily available at the point of care.

[0003] Adipose-derived stem cells (ASCs), which can be obtained in large quantities, have been utilized as cellular therapy for the induction of bone formation in tissue engineering strategies.

[0004] Allografts may be combined with stem cells. This requires a significant amount of tissue processing and cellular processing prior to seeding the allograft substrate.

[0005] Allografts seeded with living cells generally provide better surgical results.

BRIEF SUMMARY OF THE INVENTION

[0006] In an embodiment, there is provided a method of combining mesenchymal stem cells with a bone substrate, the method comprising obtaining adipose tissue having the mesenchymal stem cells together with unwanted cells; digesting the adipose tissue to form a cell suspension having the mesenchymal stem cells and the unwanted cells; adding the cell suspension with the mesenchymal stem cells to seed the bone substrate so as to form a seeded bone substrate; culturing the mesenchymal stem cells on the seeded bone substrate for a period of time to allow the mesenchymal stem cells to adhere to the bone substrate; and rinsing the bone substrate to remove the unwanted cells from the bone substrate.

[0007] In another embodiment, there is provided an allograft product including a combination of mesenchymal stem cells with a bone substrate, and the combination manufactured by obtaining adipose tissue having the mesenchymal stem cells together with unwanted cells; digesting the adipose tissue to form a cell suspension having the mesenchymal stem cells and the unwanted cells; adding the cell suspension with the mesenchymal stem cells to seed the bone substrate so as to form a seeded bone substrate; culturing the mesenchymal stem cells on the seeded bone substrate for a period of time to allow the mesenchymal stem cells to adhere to the bone substrate; and rinsing the bone substrate to remove the unwanted cells from the bone substrate.

[0008] In still another embodiment, there is provided a method of combining mesenchymal stem cells with a bone substrate, the method comprising obtaining adipose tissue having the mesenchymal stem cells together with unwanted cells; digesting the adipose tissue to form a cell suspension having the mesenchymal stem cells and the unwanted cells to acquire a stromal vascular fraction, and the digesting includes making a collagenase I solution, and filtering the solution through a 0.2 μm filter unit, mixing the adipose solution with the collagenase I solution, and adding the adipose solution mixed with the collagenase I solution to a shaker flask; placing the shaker with continuous agitation at about 75 RPM for about 45 to 60 minutes so as to provide the adipose tissue with a visually smooth appearance; aspirating a supernatant containing mature adipocytes so as to provide a pellet; adding the cell suspension with the mesenchymal stem cells to seed the bone substrate so as to form a seeded bone substrate; culturing the mesenchymal stem cells on the seeded bone substrate for a period of time to allow the mesenchymal stem cells to adhere to the bone substrate; and rinsing the bone substrate to remove the unwanted cells from the bone substrate.

[0009] In yet another embodiment, there is provided an allograft product including a combination of mesenchymal stem cells with a bone substrate, and the combination manufactured by obtaining adipose tissue having the mesenchymal stem cells together with unwanted cells; digesting the adipose tissue to form a cell suspension having the mesenchymal stem cells and the unwanted cells to acquire a stromal vascular fraction, and the digesting includes making a collagenase I solution, and filtering the solution through a 0.2 μm filter unit, mixing the adipose solution with the collagenase I solution, and adding the adipose solution mixed with the collagenase I solution to a shaker flask; placing the shaker with continuous agitation at about 75 RPM for about 45 to 60 minutes so as to provide the adipose tissue with a visually smooth appearance; aspirating a supernatant containing mature adipocytes so as to provide a pellet; adding the cell suspension with the mesenchymal stem cells to seed the bone substrate by adding the pellet onto the bone substrate so as to form a seeded bone substrate; culturing the mesenchymal stem cells on the seeded bone substrate for a period of time to allow the mesenchymal stem cells to adhere to the bone substrate; and rinsing the bone substrate to remove the unwanted cells from the bone substrate.

[0010] In an embodiment, there is provided a method of combining mesenchymal stem cells with a bone substrate, the method comprising obtaining tissue having the mesenchymal stem cells together with unwanted cells; digesting the tissue to form a cell suspension having the mesenchymal stem cells and the unwanted cells; adding the cell suspension with the mesenchymal stem cells to seed the bone substrate so as to form a seeded bone substrate; culturing the mesenchymal stem cells on the seeded bone substrate for a period of time to allow the mesenchymal stem cells to adhere to the bone substrate; and rinsing the bone substrate to remove the unwanted cells from the bone substrate.

[0011] In another embodiment, there is provided an allograft product including a combination of mesenchymal stem cells with a bone substrate, and the combination manufactured by obtaining tissue having the mesenchymal stem cells together with unwanted cells; digesting the tissue to form a cell suspension having the mesenchymal stem cells and the unwanted cells; adding the cell suspension with the mesenchymal stem cells to seed the bone substrate so as to form a seeded bone substrate; culturing the mesenchymal stem cells on the seeded bone substrate for a period of time to allow the mesenchymal stem cells to adhere to the bone substrate; and rinsing the bone substrate to remove the unwanted cells from the bone substrate.

[0012] In still another embodiment, there is provided a method of combining mesenchymal stem cells with a bone substrate, the method comprising obtaining bone marrow tissue having the mesenchymal stem cells together with unwanted cells; digesting the bone marrow tissue to form a cell suspension having the mesenchymal stem cells and the unwanted cells; adding the cell suspension with the mesenchymal stem cells to seed the bone substrate so as to form a seeded bone substrate; culturing the mesenchymal stem cells on the seeded bone substrate for a period of time to allow the mesenchymal stem cells to adhere to the bone substrate; and rinsing the bone substrate to remove the unwanted cells from the bone substrate.

[0013] In yet another embodiment, there is provided an allograft product including a combination of mesenchymal stem cells with a bone substrate, and the combination manufactured by obtaining bone marrow tissue having the mesenchymal stem cells together with unwanted cells; digesting the bone marrow tissue to form a cell suspension having the mesenchymal stem cells and the unwanted cells; adding the cell suspension with the mesenchymal stem cells to seed the bone substrate so as to form a seeded bone substrate; culturing the mesenchymal stem cells and the bone substrate for a period of time to allow the mesenchymal stem cells to adhere to the bone substrate; and rinsing the bone substrate to remove the unwanted cells from the bone substrate.

[0014] In an embodiment, there is provided a method of combining mesenchymal stem cells with a bone substrate, the method comprising obtaining muscle tissue having the mesenchymal stem cells together with unwanted cells; digesting the muscle tissue to form a cell suspension having the mesenchymal stem cells the unwanted cells; adding the cell suspension with the mesenchymal stem cells to seed the bone substrate so as to form a seeded bone substrate; culturing the mesenchymal stem cells on the seeded bone substrate for a period of time to allow the mesenchymal stem cells to adhere to the bone substrate; and rinsing the bone substrate to remove the unwanted cells from the bone substrate.

[0015] In another embodiment, there is provided an allograft product including a combination of mesenchymal stem cells with a bone substrate, and the combination manufactured by obtaining muscle tissue having the mesenchymal stem cells together with unwanted cells; digesting the muscle tissue to form a cell suspension having the mesenchymal stem cells and the unwanted cells; adding the cell suspension with the mesenchymal stem cells to seed the bone substrate so as to form a seeded bone substrate; culturing the mesenchymal stem cells on the seeded bone substrate for a period of time to allow the mesenchymal stem cells to adhere to the bone substrate; and rinsing the bone substrate to remove the unwanted cells from the bone substrate.

[0016] Other embodiments are also disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Illustrative embodiments of the invention are illustrated in the drawings, in which:

[0018] FIG. 1 illustrates a flow chart of the combination of mesenchymal stem cells with a bone substrate;

[0019] FIG. 2 illustrates a prior art example of a pellet of a stromal vascular fraction containing the desired stem cells and unwanted cells;

[0020] FIGS. 3A-3D illustrate various examples of strips (FIG. 3A and FIG. 3D) and dowels (FIG. 3C and FIG. 3D) which have a 3-D cancellous matrix structure and mesenchymal stem cells (MSCs) may adhere to;

[0021] FIG. 4 illustrates a standard curve of total live ASCs using the CCK-8 assay;

[0022] FIG. 5 illustrates mineral deposition by ASCs cultured in osteogenic medium (images shown at 20× magnification); and

[0023] FIG. 6 illustrates H&E staining showed that cells adhered to the bone surface.

DETAILED DESCRIPTION OF THE INVENTION

[0024] Unless otherwise described, human adult stem cells are generally referred to as mesenchymal stem cells or MSCs. MSCs are pluripotent cells that have the capacity to differentiate in accordance with at least two discrete development pathways. Adipose-derived stem cells or ASCs are stem cells that are derived from adipose tissue. Stromal Vascular Fraction or SVF generally refers to the centrifuged cell pellet obtained after digestion of tissue containing MScs. In one embodiment, the pellet may include multiple types of stem cells. These stem cells may include, for example, one or more of hematopoietic stem cells, epithelial progenitor cells, and mesenchymal stem cells. In an embodiment, mesenchymal stem cells are filtered from other stem cells by their adherence to a bone substrate, while the other stem cells (i.e., unwanted cells) do not adhere to the bone substrate. Other cells that do not adhere to the bone substrate may also be included in these unwanted cells.

[0025] Adipose derived stem cells may be isolated from cadavers and characterized using flow cytometry and tri-lineage differentiation (osteogenesis, chondrogenesis and adipogenesis) may be performed in vitro. The final product may be characterized using histology for microstructure and biochemical assays for cell count. This consistent cell-based product may be useful for bone regeneration.

[0026] Tissue engineering and regenerative medicine approaches offer great promise to regenerate bodily tissues. The most widely studied tissue engineering approaches, which are based on seeding and in vitro culturing of cells within the scaffold before implantation, is the cell source and the ability to control cell proliferation and differentiation. Many researchers have demonstrated that adipose tissue-derived stem cells (ASCs) possess multiple differentiation capacities. See, for example, the following, which are incorporated by reference:

[0027] Rada, T., R. L. Reis, and M. E. Gomes, Adipose Tissue-Derived Stem Cells and Their Application in Bone and Cartilage Tissue Engineering. Tissue Eng Part B Rev, 2009.
[0028] Ahn, H. H., et al., In vivo osteogenic differentiation of human adipose-derived stem cells in an injectable in situ forming gel scaffold. Tissue Eng Part A, 2009. 15(7): p. 1821-32.
[0029] Anghileri, E., et al., Neuronal differentiation potential of human adipose-derived mesenchymal stem cells. Stem Cells Dev, 2008. 17(5): p. 909-16.
[0030] Arnalich-Montiel, F., et al., Adipose-derived stem cells are a source for cell therapy of the corneal stroma. Stem Cells, 2008. 26(2): p. 570-9.
[0031] Bunnell, B. A., et al., Adipose-derived stem cells: isolation, expansion and differentiation. Methods, 2008. 45(2): p. 115-20.
[0032] Chen, R. B., et al., [Differentiation of rat adipose-derived stem cells into smooth-muscle-like cells in vitro]. Zhonghua Nan Ke Xue, 2009. 15(5): p. 425-30.
[0033] Cheng, N. C., et al., Chondrogenic differentiation of adipose-derived adult stem cells by a porous scaffold derived from native articular cartilage extracellular matrix. Tissue Eng Part A, 2009. 15(2): p. 231-41.
[0034] Cui, L., et al., Repair of cranial bone defects with adipose derived stem cells and coral scaffold in a canine model. Biomaterials, 2007. 28(36): p. 5477-86.
[0035] de Girolamo, L., et al., Osteogenic differentiation of human adipose-derived stem cells: comparison of two different inductive media. J Tissue Eng Regen Med, 2007. 1(2): p. 154-7.
[0036] Elabd, C., et al., Human adipose tissue-derived multipotent stem cells differentiate in vitro and in vivo into osteocyte-like cells. Biochem Biophys Res Commun, 2007. 361(2): p. 342-8.
[0037] Flynn, L., et al., Adipose tissue engineering with naturally derived scaffolds and adipose-derived stem cells. Biomaterials, 2007. 28(26): p. 3834-42.
[0038] Flynn, L. E., et al., Proliferation and differentiation of adipose-derived stem cells on naturally derived scaffolds. Biomaterials, 2008. 29(12): p. 1862-71.
[0039] Fraser, J. K., et al., Adipose-derived stem cells. Methods Mol Biol, 2008. 449: p. 59-67.
[0040] Gimble, J. and F. Guilak, Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy, 2003. 5(5): p. 362-9.
[0041] Gimble, J. M. and F. Guilak, Differentiation potential of adipose derived adult stem (ADAS) cells. Curr Top Dev Biol, 2003. 58: p. 137-60.
[0042] Jin, X. B., et al., Tissue engineered cartilage from hTGF beta2 transduced human adipose derived stem cells seeded in PLGA/alginate compound in vitro and in vivo. J Biomed Mater Res A, 2008. 86(4): p. 1077-87.
[0043] Kakudo, N., et al., Bone tissue engineering using human adipose-derived stem cells and honeycomb collagen scaffold. J Biomed Mater Res A, 2008. 84(1): p. 191-7.
[0044] Kim, H. J. and G. I. Im, Chondrogenic differentiation of adipose tissue-derived mesenchymal stem cells: greater doses of growth factor are necessary. J Orthop Res, 2009. 27(5): p. 612-9.
[0045] Kingham, P. J., et al., Adipose-derived stem cells differentiate into a Schwann cell phenotype and promote neurite outgrowth in vitro. Exp Neural, 2007. 207(2): p. 267-74.
[0046] Mehlhorn, A. T., et al., Chondrogenesis of adipose-derived adult stem cells in a poly-lactide-co-glycolide scaffold. Tissue Eng Part A, 2009. 15(5): p. 1159-67.
[0047] Merceron, C., et al., Adipose-derived mesenchymal stem cells and biomaterials for cartilage tissue engineering. Joint Bone Spine, 2008. 75(6): p. 672-4.
[0048] Mischen, B. T., et al., Metabolic and functional characterization of human adipose-derived stem cells in tissue engineering. Plast Reconstr Surg, 2008. 122(3): p. 725-38.
[0049] Mizuno, H., Adipose-derived stem cells for tissue repair and regeneration: ten years of research and a literature review. J Nippon Med Sch, 2009. 76(2): p. 56-66.
[0050] Tapp, H., et al., Adipose-Derived Stem Cells: Characterization and Current Application in Orthopaedic Tissue Repair. Exp Biol Med (Maywood), 2008.
[0051] Tapp, H., et al., Adipose-derived stem cells: characterization and current application in orthopaedic tissue repair. Exp Biol Med (Maywood), 2009. 234(1): p. 1-9.
[0052] van Dijk, A., et al., Differentiation of human adipose-derived stem cells towards cardiomyocytes is facilitated by laminin. Cell Tissue Res, 2008. 334(3): p. 457-67.
[0053] Wei, Y., et al., A novel injectable scaffold for cartilage tissue engineering using adipose-derived adult stem cells. J Orthop Res, 2008. 26(1): p. 27-33.
[0054] Wei, Y., et al., Adipose-derived stem cells and chondrogenesis. Cytotherapy, 2007. 9(8): p. 712-6.
[0055] Zhang, Y. S., et al., [Adipose tissue engineering with human adipose-derived stem cells and fibrin glue injectable scaffold]. Zhonghua Yi Xue Za Zhi, 2008. 88(38): p. 2705-9.

[0056] Additionally, adipose tissue is probably the most abundant and accessible source of adult stem cells. Adipose tissue derived stem cells have great potential for tissue regeneration. Nevertheless, ASCs and bone marrow-derived stem cells (BMSCs) are remarkably similar with respect to growth and morphology, displaying fibroblastic characteristics, with abundant endoplasmic reticulum and large nucleus relative to the cytoplasmic volume. See, for example, the following, which are incorporated by reference:

[0057] Gimble, J. and F. Guilak, Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy, 2003. 5(5): p. 362-9.
[0058] Gimble, J. M. and F. Guilak, Differentiation potential of adipose derived adult stem (ADAS) cells. Curr Top Dev Bioi, 2003. 58: p. 137-60.
[0059] Strem, B. M., et al., Multipotential differentiation of adipose tissue-derived stem cells. Keio J Med, 2005. 54(3): p. 132-41.
[0060] De Ugarte, D. A., et al., Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells Tissues Organs, 2003. 174(3): p. 101-9.
[0061] Hayashi, O., et al., Comparison of osteogenic ability of rat mesenchymal stem cells from bone marrow, periosteum, and adipose tissue. Calcif Tissue Int. 2008. 82(3): p. 238-47.
[0062] Kim, Y., et al., Direct comparison of human mesenchymal stem cells derived from adipose tissues and bone marrow in mediating neovascularization in response to vascular ischemia. Cell Physiol Biochem, 2007. 20(6): p. 867-76.
[0063] Lin, L., et al., Comparison of osteogenic potentials of BMP4 transduced stem cells from autologous bone marrow and fat tissue in a rabbit model of calvarial defects. Calcif Tissue Int, 2009. 85(1): p. 55-65.
[0064] Niemeyer, P., et al., Comparison of immunological properties of bone marrow stromal cells and adipose tissue-derived stem cells before and after osteogenic differentiation in vitro. Tissue Eng, 2007. 13(1): p. 111-21.
[0065] Noel, D., et al., Cell specific differences between human adipose-derived and mesenchymal-stromal cells despite similar differentiation potentials. Exp Cell Res, 2008. 314(7): p. 1575-84.
[0066] Yoo, K. H., et al., Comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues. Cell Immunol, 2009.
[0067] Yoshimura, H., et al., Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res, 2007. 327(3): p. 449-62.

[0068] Other common characteristics of ASCs and BMSCs can be found in the transcriptional and cell surface profile. Several studies have already been done in the field of bone tissue engineering using ASCs. See, for example, the following, which are incorporated by reference:

[0069] Rada, T., R. L. Reis, and M. E. Gomes, Adipose Tissue-Derived Stem Cells and Their Application in Bone and Cartilage Tissue Engineering. Tissue Eng Part B Rev, 2009.
[0070] Tapp, H., et al., Adipose-Derived Stem Cells: Characterization and Current Application in Orthopaedic Tissue Repair. Exp Bioi Med (Maywood), 2008.
[0071] Tapp, H., et al., Adipose-derived stem cells: characterization and current application in orthopaedic tissue repair. Exp Bioi Med (Maywood), 2009. 234(1): p. 1-9.
[0072] De Girolamo, L., et al., Human adipose-derived stem cells as future tools in tissue regeneration: osteogenic differentiation and cell-scaffold interaction. Int J Artif Organs, 2008. 31(6): p. 467-79.
[0073] Di Bella, C., P. Farlie, and A. J. Penington, Bone regeneration in a rabbit critical-sized skull defect using autologous adipose-derived cells. Tissue Eng Part A, 2008. 14(4): p. 483-90.
[0074] Grewal, N. S., et al., BMP-2 does not influence the osteogenic fate of human adipose-derived stem cells. Plast Reconstr Surg, 2009. 123(2 Suppl): p. 158S-65S.
[0075] Li, H., et al., Bone regeneration by implantation of adipose-derived stromal cells expressing BMP-2. Biochem Biophys Res Commun, 2007. 356(4): p. 836-42.
[0076] Yoon, E., et al., In vivo osteogenic potential of human adipose-derived stem cells/poly lactide-co-glycolic acid constructs for bone regeneration in a rat critical-sized calvarial defect model. Tissue Eng, 2007. 13(3): p. 619-27.

[0077] These studies have demonstrated that stem cells obtained from the adipose tissue exhibit good attachment properties to most of the material surfaces and the capacity to differentiate into osteoblastic-like cells in vitro and in vivo. Recently it has been shown that ASCs may stimulate the vascularization process. See, for example, the following, which are incorporated by reference:

[0078] Butt, O. I., et al., Stimulation of peri-implant vascularization with bone marrow-derived progenitor cells: monitoring by in vivo EPRoximetry. TissueEng, 2007. 13(8): p. 2053-61.
[0079] Rigotti, G., et al., Clinical treatment of radiotherapy tissue damage by lipoaspirate transplant: a healing process mediated by adipose-derived adult stem cells. Plast Reconstr Surg, 2007. 119(5): p. 1409-22; discussion 1423-4.

[0080] Demineralized bone substrate, as an allogeneic material, is a promising bone tissue-engineering scaffold due to its close relation to autologous bone in terms of structure and function. Combined with MSCs, these scaffolds have been demonstrated to accelerate and enhance bone formation within osseous defects when compared with the matrix alone. See, for example, the following, which are incorporated by reference:

[0081] Chen, L. Q., et al., [Study of MSCs in vitro cultured on demineralized bone matrix of mongrel]. Shanghai Kou Qiang Yi Xue, 2007. 16(3): p. 255-8.
[0082] Gamradt, S. C. and J. R. Lieberman, Bone graft for revision hip arthroplasty: biology and future applications. Clin Orthop Relat Res, 2003(417): p. 183-94.
[0083] Honsawek, S., D. Dhitiseith, and V. Phupong, Effects of demineralized bone matrix on proliferation and osteogenic differentiation of mesenchymal stem cells from human umbilical cord. J Med Assoc Thai, 2006. 89 Suppl 3: p. S189-95.
[0084] Kasten, P., et al., [Induction of bone tissue on different matrices: an in vitro and a in vivo pilot study in the SCID mouse]. Z Orthop Ihre Grenzgeb, 2004.142(4): p. 467-75.
[0085] Kasten, P., et al., Ectopic bone formation associated with mesenchymal stem cells in a resorbable calcium deficient hydroxyapatite carrier. Biomaterials, 2005. 26(29): p. 5879-89.
[0086] Qian, Y., Z. Shen, and Z. Zhang, [Reconstruction of bone using tissue engineering and nanoscale technology]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 2006. 20(5): p. 560-4.
[0087] Reddi, A. H., Role of morphogenetic proteins in skeletal tissue engineering and regeneration. Nat Biotechnol, 1998. 16(3): p. 247-52.
[0088] Reddi, A. H., Morphogenesis and tissue engineering of bone and cartilage: inductive signals, stem cells, and biomimetic biomaterials. Tissue Eng, 2000.6(4): p. 351-9.
[0089] Tsiridis, E., et al., In vitro and in vivo optimization of impaction allografting by demineralization and addition of rh-OP-1. J Orthop Res, 2007. 25(11): p. 1425-37.
[0090] Xie, H., et al., The performance of a bone-derived scaffold material in the repair of critical bone defects in a rhesus monkey model. Biomaterials, 2007.28(22): p. 3314-24.
[0091] Liu, G., et al., Tissue-engineered bone formation with cryopreserved human bone marrow mesenchymal stem cells. Cryobiology, 2008. 56(3): p. 209-15.
[0092] Liu, G., et al., Evaluation of partially demineralized osteoporotic cancellous bone matrix combined with human bone marrow stromal cells for tissue engineering: an in vitro and in vivo study. Calcif Tissue Int, 2008. 83(3): p. 176-85.
[0093] Liu, G., et al., Evaluation of the viability and osteogenic differentiation of cryopreserved human adipose-derived stem cells. Cryobiology, 2008. 57(1): p. 18-24.

[0094] As discussed herein, human ASCs seeded bone substrates may be characterized in terms of microstructure, cell number and cell identity using histology, biochemical assay and flow cytometry. In an embodiment, these substrates may include bone material which has been previously subjected to a demineralization process.

[0095] FIG. 1 is a flow chart of a process for making an allograft with stem cells product. In an embodiment, a stromal vascular fraction may be used to seed the allograft. It should be apparent from the present disclosure that the term “seed” relates to addition and placement of the stem cells within, or at least in attachment to, the allograft, but is not limited to a specific process. FIG. 2 illustrates a pellet of the stromal vascular fraction containing the desired stem cells.

[0096] In an exemplary embodiment, a method of combining mesenchymal stem cells with a bone substrate is provided. The method may include obtaining adipose tissue having the mesenchymal stem cells together with unwanted cells. Unwanted cells may include hematopoietic stem cells and other stromal cells. The method may further include digesting the adipose tissue to form a cell suspension having the mesenchymal stem cells and at least some or all of the unwanted cells. In another embodiment, this may be followed by negatively depleting some of the unwanted cells and other constituents to concentrate mesenchymal stem cells.

[0097] Next, the method includes adding the cell suspension with the mesenchymal stem cells to the bone substrate. This may be followed by culturing the mesenchymal stem cells and the bone substrate for a period of time to allow the mesenchymal stem cells to adhere to the bone substrate. In order to provide a desired product, the method includes rinsing the bone substrate to remove the unwanted cells from the bone substrate.

[0098] In one embodiment, an allograft product may include a combination of mesenchymal stem cells with a bone substrate such that the combination is manufactured by the above exemplary embodiment.

[0099] In an embodiment, the adipose tissue may be obtained from a cadaveric donor. A typical donor yields 2 liters of adipose containing 18 million MSCs. In one embodiment, a bone substrate may be from the same cadaveric donor as the adipose tissue. In another embodiment, the adipose tissue may be obtained from a patient. In addition, both the bone substrate and the adipose tissue may be obtained from the same patient. This may include, but is not limited to, removal of a portion of the ilium (e.g., the iliac crest) may be removed from the patient by a surgical procedure and adipose cells may be removed using liposuction. Other sources, and combination of sources, of adipose tissue, other tissues, and bone substrates may be utilized.

[0100] Optionally, the adipose tissue may be washed prior to or during digestion. Washing may include using a thermal shaker at 75 RPM at 37° C. for at least 10 minutes. Washing the adipose tissue may include washing with a volume of PBS substantially equal to the adipose tissue. In an embodiment, washing the adipose tissue includes washing with the PBS with 1% penicillin and streptomycin at about 37° C.

[0101] For example, washing the adipose tissue may include agitating the tissue and allowing phase separation for about 3 to 5 minutes. This may be followed by aspirating off an infranatant solution. The washing may include repeating washing the adipose tissue multiple times until a clear infranatant solution is obtained. In one embodiment, washing the adipose tissue may include washing with a volume of growth media substantially equal to the adipose tissue.

[0102] In another exemplary embodiment, a method of combining mesenchymal stem cells with a bone substrate is provided. The method may include obtaining bone marrow tissue having the mesenchymal stem cells together with unwanted cells. Unwanted cells may include hematopoietic stem cells and other stromal cells. The method may further include digesting the bone marrow tissue to form a cell suspension having the mesenchymal stem cells and the unwanted cells. In another embodiment, this may be followed by naturally selecting MSCs and depleting some of the unwanted cells and other constituents to concentrate mesenchymal stem cells.

[0103] Next, the method includes adding the cell suspension with the mesenchymal stem cells to the bone substrate. This may be followed by culturing the mesenchymal stem cells and the bone substrate for a period of time to allow the mesenchymal stem cells to adhere to the bone substrate. In order to provide a desired product, the method includes rinsing the bone substrate to remove the unwanted cells from the bone substrate.

[0104] In one embodiment, an allograft product may include a combination of mesenchymal stem cells with a bone substrate such that the combination is manufactured by the above exemplary embodiment.

[0105] In another exemplary embodiment, a method of combining mesenchymal stem cells with a bone substrate is provided. The method may include obtaining muscle tissue having the mesenchymal stem cells together with unwanted cells. Unwanted cells may include hematopoietic stem cells and other stromal cells. The method may further include digesting the muscle tissue to form a cell suspension having the mesenchymal stem cells and the unwanted cells. In another embodiment, this may be followed by naturally selecting MSCs to concentrate mesenchymal stem cells.

[0106] Next, the method includes adding the cell suspension with the mesenchymal stem cells to the bone substrate. This may be followed by culturing the mesenchymal stem cells and the bone substrate for a period of time to allow the mesenchymal stem cells to adhere to the bone substrate. In order to provide a desired product, the method includes rinsing the bone substrate to remove the unwanted cells from the bone substrate.

[0107] In one embodiment, an allograft product may include combination of mesenchymal stem cells with a bone substrate such that the combination is manufactured by the above exemplary embodiment.

[0108] In another exemplary embodiment, a method of combining mesenchymal stem cells with a bone substrate is provided. The method may include obtaining tissue having the mesenchymal stem cells together with unwanted cells. Unwanted cells may include hematopoietic stem cells and other stromal cells.

[0109] The method may further include digesting the tissue to form a cell suspension having the mesenchymal stem cells and at least some of the unwanted cells. In another embodiment, this may be followed by negatively depleting some of the unwanted cells and other constituents to concentrate mesenchymal stem cells.

[0110] Next, the method includes adding the cell suspension with the mesenchymal stem cells to the bone substrate. In an embodiment, this substrate may include a bone material which has been subjected to a demineralization process. In another embodiment, this substrate may be a non-bone material, which may include (but is not limited to) a collagen based material. This may be followed by culturing the mesenchymal stem cells and the bone substrate for a period of time to allow the mesenchymal stem cells to adhere to the bone substrate. In order to provide a desired product, the method includes rinsing the bone substrate to remove the unwanted cells from the bone substrate.

[0111] In one embodiment, an allograft product may include a combination of mesenchymal stem cells with a bone substrate such that the combination is manufactured by the above exemplary embodiment.

[0112] Digesting the cell suspension may include making a collagenase I solution, and filtering the solution through a 0.2 μm filter unit, mixing the adipose tissue with the collagenase I solution, and adding the cell suspension mixed with the collagenase I solution to a shaker flask. Digesting the cell suspension may further include placing the shaker with continuous agitation at about 75 RPM for about 45 to 60 minutes so as to provide the adipose tissue with a visually smooth appearance.

[0113] Digesting the cell suspension may further include aspirating supernatant containing mature adipocytes so as to provide a pellet, which may be referred to as a stromal vascular fraction. (See, for example, FIG. 2.) Prior to seeding, a lab sponge or other mechanism may be used to pat dry bone substrate.

[0114] In one embodiment, adding the cell suspension with the mesenchymal stem cells to the bone substrate may include using a cell pellet for seeding onto the bone substrate. In an embodiment, adding the cell suspension with the mesenchymal stem cells to the bone substrate may include using a cell pellet for seeding onto the bone substrate. In another embodiment, adding the cell suspension with the mesenchymal stem cells to the bone substrate may include using a cell pellet for seeding onto the bone substrate of cortical bone. In another embodiment, adding the cell suspension with the mesenchymal stem cells to the bone substrate may include adding the cell pellet onto the bone substrate of cancellous bone. In another embodiment, adding the cell suspension with the mesenchymal stem cells to the bone substrate may include adding the cell pellet onto the bone substrate of ground bone. In another embodiment, adding the cell suspension with the mesenchymal stem cells to the bone substrate may include adding the cell pellet onto the bone substrate of cortical/cancellous bone. In another embodiment, adding the cell suspension with the mesenchymal stem cells to the bone substrate may include adding the cell pellet onto the bone substrate of demineralized cancellous bone.

[0115] In an embodiment, the method may include placing the bone substrate into a cryopreservation media after rinsing the bone substrate. This cryopreservation media may be provided to store the final products. For example, the method may include maintaining the bone substrate into a frozen state after rinsing the bone substrate to store the final products. The frozen state may be at about negative 80° C.

[0116] In another embodiment, Ficoll density solution may be utilized. For example, negatively depleting the concentration of the mesenchymal stem cells may include adding a volume of PBS and a volume of Ficoll density solution to the adipose solution. The volume of PBS may be 5 ml and the volume of Ficoll density solution may be 25 ml with a density of 1.073 g/ml. Negatively depleting the concentration of the mesenchymal stem cells may also include centrifuging the adipose solution at about 1160 g for about 30 minutes at about room temperature. In one embodiment, the method may include stopping the centrifuging the adipose solution without using a brake.

[0117] Negatively depleting the concentration of the mesenchymal stem cells is optional and may next include collecting an upper layer and an interface containing nucleated cells, and discarding a lower layer of red cells and cell debris. Negatively depleting the concentration of the mesenchymal stem cells may also include adding a volume of D-PBS of about twice an amount of the upper layer of nucleated cells, and inverting a container containing the cells to wash the collected cells. Negatively depleting the concentration of the mesenchymal stem cells may include centrifuging the collected cells to pellet the collected cells using the break during deceleration.

[0118] In an embodiment, negatively depleting the concentration of the mesenchymal stem cells may further include centrifuging the collected cells at about 900 g for about 5 minutes at about room temperature. Negatively depleting some of the unwanted cells may include discarding a supernatant after centrifuging the collected cells, and resuspending the collected cells in a growth medium.

[0119] In one embodiment, adding the cell suspension with the mesenchymal stem cells to the bone substrate may include adding the cell pellet onto the bone substrate. Adding the solution with the mesenchymal stem cells to the bone substrate may include adding cell pellet onto the bone substrate which was subjected to a demineralization process. In another embodiment, adding the cell suspension with the mesenchymal stem cells to the bone substrate may include adding the cell pellet onto the bone substrate of cortical bone. In an embodiment, adding the cell suspension with the mesenchymal stem cells to the bone substrate includes adding the cell pellet onto the bone substrate of cancellous bone. In another embodiment, adding the cell suspension with the mesenchymal stem cells to the bone substrate may include adding the cell pellet onto the bone substrate of ground bone. In another embodiment, adding the cell suspension with the mesenchymal stem cells to the bone substrate may include adding the cell pellet onto the bone substrate of cortical/cancellous bone. In another embodiment, adding the cell suspension with the mesenchymal stem cells to the bone substrate may include adding the cell pellet onto the bone substrate of demineralized cancellous bone.

[0120] In an embodiment, the method may further include placing the bone substrate into a cryopreservation media after rinsing the bone substrate. This cryopreservation media may be provided to store the final products. The method may include maintaining the bone substrate into a frozen state after rinsing the bone substrate to store the final products. The frozen state may be at about negative 80° C.

[0121] The seeded allografts are cultured for a period of time to allow the mesenchymal stem cells to adhere to the bone substrate. The unwanted cells were rinsed and removed from the bone substrate. After culturing, a lab sponge or other mechanism may be used to pat dry the bone substrate.

[0122] The mesenchymal stem cells are anchorage dependent. The mesenchymal stem cells naturally adhere to the bone substrate. The mesenchymal stem cells are non-immunogenic and regenerate bone. The unwanted cells are generally anchorage independent. This means that the unwanted cells generally do not adhere to the bone substrate. The unwanted cells may be immunogenic and may create blood and immune system cells. For cell purification during a rinse, mesenchymal stem cells adhere to the bone while unwanted cells, such as hematopoietic stem cells, are rinsed away leaving a substantially uniform population of mesenchymal stem cells on the bone substrate.

[0123] The ability to mineralize the extracellular matrix and to generate bone is not unique to MSCs. In fact, ASCs possess a similar ability to differentiate into osteoblasts under similar conditions. Human ASCs offer a unique advantage in contrast to other cell sources. The multipotent characteristics of ASCs, as wells as their abundance in the human body, make these cells a desirable source in tissue engineering applications.

[0124] In various embodiments, bone substrates (e.g., cortical cancellous dowels, strips, cubes, blocks, discs, and granules, as well as other substrates formed in dowels, strips, cubes, blocks, discs, and granules) may be subjected to a demineralization process to remove blood, lipids and other cells so as to leave a matrix. FIGS. 3A-3D illustrate various examples of strips (FIGS. 3A and 3B) and dowels (FIGS. 3C and 3D). Generally, these substrates may have a 3-D cancellous matrix structure, which MSCs may adhere to.

[0125] In addition, this method and combination product involve processing that does not alter the relevant biological characteristics of the tissue. Processing of the adipose/stem cells may involve the use of antibiotics, cell media, collagenase. None of these affects the relevant biological characteristics of the stem cells. The relevant biological characteristics of these mesenchymal stem cells are centered on renewal and repair. The processing of the stem cells does not alter the cell's ability to continue to differentiate and repair.

[0126] In the absence of stimulation or environmental cues, mesenchymal stem cells (MSCs) remain undifferentiated and maintain their potential to form tissue such as bone, cartilage, fat, and muscle. Upon attachment to an osteoconductive matrix, MSCs have been shown to differentiate along the osteoblastic lineage in vivo. See, for example, the following, which are incorporated by reference:

[0127] Arinzeh T L, Peter S J, Archambault M P, van den Bas C, Gordon S, Kraus K, Smith A, Kadiyala S. Allogeneic mesenchymal stem cells regenerate bone in a critical sized canine segmental defect. J Bone Joint Surg Am. 2003; 85-A:1927-35.
[0128] Bruder S P, Kurth A A, Shea M, Hayes W C, Jaiswal N, Kadiyala S. Bone regeneration by implantation of purified, culture-expanded human mesenchymal stem cells, J Orthop Res. 1998; 16:155-62.

Example 1

Adipose Recovery

[0129] Adipose was recovered from cadaveric donors. Adipose aspirate may be collected using liposuction machine and shipped on wet ice.

Washing

[0130] Adipose tissue was warmed up in a thermal shaker at RPM=75, 37° C. for 10 min. Adipose was washed with equal volume of pre-warmed phosphate buffered saline (PBS) at 37° C., 1% penicillin/streptomycin. Next, the adipose was agitated to wash the tissue. Phase separation was allowed for about 3 to 5 minutes. The infranatant solution was aspirated. The wash was repeated 3 to 4 times until a clear infranatant solution was obtained.

[0131] The solution was suspended in an equal volume of growth media (DMEM/F12, 10% FBS, 1% penicillin/streptomycin) and stored in a refrigerator at about 4° C.

Digestion and Combining of Cell Suspension with Allografts

[0132] Digestion of the adipose was undertaken to acquire a stromal vascular fraction (SVF) followed by combining the solution onto an allograft.

[0133] Digestion involved making collagenase I solution, including 1% fetal bovine serum (FBS) and 0.1% collagenase I. The solution was filtered through a 0.2 urn filter unit. This solution should be used within 1 hour of preparation.

[0134] Next, take out the washed adipose and mix with collagenase I solution at 1:1 ratio. Mixture was added to a shaker flask.

[0135] The flask was placed in an incubating shaker at 37° C. with continuous agitation (at about RPM=75) for about 45 to 60 minutes until the tissue appeared smooth on visual inspection.

[0136] The digestate was transferred to centrifuge tubes and centrifuged for 5 minutes at about 300-500 g at room temperature. The supernatant, containing mature adipocytes, was then aspirated. The pellet was identified as the stromal vascular fraction (SVF).

[0137] Growth media was added into every tube (i.e., 40 ml total was added into the 4 tubes) followed by gentle shaking.

[0138] All of the cell mixtures were transferred into a 50 ml centrifuge tube. A 200 μl sample was taken, 50 μl is for initial cell count, and the remainder of the 150 μl was used for flow cytometry.

[0139] Aliquot cell mixtures were measured into 2 centrifuge tubes (of 10 ml each) and centrifuged at about 300 g for 5 minutes. The supernatant was aspirated.

[0140] A cell pellet obtained from one tube was used for seeding onto allografts. The allografts may include cortical/cancellous or both which was subjected to a demineralization process.

[0141] Certain volume of growth medium was added into the cell pellets and shaken to break the pellets. A very small volume of cell suspension was added onto allografts. After culturing in C0₂incubator at 37° C. for a few hours, more growth medium (DMEM/F12, 10% FBS with antibiotics) was added. This was astatic “seeding” process. A dynamic “seeding” process can be used for particular bone substrate. 10 ml of a cell suspension and bone substrate were placed in a 50 ml centrifuge tube on an orbital shaker and agitated at 100 to 300 rpm for 6 hours.

[0142] After a few days (about 1 to 3 days), the allograft was taken out and rinsed thoroughly in PBS and sonicated to remove unwanted cells. The allograft was put into cryopreservation media (10% DMSO, 90% serum) and kept frozen at −80° C. The frozen allograft combined with the mesenchymal stem cells is a final product.

Example 2

Adipose Recovery

[0143] Adipose was recovered from cadaveric donors. Adipose aspirate may be collected using liposuction machine and shipped on wet ice.

Washing

[0144] Adipose tissue was processed in a thermal shaker at RPM=75, 37° C. for 10 min. Adipose was washed with equal volume of pre-warmed phosphate buffered saline (PBS) at 37° C., 1% penicillin/streptomycin. Next, the adipose was agitated to wash the tissue. Phase separation was allowed for about 3 to 5 minutes. The supernatant solution was sucked off. The wash was repeated 3 to 4 times until a clear infranatant solution was obtained.

Acquire Ficoll Concentrated Stem Cells and Combine onto Allograft

[0145] Ficoll concentrated stem cells were acquired and seeded onto an allograft. 5 ml PBS was placed into the 50 ml tube with cells and 25 ml of 1.073 g/ml Ficoll density solution was added to the bottom of the tube with a pipet.

[0146] The tubes were subjected to centrifugation at 1160 g for 30 min at room temperature and stopped with the brake off. The upper layer and interface, approximately 15 to 17 ml containing the nucleated cells were collected with a pipet and transferred to a new 50 ml disposable centrifuge tube. The lower layer contained red cells and cell debris and was discarded.

[0147] Next, 2 volumes of 0-PBS were added. The tubes were capped and mix gently by inversion to wash the cells.

[0148] The tubes with the diluted cells were then subjected to centrifugation at 900 g for 5 minutes at room temperature to pellet the cells with the brake on during deceleration.

[0149] The supernatant was discarded and the washed cells were resuspended in 10 ml of growth medium. 10 ml of growth media was added into the tube and it was shaken gently. A 1 ml sample was taken with 100 μl is for cell count, and the remainder of 900 μl was used for flow cytometry.

[0150] The remainder of the cell mixtures were centrifuged at about 300 g for about 5 minutes. The supernatant was aspirated.

[0151] A cell pellet was used for “seeding” onto allografts. Allografts may include demineralized bone, cortical/cancellous bone, or both. A very small volume of medium was added into the cell pellet and shaken. 100 μl of cell mixtures were added onto a 15 mm disc within a 24-well culture plate.

[0152] After culturing the allograft in a C02 incubator at about 37° C., 1 ml growth medium (DMEM/F12, 10% FBS with antibiotics) was added. This was a static “seeding” process. A dynamic “seeding” process can be used for a particular bone substrate.

[0153] After a few days (about 1 to 3 days), the allograft was taken out and rinsed thoroughly in PBS to remove unwanted cells. The allograft was put into cryopreservation media (10% DMSO, 90% serum) and kept frozen at −80° C. The frozen allograft combined with the stem cells is a final product.

Example 3

Bone Marrow Recovery

[0154] Adipose was recovered from cadaveric donors. Adipose aspirate may be collected using liposuction machine and shipped on wet ice.

Washing

[0155] The bone marrow sample is washed by adding 6 to 8 volumes of Dulbecco's phosphate buffered saline (D-PBS) in a 50 ml disposable centrifuge, inverting gently and subjecting to centrifugation (800 g for 10 min) to pellet cells to the bottom of the tube.

Acquire Stem Cells and Combine onto Allograft

[0156] The supernatant is discarded and the cell pellets from all tubes are resuspended in 1-2 ml of growth medium (DMEM, low glucose, with 10% FBS and 1% pen/strap). The cell mixtures are seeded onto allografts. With a few hours of culture in C02 incubator at 37° C., more growth medium is added. A few days later, the allograft is taken out and rinsed thoroughly in PBS and put into cryopreservation media (10% DMSO, 90% serum) and kept frozen.

Example 4

Skeletal Muscle Recovery

[0157] Skeletal muscle may be recovered from cadaveric donors.

Washing

[0158] Minced skeletal muscle (1-3 mm cube) is digested in a 3 mg/ml collagenase D solution in α-MEM at 37° C. for 3 hours. The solution is filtered with 100 um nylon mesh. The solution is centrifuged at 500 g for 5 min.

Acquire Stem Cells and Combine onto Allograft

[0159] The supernatant is discarded and the cell pellets from all tubes are resuspended in 1-2 ml of growth medium (DMEM, low glucose, with 10% FBS and 1% pen/strap). The cell mixtures are seeded onto allografts. With a few hours of culture in C02 incubator at 37° C., more growth medium will be added. A few days later, the allograft is taken out and rinsed thoroughly in PBS and put into cryopreservation media (10% DMSO, 90% serum) and kept frozen.

Example 5

Adipose Recovery

[0160] Adipose was recovered from a cadaveric donor within 24 hours of death and shipped in equal volume of DMEM in wet ice.

Washing

[0161] Adipose were washed 3 times with PBS and suspended in an equal volume of PBS supplemented with Collagenase Type I prewarmed to 37° C. The tissue was placed in a shaking water bath at 37° C. with continuous agitation for 45 to 60 minutes and centrifuged for 5 minutes at room temperature. The supernatant, containing mature adipocytes, was aspirated. The pellet was identified as the SVF (stromal vascular fraction).

Cortical Cancellous Bone Recovery

[0162] Human cortical cancellous bone was recovered from ilium crest from the same donor. The samples were sectioned into strips (20×50×5 mm), and then they were subjected to a demineralization process with HCl for 3 hours, rinsed with PBS until the pH is neutral.

Digestion and Combining of Cell Suspension with Allograft

[0163] The adipose-derived stem cells (ASCs) were added onto the grafts and cultured in C02 incubator at 37° C. Then the allografts were rinsed thoroughly in PBS to remove antibiotics and other debris. At the end, the allografts were put into cryopreservation media and kept frozen at −80° C.

Example 6

Adipose-Derived Stem Cell Characterization

Flow Cytometry Analysis

[0164] The following antibodies were used for flow cytometry. PE anti-CD73 (clone AD2) Becton Dickinson, PE anti-CD90 (clone F15-42-1) AbD SeroTec, PE anti-CD105 (clone SN6) AbD SeroTec, PE anti-Fibroblasts/Epithelial Cells (clone 07-FIB) AbD SeroTec, FITC anti-CD34 (clone 8G12) Becton Dickinson, FITC Anti-CD45 (clone 2D1) Becton Dickinson, and PE anti-CD271 (clone ME20.4-1.H4) Miltenyi BioTec. The Isotype controls were FITC Mouse IgG1 Kappa (clone MOPC-21) Becton Dickinson, PE Mouse IgG1 Kappa (clone MOPC-21) Becton Dickinson, and PE Mouse IgG2a Kappa (clone G155-178) Becton Dickinson.

[0165] A small aliquot of the cells were stained with a propidium iodide/detergent solution and fluorescent nuclei were counted using a hemocytometer on a fluorescent microscope. This total cell count was used to adjust the number of cells per staining tube to no more than 5.0×105 cells. The cells were washed with flow cytometric wash buffer (PBS supplemented with 2% FBS and 0.1% NaN3), stained with the indicated antibodies and washed again before acquisition. Staining was for 15 minutes at room temperature (15-30DC).

[0166] At least 20,000 cells were acquired for each sample on a FACScan flow cytometer equipped with a 15-mW, 488-nm, argon-ion laser (BD Immunocytometry Systems, San Jose, Calif.). The cytometer QC and setup included running SpheroTech rainbow (3 μm, 6 peaks) calibration beads (SpheroTech Inc.) to confirm instrument functionality and linearity. Flow cytometric data were collected and analyzed using Cell Quest software (BD Immunocytometry Systems). The small and large cells were identified by forward (FSC) and side-angle light scatter (SSC) characteristics. Autofluorescence was assessed by acquiring cells on the flow cytometer without incubating with fluorochrome labeled antibodies. Surface antigen expression was determined with a variety of directly labeled antibodies according to the supplier's recommendations. Antibodies staining fewer than 20% of the cells relative to the Isotype-matched negative control were considered negative (this is standard-of-practice for immunophenotyping leukocytes for leukemia lymphoma testing). The viability of the small and large cells was determined using the Becton Dickinson Via-Probe (7-AAD).

In Vitro Tri-Lineage Differentiation

[0167] Osteogenesis: Confluent cultures of primary ASCs were induced to undergo osteogenesis by replacing the stromal medium with osteogenic induction medium (® osteogenesis differentiation kit STEMPRO® Osteogeneis Differentiation Kit (ThermoFisher Scientific, previously Invitrogen, Cat # A1007201), a kit used for the osteogenic differentiation of mesenchymal stem cells in tissue culture vessels that contains STEMPRO® Osteocyte/Chondrocyte Differentiation Basal Medium (Gibco® Cat. # A10069) and STEMPRO® Osteogenesis Supplement (Gibco® Cat. # A10066-01). Cultures were fed with fresh osteogenic differentiation medium every 3 to 4 days for a period of up to 3 weeks. Cells were then fixed in 10% neutral buffered formalin and rinsed with Dl water. Osteogenic differentiation was determined by staining for calcium phosphate with Alizarin red (Sigma).

[0168] Adipogenesis: Confluent cultures of primary ASCs were induced to undergo adipogenesis by replacing the stromal medium with adipogenic induction medium (STEMPRO® Adipogenesis Differentiation Kit (ThermoFisher Scientific, previously Invitrogen, Cat. # A1007001), a kit used for the adipogenic differentiation of mesenchymal stem cells in tissue culture vessels that contains STEMPRO® Adipocyte Differentiation Basal Medium (Gibco® Cat. # A10410) and STEMPRO® Adipogenesis Supplement (Gibco® Cat. # A10065-01). Cultures were fed with fresh adipogenic differentiation medium every 3 to 4 days for a period of up to 2 weeks. Cells were then fixed in 10% neutral buffered formalin and rinsed with PBS. Adipogenic differentiation was determined by staining for fat globules with oil red O (Sigma).

[0169] Chondrogenesis: Confluent cultures of primary ASCs were induced to undergo chondrogenesis by replacing the stromal medium with chondrogenic induction medium (STEMPRO® Chondrogenesis Differentiation Kit (ThermoFisher Scientific, previously Invitrogen), a kit used for the chondrogenic differentiation of mesenchymal stem cells in tissue culture vessels that contains STEMPRO® Chondrocyte Differentiation Basal Medium (Gibco® Cat. # A10069) and STEMPRO® Chondrogenesis Supplement (Gibco® Cat. # A10064-01). Cultures were fed with fresh chondrogenic induction medium every 3 to 4 days for a period of up to 3 weeks. Cells were then fixed in 10% neutral buffered formalin and rinsed with PBS. Chondrogenic differentiation was determined by staining for proteoglycans with Alcian blue (Sigma).

Final Product Characterization

[0170] Cell count may be preformed [sic] with a CCK-8 Assay. Cell Counting Kit 8 (CCK-8, Dojindo Molecular Technologies, Maryland) allows sensitive colorimetric assays for the determination of the number of viable cells in cell proliferation assays. With reference to FIG. 4, there is illustrated a standard curve of total live ASCs using the CCK-8 assay. WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] is reduced by dehydrogenases in cells to give a yellow colored product (formazan), which is soluble in the tissue culture medium. The amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells. The allografts were thawed and rinsed with PBS and then patted dry. Growth medium and CCK-8 solution were added into the allografts at a ratio of 10:1 cultured at 37° C. for 2 hours and evaluated in a plate reader with excitation set to 460 nm and emission set to 650 nm. The results were interpolated from a standard curve (FIG. 4) based on ASCs only (passage=3).

Histology

[0171] When the cultures were terminated, the constructs were fixed in 10% neutral buffered formal in (Sigma, St. Louis, Mo.) for 48 h, put in a processor (Citadel 2000; Thermo Shandon, Pittsburgh, Pa.) overnight, and embedded in paraffin. Sections were cut to 8 μm and mounted onto glass slides and stained with hematoxylin and eosin (H&E). Conventional light microscopy was used to analyze sections for matrix and cell morphology.

Statistical Analysis

[0172] All quantitative data were expressed as the mean±standard deviation. Statistical analysis was performed with one-way analysis of variance. A value of p<0.05 was considered statistically significant.

Results

Final Product Appearance

[0173] FIGS. 3A-3D illustrate an appearance of strips, dowels and disks. In these embodiments, all have a cortical bottom and cancellous top. Other embodiments may be used.

ASC Characterization

Flow Cytometry—Immunophenotype of SVF

[0174] The SVF were stained with CD105, CD90 and CD73 to determine if there were significant numbers of MSC present. The immunophenotype of the stromal vascular fraction was consistent from donor to donor. The large cells (mean 3%) have the following immunophenotype and mean percentage: D7-FIB+ (36%), CD105+ (43%), CD90+(63%), CD73+ (28%) and CD34+ (62%). The small cells (mean 97%) contain only a small percentage of the markers tested and therefore could not be immunophenotyped with this method: D7-FIB (5%), CD105 (6%), CD90 (15%), CD73 (6%) and CD34 (10%). The SVF contained a significant population of CD34+ cells (Large CDC34+ 62% and small CD34+ 10%). The paucity of CD45+ cells (Large 15% and small 3%) would suggest that the SVF does not contain significant numbers of WBC (CD45+, low FSC, low SSC) or hematopoietic stem cells (CD34+, low CD45+, medium FSC, low SSC). The anti-Fibroblasts/Epithelial Cells (clone D7-FIB) antibody has been reported to be a good marker for MSC. The large cells were D7-FIB+36% and the small cells were D7-FIB+5%. CD271 should be negative on SVF cells and the large cells were CD271+10% and the small cells were CD271+0%. Following adherence of the SVF (ASCs, P1), the immunophenotype became more homogenous for both the large and small cells. The large cells (53%) have the following immunophenotype and percentage: D7-FIB+(93%), CD105+(98%), CD90+(96%) and CD73+(99%). The small cells (47%) have the following immunophenotype and percentage: D7-FIB+(77%), CD105+(75%), CD90+(58%) and CD73+(83%). The ASCs has lost CD34 marker expression (P3: large 4% and small 1%) (P1: large 8% and small 6%) and the CD45+ cells remained low (P3: large 2% and small 2%) (P1: large 3% and small 1%). This would suggest that there are few WBC (CD45+, low FSC, low SSC) or hematopoietic stem cells (CD34+, low CD45+, medium FSC, low SSC) present. The anti-Fibroblasts/Epithelial Cell (clone D7-FIB) antibody for the adherent and cultured cells showed an increased expression. The large cells were D7-FIB+93% and the small cells were D7-FIB+77%. CD271 should become positive following adherence and culture of the SVF. For P3 the large cells were CD271+4% and the small cells were CD271+1%. For P1 the large cells were CD271+27% and the small cells were CD271+3%. CD271 does not seem to be a useful marker for cultured MSC but more data is required.

Estimated Mean Total Percentage of MSC

[0175] CD105 was chosen to estimate the mean total percentage of MSC; although there is no single surface marker that can discern MSC in a mixed population. For the SVF with a mean of 3% large cells, a mean of 43% CD105+ cells, the mean total percentage would be 1.3%. For the SVF with a mean of 98% small cells, a mean of 6% CD105+ cells, the mean total percentage would be 5.9%. Combining the large and small totals gives a mean total of 7.2% MSC for the SVF.

In Vitro Tri-Lineage Differentiation

[0176] FIG. 5 illustrates mineral deposition by ASCs cultured in osteogenic medium (A) indicating early stages of bone formation. The samples were stained with alizarin red S. Negative control (D) showed no sign of bone formation. Fat globules seen in ASCs cultured in adipogenic medium (B) indicating differentiation into adipocytes. The samples were stained with Oil red O. The picture (E) is negative control. Proteoglycans produced by ASCs cultured in chondrogenic medium (C) indicating early stages of chondrogenesis. The samples were stained with alcian blue. The negative control (F) showed no sign of chondrogenesis.

[0177] For the osteogenic differentiation, morphological changes appeared during the second week of the culture. At the end of the 21-day induction period, some calcium crystals were clearly visible. Cell differentiation was confirmed by alizarin red staining (FIG. 5 image (A)).

[0178] The adipogenic potential was assessed by induction of confluent ASCs. At the end of the induction cycles (7 to 14 days), a consistent cell vacuolation was evident in the induced cells. Vacuoles brightly stained for fatty acid with oil red O staining (FIG. 5 image (B)). Chondrogenic potential was assessed by induction of confluent ASCs. At the end of the induction cycles (14 to 21 days), the induced cells were clearly different from non-induced control cells. Cell differentiation was confirmed with Alcian blue staining (FIG. 5 image (C)).

Final Product Characterization

Cell Count: CCK-8 Assay

[0179] 28 grafts were tested from 8 donors and had an average of 50,000 live cells/graft.

Histology

[0180] H&E was performed to demonstrate cell morphology in relation to the underlying substrate (cancellous bone matrix). The stem cells are elongated and adhere to the surface of cancellous bone. FIG. 6 is an illustration of H&E staining that showed that stem cells adhered to the bone surface.

CONCLUSIONS

[0181] The ability of DBM to enhance osteogenesis of ASCs in vitro and in vivo is believed to be due to the interaction of osteoprogenitors with these matrix incorporated osteoinductive factors, which can induce MSCs into osteoblasts. In turn, the incorporation of an osteogenic cell source into DBM can potentially limit the need for the migration and expansion of indigenous osteoprogenitors within defect sites, allowing for an increased rate of bone formation and osseointegration. See, for example, the following, which are incorporated by reference:

[0182] Liu, G., et al., Evaluation of partially demineralized osteoporotic cancellous bone matrix combined with human bone marrow stromal cells for tissue engineering: an in vitro and in vivo study. Calcif Tissue Int, 2008. 83(3): p. 176-85.
[0183] Wang, J. and M. J. Glimcher, Characterization of matrix-induced osteogenesis in rat calvarial bone defects: II. Origins of bone forming cells. Calcif Tissue Int, 1999. 65(6): p. 486-93.
[0184] Wang, J. and M. J. Glimcher, Characterization of matrix-induced osteogenesis in rat calvarial bone defects: I. Differences in the cellular response to demineralized bone matrix implanted in calvarial defects and insubcutaneous sites. Calcif Tissue Int, 1999. 65(2): p. 156-65.
[0185] Wang, J., et al., Characterization of demineralized bone matrix-induced osteogenesis in rat calvarial bone defects: III. Gene and protein expression. Calcif Tissue Int, 2000. 67(4): p. 314-20.
[0186] Bruder, S. P. and B. S. Fox, Tissue engineering of bone. Cell based strategies. Clin Orthop Relat Res, 1999(367 Suppl): p. S68-83.

[0187] Many studies have demonstrated that purified, culture-expanded human MSCs can be directed into the osteogenic lineage in vitro, culminating in a mineralized matrix production. See, for example, the following, which are incorporated by reference:

[0188] Chen, L. Q., et al., [Study of MSCs in vitro cultured on demineralized bone matrix of mongrel]. Shanghai Kou Qiang Yi Xue, 2007. 16(3): p. 255-8.
[0189] Honsawek, S., D. Dhitiseith, and V. Phupong, Effects of demineralized bone matrix on proliferation and osteogenic differentiation of mesenchymal stem cells from human umbilical cord. J Med Assoc Thai, 2006. 89 Suppl 3: p. 8189-95.
[0190] Kasten, P., et al., Ectopic bone formation associated with mesenchymal stem cells in a resorbable calcium deficient hydroxyapatite carrier. Biomaterials, 2005. 26(29): p. 5879-89.
[0191] Qian, Y., Z. Shen, and Z. Zhang, [Reconstruction of bone using tissue engineering and nanoscale technology]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 2006. 20(5): p. 560-4.
[0192] Liu, G., et al., Tissue-engineered bone formation with cryopreserved human bone marrow mesenchymal stem cells. Cryobiology, 2008. 56(3): p. 209-15.
[0193] Ko, E. K., et al., In vitro osteogenic differentiation of human mesenchymal stem cells and in vivo bone formation in composite nanofiber meshes. Tissue Eng Part A, 2008. 14(12): p. 2105-19.

[0194] The ability to mineralize the extracellular matrix and to generate bone is not unique to MSCs. In fact, ASCs possess a similar ability to differentiate into osteoblasts under similar conditions. Human ASCs offer a unique advantage in contrast to other cell sources. The multipotent characteristics of ASCs, as wells as their abundance in the human body, make these cells a popular source in tissue engineering applications. This consistent cell-based new product has the potential to be effective for bone regeneration.

Claims

What is claimed is:

1. A method of making an allograft product for enhancing bone formation, the method consisting of:

providing a bone substrate obtained from a human, cadaveric donor;

providing a tissue comprising mesenchymal stem cells and unwanted cells from the human, cadaveric donor;

processing the tissue to form a cell suspension comprising the mesenchymal stem cells and the unwanted cells;

adding the cell suspension to the bone substrate to form a seeded bone substrate;

incubating the seeded bone substrate for a period of time to allow the mesenchymal stem cells to adhere to the bone substrate; and

rinsing the seeded bone substrate to remove the unwanted cells from the bone substrate;

thereby making the allograft product for enhancing bone formation, wherein the allograft product comprises bone substrate with mesenchymal stem cells adhered thereto.

2. The method in accordance with claim 1, wherein the tissue is selected from the group consisting of adipose tissue, muscle tissue, and bone marrow tissue.

3. A method in accordance with claim 1, wherein the bone substrate comprises bone tissue that has been subjected to a demineralization process.

4. A method in accordance with claim 1, wherein the bone substrate comprises cortical bone.

5. A method in accordance with claim 1, wherein the bone substrate comprises cancellous bone.

6. A method in accordance with claim 1, wherein the bone substrate comprises ground bone.

7. A method in accordance with claim 1, wherein the bone substrate comprises both cortical and cancellous bone.

8. A method in accordance with claim 1, wherein the bone substrate comprises demineralized cancellous bone.

9. A method in accordance with claim 1, wherein the bone substrate comprises fully demineralized bone, partially demineralized bone, or a combination thereof.

10. A method in accordance with claim 1, wherein the incubating is performed for about 1-3 days.

11. A method in accordance with claim 1, wherein the incubating is performed for no more than about 3 days.

12. A method in accordance with claim 1, wherein the incubating consists of incubating the seeded bone substrate in growth medium.

13. A method of making an allograft product for enhancing bone formation, the method consisting of:

providing a bone substrate obtained from a human, cadaveric donor;

providing a tissue comprising mesenchymal stem cells and unwanted cells from the human, cadaveric donor;

processing the tissue to form a cell suspension comprising the mesenchymal stem cells and the unwanted cells;

adding the cell suspension to the bone substrate to form a seeded bone substrate;

incubating the seeded bone substrate for a period of time to allow the mesenchymal stem cells to adhere to the bone substrate;

rinsing the seeded bone substrate to remove the unwanted cells from the bone substrate; and

placing the seeded bone substrate into a cryopreservation medium;

thereby making the allograft product for enhancing bone formation, wherein the allograft product comprises bone substrate with mesenchymal stem cells adhered thereto.

14. The method in accordance with claim 13, wherein the tissue is selected from the group consisting of adipose tissue, muscle tissue, and bone marrow tissue.

15. A method in accordance with claim 13, wherein the bone substrate comprises bone tissue that has been subjected to a demineralization process.

16. A method in accordance with claim 13, wherein the bone substrate comprises cortical bone.

17. A method in accordance with claim 13, wherein the bone substrate comprises cancellous bone.

18. A method in accordance with claim 13, wherein the bone substrate comprises ground bone.

19. A method in accordance with claim 13, wherein the bone substrate comprises both cortical and cancellous bone.

20. A method in accordance with claim 13, wherein the bone substrate comprises demineralized cancellous bone.

21. A method in accordance with claim 13, wherein the bone substrate comprises fully demineralized bone, partially demineralized bone, or a combination thereof.

22. A method in accordance with claim 13, wherein the incubating is performed for about 1-3 days.

23. A method in accordance with claim 13, wherein the incubating is performed for no more than about 3 days.

24. A method in accordance with claim 13, wherein the incubating consists of incubating the seeded bone substrate in growth medium.

Patent Citations (305)

Patent	Date	Inventor	Cited By
US4627853(A)	1986-12-01	Campbell et al.	Applicant
US5073373(A)	1991-12-01	O'Leary et al.	Applicant
US5197985(A)	1993-03-01	Caplan et al.	Applicant
US5284655(A)	1994-02-01	Bogdansky et al.	Applicant
US5298254(A)	1994-03-01	Prewett et al.	Applicant
US5306304(A)	1994-04-01	Gendler	Applicant
US5314476(A)	1994-05-01	Prewett et al.	Applicant
US5405390(A)	1995-04-01	O'Leary et al.	Applicant
US5439684(A)	1995-08-01	Prewett et al.	Applicant
US5486359(A)	1996-01-01	Caplan	Applicant
US5507813(A)	1996-04-01	Dowd et al.	Applicant
US5510396(A)	1996-04-01	Prewett et al.	Applicant
US5516532(A)	1996-05-01	Atala et al.	Applicant
US5531791(A)	1996-07-01	Wolfinbarger	Applicant
US5556379(A)	1996-09-01	Wolfinbarger	Applicant
US5676146(A)	1997-10-01	Scarborough	Applicant
US5707962(A)	1998-01-01	Chen et al.	Applicant
US5736396(A)	1998-04-01	Bruder et al.	Applicant
US5749874(A)	1998-05-01	Schwartz	Applicant
US5769899(A)	1998-06-01	Schwartz et al.	Applicant
US5786207(A)	1998-07-01	Katz et al.	Applicant
US5788941(A)	1998-08-01	Dalmasso et al.	Applicant
US5797871(A)	1998-08-01	Wolfinbarger	Applicant
US5811094(A)	1998-09-01	Caplan	Applicant
US5820581(A)	1998-10-01	Wolfinbarger	Applicant
US5827740(A)	1998-10-01	Pittenger	Applicant
US5895426(A)	1999-04-01	Scarborough et al.	Applicant
US5899939(A)	1999-05-01	Boyce et al.	Applicant
US5906934(A)	1999-05-01	Grande et al.	Applicant
US5908782(A)	1999-06-01	Marshak et al.	Applicant
US5908784(A)	1999-06-01	Johnstone et al.	Applicant
US5942225(A)	1999-08-01	Bruder et al.	Applicant
US5948428(A)	1999-09-01	Lee et al.	Applicant
US5962325(A)	1999-10-01	Naughton et al.	Applicant
US5972368(A)	1999-10-01	McKay	Applicant
US5976104(A)	1999-11-01	Wolfinbarger	Applicant
US5977034(A)	1999-11-01	Wolfinbarger	Applicant
US5977432(A)	1999-11-01	Wolfinbarger	Applicant
US6022540(A)	2000-02-01	Bruder et al.	Applicant
US6024735(A)	2000-02-01	Wolfinbarger	Applicant
US6030635(A)	2000-02-01	Gertzman et al.	Applicant
US6030836(A)	2000-02-01	Thiede	Applicant
US6090998(A)	2000-07-01	Grooms et al.	Applicant
US6143012(A)	2000-11-01	Gausepohl	Applicant
US6174333(B1)	2001-01-01	Kadiyala	Applicant
US6180606(B1)	2001-01-01	Chen et al.	Applicant
US6189537(B1)	2001-02-01	Wolfinbarger	Applicant
US6200347(B1)	2001-03-01	Anderson et al.	Applicant
US6200606(B1)	2001-03-01	Peterson	Applicant
US6214369(B1)	2001-04-01	Grande	Applicant
US6225119(B1)	2001-05-01	Qasba et al.	Applicant
US6235316(B1)	2001-05-01	Adkisson	Applicant
US6291240(B1)	2001-09-01	Mansbridge et al.	Applicant
US6294041(B1)	2001-09-01	Boyce et al.	Applicant
US6294187(B1)	2001-09-01	Boyce et al.	Applicant
US6305379(B1)	2001-10-01	Wolfinbarger	Applicant
US6315795(B1)	2001-11-01	Scarborough et al.	Applicant
US6322784(B1)	2001-11-01	Pittenger et al.	Applicant
US6326018(B1)	2001-12-01	Gertzman et al.	Applicant
US6340477(B1)	2002-01-01	Anderson	Applicant
US6355239(B1)	2002-03-01	Bruder	Applicant
US6371988(B1)	2002-04-01	Pafford et al.	Applicant
US6379953(B1)	2002-04-01	Bruder et al.	Applicant
US6387367(B1)	2002-05-01	Davis-Sproul et al.	Applicant
US6391297(B1)	2002-05-01	Halvorsen	Applicant
US6436138(B1)	2002-08-01	Dowd	Applicant
US6464983(B1)	2002-10-01	Grotendorst	Applicant
US6478825(B1)	2002-11-01	Winterbottom et al.	Applicant
US6482231(B1)	2002-11-01	Abatangelo	Applicant
US6482233(B1)	2002-11-01	Aebi et al.	Applicant
US6511509(B1)	2003-01-01	Ford et al.	Applicant
US6534095(B1)	2003-03-01	Moore-Smith et al.	Applicant
US6541024(B1)	2003-04-01	Kadiyala et al.	Applicant
US6555374(B1)	2003-04-01	Gimble et al.	Applicant
US6576015(B2)	2003-06-01	Geistlich et al.	Applicant
US6616698(B2)	2003-09-01	Scarborough	Applicant
US6645764(B1)	2003-11-01	Adkisson	Applicant
US6652593(B2)	2003-11-01	Boyer, II et al.	Applicant
US6685626(B2)	2004-02-01	Wironen	Applicant
US6696073(B2)	2004-02-01	Boyce et al.	Applicant
US6716245(B2)	2004-04-01	Pasquet et al.	Applicant
US6746484(B1)	2004-06-01	Liu et al.	Applicant
US6777231(B1)	2004-08-01	Katz	Applicant
US6800480(B1)	2004-10-01	Bodnar et al.	Applicant
US6805713(B1)	2004-10-01	Carter et al.	Applicant
US6808585(B2)	2004-10-01	Boyce et al.	Applicant
US6835377(B2)	2004-12-01	Goldberg et al.	Applicant
US6837907(B2)	2005-01-01	Wolfinbarger	Applicant
US6855169(B2)	2005-02-01	Boyer et al.	Applicant
US6863694(B1)	2005-03-01	Boyce et al.	Applicant
US6863900(B2)	2005-03-01	Kadiyala	Applicant
US6902578(B1)	2005-06-01	Anderson et al.	Applicant
US6911045(B2)	2005-06-01	Shimp	Applicant
US6911212(B2)	2005-06-01	Gertzman et al.	Applicant
US6989030(B1)	2006-01-01	Ohgushi	Applicant
US6998135(B1)	2006-02-01	Sunwoo et al.	Applicant
US7018412(B2)	2006-03-01	Ferreira et al.	Applicant
US7029666(B2)	2006-04-01	Bruder et al.	Applicant
US7048765(B1)	2006-05-01	Grooms et al.	Applicant
US7115146(B2)	2006-10-01	Boyer, II et al.	Applicant
US7132110(B2)	2006-11-01	Kay et al.	Applicant
US7153500(B2)	2006-12-01	Qasba et al.	Applicant
US7163691(B2)	2007-01-01	Knaack et al.	Applicant
US7172629(B2)	2007-02-01	McKay	Applicant
US7179649(B2)	2007-02-01	Halvorsen	Applicant
US7241874(B2)	2007-07-01	Thorne	Applicant
US7259011(B2)	2007-08-01	Lucas	Applicant
US7291450(B2)	2007-11-01	Sowemimo-Coker et al.	Applicant
US7297540(B2)	2007-11-01	Mitrani	Applicant
US7316822(B2)	2008-01-01	Binette et al.	Applicant
US7323011(B2)	2008-01-01	Shepard et al.	Applicant
US7335381(B2)	2008-02-01	Malinin	Applicant
US7338517(B2)	2008-03-01	Yost et al.	Applicant
US7361195(B2)	2008-04-01	Schwartz et al.	Applicant
US7371409(B2)	2008-05-01	Petersen et al.	Applicant
US7470537(B2)	2008-12-01	Hedrick et al.	Applicant
US7473420(B2)	2009-01-01	Fraser et al.	Applicant
US7498040(B2)	2009-03-01	Masinaei et al.	Applicant
US7501115(B2)	2009-03-01	Fraser et al.	Applicant
US7582309(B2)	2009-09-01	Rosenberg et al.	Applicant
US7595043(B2)	2009-09-01	Hedrick et al.	Applicant
US7608113(B2)	2009-10-01	Boyer et al.	Applicant
US7622562(B2)	2009-11-01	Thorne et al.	Applicant
US7625581(B2)	2009-12-01	Laredo et al.	Applicant
US7662185(B2)	2010-02-01	Alfaro et al.	Applicant
US7662625(B2)	2010-02-01	Stern et al.	Applicant
US7687059(B2)	2010-03-01	Fraser et al.	Applicant
US7732126(B2)	2010-06-01	Zhang et al.	Applicant
US7753963(B2)	2010-07-01	Boyer et al.	Applicant
US7763071(B2)	2010-07-01	Bianchi et al.	Applicant
US7776089(B2)	2010-08-01	Bianchi et al.	Applicant
US7785634(B2)	2010-08-01	Borden	Applicant
US7807458(B2)	2010-10-01	Schiller	Applicant
US7811608(B2)	2010-10-01	Kay et al.	Applicant
US7815926(B2)	2010-10-01	Syring et al.	Applicant
US7837740(B2)	2010-11-01	Semler et al.	Applicant
US7863043(B2)	2011-01-01	Gentry et al.	Applicant
US7875296(B2)	2011-01-01	Binette et al.	Applicant
US7879103(B2)	2011-02-01	Gertzman et al.	Applicant
US7883511(B2)	2011-02-01	Fernyhough	Applicant
USRE42208(E)	2011-03-01	Truncale et al.	Applicant
US7914779(B2)	2011-03-01	Hariri	Applicant
US7931692(B2)	2011-04-01	Sybert et al.	Applicant
US8002813(B2)	2011-08-01	Scarborough et al.	Applicant
US8002837(B2)	2011-08-01	Stream et al.	Applicant
US8021692(B2)	2011-09-01	Hiles	Applicant
US8025896(B2)	2011-09-01	Malaviya et al.	Applicant
US8039016(B2)	2011-10-01	Drapeau et al.	Applicant
USRE43258(E)	2012-03-01	Truncale et al.	Applicant
US8133421(B2)	2012-03-01	Boyce et al.	Applicant
US8137702(B2)	2012-03-01	Binette et al.	Applicant
US8163549(B2)	2012-04-01	Yao et al.	Applicant
US8167943(B2)	2012-05-01	Carter et al.	Applicant
US8197474(B2)	2012-06-01	Scarborough et al.	Applicant
US8202539(B2)	2012-06-01	Behnam et al.	Applicant
US8221500(B2)	2012-07-01	Truncale et al.	Applicant
US8268008(B2)	2012-09-01	Betz et al.	Applicant
US8292968(B2)	2012-10-01	Truncale et al.	Applicant
US8328876(B2)	2012-12-01	Behnam et al.	Applicant
US8334135(B2)	2012-12-01	Rodriguez et al.	Applicant
US8343229(B2)	2013-01-01	Coale	Applicant
US8357384(B2)	2013-01-01	Behnam et al.	Applicant
US8389017(B1)	2013-03-01	Starling et al.	Applicant
US8399010(B2)	2013-03-01	McKay	Applicant
US8403991(B2)	2013-03-01	Ullrich, Jr. et al.	Applicant
US8409623(B2)	2013-04-01	Shim et al.	Applicant
US8435566(B2)	2013-05-01	Behnam et al.	Applicant
US8496970(B2)	2013-07-01	Binette et al.	Applicant
US8506632(B2)	2013-08-01	Ganem et al.	Applicant
US8551176(B2)	2013-10-01	Ullrich, Jr. et al.	Applicant
US8563040(B2)	2013-10-01	Marchosky	Applicant
US8574825(B2)	2013-11-01	Shelby et al.	Applicant
US8585766(B2)	2013-11-01	Ullrich, Jr. et al.	Applicant
US8637004(B2)	2014-01-01	Danilkovich et al.	Applicant
US8652214(B2)	2014-02-01	Fritz et al.	Applicant
US8652458(B2)	2014-02-01	Jackson et al.	Applicant
US8722075(B2)	2014-05-01	Shimp et al.	Applicant
US8734525(B2)	2014-05-01	Behnam et al.	Applicant
US8758791(B2)	2014-06-01	McKay	Applicant
US8771368(B2)	2014-07-01	McKay	Applicant
US8834928(B1)	2014-09-01	Truncale et al.	Applicant
US8859007(B2)	2014-10-01	Carter et al.	Applicant
US8865199(B2)	2014-10-01	Coleman et al.	Applicant
US8883210(B1)	2014-11-01	Truncale et al.	Applicant
US8945535(B2)	2015-02-01	Steinwachs et al.	Applicant
US8992964(B2)	2015-03-01	Shelby et al.	Applicant
US9029077(B2)	2015-05-01	Song et al.	Applicant
US9050178(B2)	2015-06-01	Barry et al.	Applicant
US9095524(B2)	2015-08-01	Warnke et al.	Applicant
US9162012(B2)	2015-10-01	Benham et al.	Applicant
US9180166(B2)	2015-11-01	Arinzeh et al.	Applicant
US9192695(B2)	2015-11-01	Shi	Applicant
US9193948(B2)	2015-11-01	Nicoll et al.	Applicant
US2002/0192263(A1)	2002-12-01	Merboth et al.	Applicant
US2003/0050709(A1)	2003-03-01	Noth et al.	Applicant
US2003/0161817(A1)	2003-08-01	Young et al.	Applicant
US2004/0043006(A1)	2004-03-01	Badylak et al.	Applicant
US2004/0078090(A1)	2004-04-01	Binette et al.	Applicant
US2005/0048036(A1)	2005-03-01	Hedrick et al.	Applicant
US2006/0134781(A1)	2006-06-01	Yang et al.	Applicant
US2006/0210643(A1)	2006-09-01	Truncale et al.	Applicant
US2007/0014729(A1)	2007-01-01	Farhat et al.	Applicant
US2007/0020248(A1)	2007-01-01	Everaerts et al.	Applicant
US2007/0134216(A1)	2007-06-01	Harlow et al.	Applicant
US2007/0179607(A1)	2007-08-01	Hodorek et al.	Applicant
US2007/0185231(A1)	2007-08-01	Liu et al.	Applicant
US2007/0212676(A1)	2007-09-01	Takakura	Applicant
US2007/0249044(A1)	2007-10-01	Desai et al.	Applicant
US2007/0258963(A1)	2007-11-01	Danilkovitch et al.	Applicant
US2007/0265705(A1)	2007-11-01	Gaissmaier et al.	Applicant
US2008/0058953(A1)	2008-03-01	Scarborough	Applicant
US2008/0249632(A1)	2008-10-01	Stone et al.	Applicant
US2008/0249638(A1)	2008-10-01	Asgari	Applicant
US2008/0269895(A1)	2008-10-01	Steinwachs et al.	Applicant
US2008/0286268(A1)	2008-11-01	Johnson	Applicant
US2008/0305145(A1)	2008-12-01	Shelby et al.	Applicant
US2008/0306610(A1)	2008-12-01	Wang et al.	Applicant
US2009/0024223(A1)	2009-01-01	Chen et al.	Applicant
US2009/0041730(A1)	2009-02-01	Barry et al.	Applicant
US2009/0053279(A1)	2009-02-01	Badylak et al.	Applicant
US2009/0202977(A1)	2009-08-01	Ott et al.	Applicant
US2010/0049322(A1)	2010-02-01	McKay	Applicant
US2010/0124776(A1)	2010-05-01	Shi	Applicant
US2010/0168869(A1)	2010-07-01	Long et al.	Applicant
US2010/0196333(A1)	2010-08-01	Gaskins et al.	Applicant
US2010/0241228(A1)	2010-09-01	Syring et al.	Applicant
US2010/0247494(A1)	2010-09-01	Gregory et al.	Applicant
US2011/0045044(A1)	2011-02-01	Masinaei et al.	Applicant
US2011/0070271(A1)	2011-03-01	Truncale et al.	Applicant
US2011/0182961(A1)	2011-07-01	McKay	Applicant
US2011/0262404(A1)	2011-10-01	Badoer et al.	Applicant
US2011/0262516(A1)	2011-10-01	Zheng et al.	Applicant
US2011/0293577(A1)	2011-12-01	Vesey	Applicant
US2011/0311496(A1)	2011-12-01	Pittenger et al.	Applicant
US2011/0318314(A1)	2011-12-01	Aggarwal et al.	Applicant
US2012/0035276(A1)	2012-02-01	Spievack	Applicant
US2012/0082704(A1)	2012-04-01	Phillips et al.	Applicant
US2012/0087958(A1)	2012-04-01	Dufrane et al.	Applicant
US2012/0148537(A1)	2012-06-01	Chan et al.	Applicant
US2012/0148548(A1)	2012-06-01	Barry et al.	Applicant
US2012/0189707(A1)	2012-07-01	Chun et al.	Applicant
US2012/0213859(A1)	2012-08-01	Shelby et al.	Applicant
US2012/0251609(A1)	2012-10-01	Huang et al.	Applicant
US2012/0259415(A1)	2012-10-01	Van Dyke et al.	Applicant
US2012/0297550(A1)	2012-11-01	Ngo et al.	Applicant
US2012/0308529(A1)	2012-12-01	Zanotti et al.	Applicant
US2013/0004464(A1)	2013-01-01	Nadal-Ginard	Applicant
US2013/0013068(A1)	2013-01-01	Forsell et al.	Applicant
US2013/0122095(A1)	2013-05-01	Kestler et al.	Applicant
US2013/0131804(A1)	2013-05-01	Barry et al.	Applicant
US2013/0149294(A1)	2013-06-01	Rueger et al.	Applicant
US2013/0189338(A1)	2013-07-01	Drapeau et al.	Applicant
US2013/0195809(A1)	2013-08-01	Crawford et al.	Applicant
US2013/0195810(A1)	2013-08-01	Crawford et al.	Applicant
US2013/0197654(A1)	2013-08-01	Samuelson et al.	Applicant
US2013/0316454(A1)	2013-11-01	Lu et al.	Applicant
US2014/0024115(A1)	2014-01-01	Bogdansky et al.	Applicant
US2014/0037598(A1)	2014-01-01	Jansen et al.	Applicant
US2014/0093543(A1)	2014-04-01	Morreale	Applicant
US2014/0105872(A1)	2014-04-01	Danilkovich et al.	Applicant
US2014/0112894(A1)	2014-04-01	Zheng et al.	Applicant
US2014/0121772(A1)	2014-05-01	Emerton et al.	Applicant
US2014/0127177(A1)	2014-05-01	Tom et al.	Applicant
US2014/0161776(A1)	2014-06-01	Aggarwal et al.	Applicant
US2014/0170232(A1)	2014-06-01	Shelby et al.	Applicant
US2014/0205674(A1)	2014-07-01	Wei	Applicant
US2014/0208980(A1)	2014-07-01	Song et al.	Applicant
US2014/0212471(A1)	2014-07-01	Drapeau et al.	Applicant
US2014/0212499(A1)	2014-07-01	Cooper et al.	Applicant
US2014/0220142(A1)	2014-08-01	Song et al.	Applicant
US2014/0227339(A1)	2014-08-01	Jackson et al.	Applicant
US2014/0234272(A1)	2014-08-01	Vesey et al.	Applicant
US2014/0255356(A1)	2014-09-01	Victor et al.	Applicant
US2014/0255506(A1)	2014-09-01	Behnam et al.	Applicant
US2014/0277570(A1)	2014-09-01	Behnam et al.	Applicant
US2014/0286911(A1)	2014-09-01	Shi et al.	Applicant
US2014/0314822(A1)	2014-10-01	Carter et al.	Applicant
US2014/0342013(A1)	2014-11-01	He et al.	Applicant
US2015/0004247(A1)	2015-01-01	Carter et al.	Applicant
US2015/0010506(A1)	2015-01-01	Jansen et al.	Applicant
US2015/0012107(A1)	2015-01-01	Koford et al.	Applicant
US2015/0030571(A1)	2015-01-01	Tremolada	Applicant
US2015/0037387(A1)	2015-02-01	Coleman et al.	Applicant
US2015/0093429(A1)	2015-04-01	Carter et al.	Applicant
US2015/0174295(A1)	2015-06-01	Woodell-May et al.	Applicant
US2015/0182667(A1)	2015-07-01	Guelcher et al.	Applicant
US2015/0202345(A1)	2015-07-01	Shelby et al.	Applicant
US2015/0202346(A1)	2015-07-01	Shelby et al.	Applicant
US2015/0251361(A1)	2015-09-01	Meyer et al.	Applicant
US2015/0258244(A1)	2015-09-01	Shelby et al.	Applicant
US2015/0306278(A1)	2015-10-01	McKay	Applicant
US2016/0030639(A1)	2016-02-01	Shi	Applicant
US2016/0067377(A1)	2016-03-01	Shi et al.	Applicant
US2016/0144076(A1)	2016-05-01	Matheny et al.	Applicant
US2016/0263284(A1)	2016-09-01	Shi	Applicant
WO9726326	1997-07-01		Applicant
WO200272(A2)	2002-01-01		Applicant
WO2006062989(A1)	2006-06-01		Applicant
WO2006112390(A1)	2006-10-01		Applicant
WO2008036374(A2)	2008-03-01		Applicant
WO2008041854(A1)	2010-04-01		Applicant
WO2010059565	2010-05-01		Applicant
WO2012135205	2012-10-01		Applicant
WO2013047936	2013-04-01		Applicant
WO2015053739(A1)	2015-04-01		Applicant

Non-Patent Literature (140)

Yin et al., Vitreous cryopreservation of tissue engineered bone composed of bone marrow mesenchymal stem cells and partially demineralized bone matrix, Cryobiology, vol. 59 (2009), pp. 180-187.Examiner
Ahn, et al., “In Vivo Osteogenic Differentiation of Human Adipose-Derived Stem Cells in an Injectable in Situ-Forming D Gel Scaffold”, Tissue Eng Part A, 2009, vol. 15, No. 7, pp. 1821-1832.Applicant
Angelo, “Micronutrients and Bone Health”, Linus Pauling Institute, [accessed on Oct. 20, 2015, retrieved from the Internet: <http://lpi.oregonstate.edu/infocenter/bonehealth.html>].Applicant
Anghileri, et al., “Neuronal Differentiation Potential of Human Adipose-Derived Mesenchymal Stem Cells”, Stem Cells Dev., 2008, vol. 17, No. 5, pp. 909-916.Applicant
Arnalich-Montiel, et al., “Adipose-Derived Stem Cells Are a Source for Cell Therapy of the Corneal Stroma”, Stem Cells, 2008, vol. 26, pp. 570-579.Applicant
Aubin, et al., Isolation of Bone Cell Clones with Differences in Growth, Hormone Responses, and Extracellular Matrix Production, 92 J. Cell. Biol. 452-61 (1982).Applicant
Baer, et al., “Adipose-Derived Mesenchymal Stromal/Stem Cells: Tissue Localization, Characterization, and Heterogeneity,” Stem Cells International, Hidsawi Publishing, vol. 2012, Article ID 812693, 11 pages.Applicant
Barry, , “Mesenchymal stem cell therapy in joint disease,” Tissue engineering of cartilage and bone: Novartis Foundation Symposium, 249, 86-102 (2003).Applicant
Bennett, et al., Adipocytic cells cultured from marrow have osteogenic potential, 99 J. Cell. Sci. 131 (1991).Applicant
Bruder, et al., “Tissue Engineering of Bone. Cell Based Strategies”, Clin Orthop Relat Res., 1999, vol. 367 Suppl., pp. S68-S83.Applicant
Bunnell, et al., “Adipose-Derived Stem Cells: Isolation, Expansion and Differentiation”, Methods, 2008, vol. 45, No. 2, pp. 115-120.Applicant
Butt, et al., “Stimulation of Peri-Implant Vascularization with Bone Marrow-Derived Progenitor Cells: Monitoring by in Vivo EPR Oximetry”, Tissue Eng., 2007, vol. 13, No. 8, pp. 2053-2061.Applicant
Cha, et al., “Stem cells in cutaneous wound healing,” Clinics in Dermatology 25:73-78 (2007).Applicant
Chamberlain, et al., “Concise Review: Mesenchymal Stem Cells: Their Phenotype, Differentiation Capacity, Immunological Features, and Potential for Homing,” Stem Cells (2007) 25:2739-2749.Applicant
Chen, et al., “Differentiation of Rat Adipose-Derived Stem Cells into Smooth-Muscle-Like Cells in Vitro”, Zhonghua Nan Ke Xue, 2009, vol. 15, No. 5, pp. 425-430.Applicant
Chen, et al., “Study of MSCs in Vitro Cultured on Demineralized Bone Matrix of Mongrel”, Shanghai Kou Qiang Yi Xue, 2007, vol. 16, No. 3, pp. 255-258.Applicant
Cheng, et al., “Chondrogenic Differentiation of Adipose-Derived Adult Stem Cells by a Porous Scaffold Derived from Native Articular Cartilage Extracellular Matrix”, Tissue Eng Part A, 2009, vol. 15, No. 2, pp. 231-241.Applicant
Chu, et al. “Leukocytes in blood transfusion: adverse effects and their prevention,” HKMJ (1999) 5:280-284.Applicant
Chung, et al., “Bladder Reconstitution with Bone Marrow Derived Stem Cells Seeded on Small Intestinal Submucosa Improves Morphological and Molecular Composition,” J. Urology 174:353-359 (2005).Applicant
Cui, et al., “Repair of Cranial Bone Defects with Adipose Derived Stem Cells and Coral Scaffold in a Canine Model”, Biomaterials, 2007, vol. 28, pp. 5477-5486.Applicant
Damien, et al., “Bone Graft and Bone Graft Substitutes: A Review of Current Technology and Applications”, Journal Applied Biomaterials, 1991, vol. 2, pp. 187-208.Applicant
De Girolamo, et al., “Human Adipose-Derived Stem Cells as Future Tools in Tissue Regeneration: Ostegenic Differentiation and Cell-Scaffold Interaction”, Int J Artif Organs, 2008, vol. 31, No. 6, pp. 467-479.Applicant
De Girolamo, et al. “Osteogenic Differentiation of Human Adipose-Derived Stem Cells: Comparison of Two Different Inductive Media”, J Tissue Eng Regen Med, 2007, vol. 1, No. 2, pp. 154-157.Applicant
De Long, et al., “Bone Grafts and Bone Graft Substitutes in Orthpaedic Trauma Surgery. A Critical Analysis”, The Journal of Bone & Joint Surgery, 2007, vol. 89, pp. 649-658.Applicant
De Ugarte, et al., “Comparison of Multi-Lineage Cells from Human Adipose Tissue and Bone Marrow”, Cells Tissues Organs, 2003, vol. 174, No. 3, pp. 101-109.Applicant
Diekman, et al., “Chondrogenesis of adult stem cells from adipose tissue and bone marrow: Induction by growth factors and cartilage-derived matrix,” Tissue Engineering, 16, 523-533 (2010); pub. online Sep. 2009.Applicant
Di Bella, et al., “Bone Regeneration in a Rabbit Critical-Sized Skull Defect Using Autologous Adipose-Derived Cells”, Tissue Eng Part A., 2008, vol. 14, No. 4, pp. 483-490.Applicant
Dubois, et al., Methods in Molecular Biology, vol. 449, 2008, pp. 69-79.Applicant
Elabd, et al., “Human Adipose Tissue-Derived Multipotent Stem Cells Differentiate in Vitro and in Vivo into Ostecyte-Like Cells”, Biochem Biophys Res Commun., 2007, vol. 361, No. 2, pp. 342-348.Applicant
Eyre, D. Collagen of articular cartilage, Arthritis Res. 4:30-35 (2002).Applicant
Filipak, Michiko et al., Tumor Necrosis Factor Inhibits the Terminal Event in Mesenchymal Stem Cell Differentiation, 137, J. Cell. Phys. 368, 367-73 (1988).Applicant
Flynn, L., et al., “Adipose Tissue Engineering With Naturally Derived Scaffolds and Adipose-Derived Stem Cells”, Biomaterials, 2007, vol. 28, No. 26, pp. 3834-3842.Applicant
Flynn, L., et al., “Proliferation and Differentiation of Adipose-Derived Stem Cells on Naturally Derived Scaffolds”, Biomaterials, 2008, vol. 29, No. 12, pp. 1862-1871.Applicant
Fraser, JK, et al., “Adipose-Derived Stem Cells”, Methods Mol Bioi., 2008, vol. 449, pp. 59-67.Applicant
Freed, L.E. et al., Joint resurfacing using allograft chondrocytes and synthetic biodegradable polymer scaffolds, 28 J. Biomed. Mater. Res. 891 (1994).Applicant
Gamradt, et al., “Bone Graft for Revision Hip Arthroplasty: Biology and Future Applications”, Clin Orthop Relat D Res., 2003, vol. 417, pp. 183-194.Applicant
Gimble, et al., “Differentiation Potential of Adipose Derived Adult Stem (ADAS) Cells”, Curr Top Dev Bioi., 2003, vol. 58, pp. 137-160.Applicant
Gimble, J., et al., “Adipose-Derived Adult Stem Cells: Isolation, Characterization, and Differentiation Potential”, Cytotherapy, 2003, vol. 5, No. 5, pp. 362-369.Applicant
Glowacki, et al., “Application of the Biological Principle of Induced Osteogenesis for Craniofacial Defects”, The Lancet, 1981, pp. 959-962.Applicant
Goh, et al., “Cryopreservation Characteristics of Adipose Derived Stem Cells: Maintenance of 6 Differentiation Potential and Viability”, Journal of Tissue Engineering and Regenerative Medicine, 2007, vol. 1, pp. 322-324.Applicant
Gomes, et al., “Tissue Engineering: Key Elements and Some Trends”, Macromolecular Bioscience, 2004, vol. 4, pp. 737-742.Applicant
Greenwald, et al., “Bone-Graft Substitutes: Facts, Fictions, and Applications”, The Journal of Bone & Joint Surgery, 2001, vol. 83, pp. 98-103.Applicant
Grewal, et al., “BMP-2 Does Not Influence the Osteogenic Fate of Human Adipose-Derived Stem Cells”, Plast Reconstr Surg., 2009, vol. 123(2 Suppl), pp. 158S-165S.Applicant
Grigoriadis, A.E. et al., Differentiation of Muscle, Fat, Cartilage, and Bone from Progenitor Cells Present in a Bone-derived Clonal Cell Population: Effect of Dexamethasone, 106, J. Cell. Biol. 2144, 2139-51 (1988).Applicant
Hayashi, et al., “Comparison of Osteogenic Ability of Rat Mesenchymal Stem Cells from Bone Marrow, Periosteum, and Adipose Tissue”, CalcifTissue Int., 2008, vol. 82, No. 3, pp. 238-247.Applicant
Hicok et al., Tissue Engineering, vol. 10, No. 3/4, 2004, pp. 371-382.Applicant
Honsawek, S., et al., Effects of Demineralized Bone Matrix on Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cells from Human Umbilical Cord, J Med Assoc Thai, 2006, vol. 89, Suppl 3, pp. 189-195.Applicant
Hutmacher, et al., “State of the Art and Future Directions of Scaffold-Based Bone Engineering from a Biomaterials Perspective”, Journal of Tissue Engineering and Regenerative Medicine, 2007, vol. 1, pp. 245-260.Applicant
International Search Report and Written Opinion dated Jun. 29, 2010 for application No. PCT/US2009/064611, 11 pages.Applicant
International Search Report issued in connection with corresponding International Application No. PCT/US13/63674, dated Jan. 29, 2014, 9 pages.Applicant
Jin, et al., “Tissue Engineered Cartilage from hTGF beta2 Transduced Human Adipose Derived Stem Cells Seeded in PLGA/Aiginate Compound in Vitro and in Vivo”, J Biomed Mater Res A, 2008, vol. 86, No. 4, pp. 1077-1087.Applicant
Jurgens, W.J.F. et al., “Effect of tissue-harvesting site on yield of stem cells derived from adipose tissue: implications for cell-based therapies,” Cell Tissue Res (2008) 332:415-426.Applicant
Kakudo, et al., “Bone Tissue Engineering Using Human Adipose-Derived Stem Cells and Honeycomb Collagen Scaffold”, J Biomed Mater Res A, 2008, vol. 84, No. 1, pp. 191-197.Applicant
Kasten, P., et al., “Ectopic Bone Formation Associated With Mesenchymal Stem Cells in a Resorbably Calcium D Deficient Hydroxyapatite Carrier”, Biomaterials, 2005, vol. 26, No. 29, pp. 5879-5889.Applicant
Kasten, P., et al., “Induction of Bone Tissue on Different Matrices: An In Vitro and a In Vivo Pilot Study in the SCID Mouse”, Z Orthop Ihre Grenzgeb., 2004, vol. 142, No. 4, pp. 467-475.Applicant
Kim, et al., “Chondrogenic Differentiation of Adipose Tissue-Derived Mesenchymal Stem Cells: Greater Doses of Growth Factor are Necessary”, J Orthop Res, 2009, vol. 27, No. 5, pp. 612-619.Applicant
Kim, et al., “Direct Comparison of Human Mesenchymal Stem Cells Derived from Adipose Tissues and Bone Marrow in Mediating Neovascularization in Response to Vascular Ischemia”, Cell Physiol Biochem, 2007, vol. 20, No. 6, pp. 867-876.Applicant
Kingham, et al., “Adipose-Derived Stem Cells Differentiate into a Schwann Cell Phenotype and Promote Neurite Outgrowth in Vitro”, Exp Neurol., 2007, vol. 207, No. 2, pp. 267-274.Applicant
Ko, et al., “In Vitro Osteogenic Differentiation of Human Mesenchymal Stem Cells and In Vivo Bone Formation in Composite Nanofiber Meshes”, Tissue Eng Part A., 2008, vol. 14, No. 12, pp. 2105-2119.Applicant
Langer, et al., “Tissue Engineering”, Science, 1993, vol. 260, No. 5110, pp. 920-926.Applicant
Le Blanc, K., et al., “HLA Expression and Immunologic Properties of Differentiated and Undifferentiated Mesenchymal Stem Cells”, Experimented Hematology, 2003, pp. 890-896.Applicant
Lecoeur, L.et al., In vitro induction of osteogenic differentiation from non-osteogenic mesenchymal cells, 18 Biomaterials 989 (1997).Applicant
Lin, et al., “Comparison of Osteogenic Potentials of BMP4 Transduced Stem Cells from Autologous Bone Marrow and Fat Tissue in a Rabbit Model of Calvarial Defects”, CalcifTissue Int., 2009, vol. 85, No. 1, pp. 55-56.Applicant
Liu, et al., “Evaluation of Partially Demineralized Osteoporotic Cancellous Bone Matrix Combined with 4 Human Bone Marrow Stromal Cells for Tissue Engineering: An In Vitro and In Vivo Study”, CalcifTissue Int., 2008, vol. 83, No. 3, pp. 176-185.Applicant
Liu, et al., “Evaluation of the Viability and Osteogenic Differentiation of Cryopreserved Human Adipose-Derived Stem Cells”, Cryobiology, 2008, vol. 57, pp. 18-24.Applicant
Liu, G., et al., “Tissue-Engineered Bone Formation with Cryopreserved Human Bone Marrow Mesenchymal Stem D Cells”, Cryobiology, 2008, vol. 56, No. 3, pp. 209-215.Applicant
Li, et al., “Bone Regeneration by Implantation of Adipose-Derived Stromal Cells Expressing BMP-2”, Biochem Biophys Res Commun, 2007, vol. 356, No. 4, pp. 836-842.Applicant
Masuno, H. et al., Synthesis of inactive nonsecretable high mannose-type lipoprotein lipase by cultured brown adipocytes of combined lipase-deficient cld/cld mice, 265 J. Biol. Chem. 1628 (1990).Applicant
Mauney, et al., “In Vitro and in Vivo Evaluation of Differentially Demineralized Cancellous Bone Scaffolds Combined with Human Bone Marrow Stromal Cells for Tissue Engineering”, Biomaterials, 2005, vol. 26, pp. 3173-3185.Applicant
Melhorn, et al., “Chondrogenesis of Adipose-Derived Adult Stem Cells in a Poly-Lactide-Co-Giycolide Scaffold”, 0 Tissue Eng Part A, 2009, vol. 15, No. 5, pp. 1159-1167.Applicant
Merceron, et al., “Adipose-Derived Mesenchymal Stem Cells and Biomaterials for Cartilage Tissue Engineering”, Joint Bone Spine, 2008, vol. 75, No. 6, pp. 672-674.Applicant
Minana, M-D et al., “IFATS Collection: Identification of Hemangioblasts in the Adult Human Adipose Tissue,” Stem Cells, 2008, 26:2696-2704.Applicant
Mischen, et al., “Metabolic and Functional Characterization of Human Adipose-Derived Stem Cells in Tissue Engineering”, Plast Reconstr Surg., 2008, vol. 22, No. 3, pp. 725-738.Applicant
Mitchell, J.B., e al. “Immunophenotype of Human Adipose-Derived Cells: Temporal Changes in Stromal-Associated and Stem Cell-Associated Markers”, Stem Cells, 2006, vol. 24, pp. 376-385.Applicant
Mizuno, “Adipose-Derived Stem Cells for Tissue Repair and Regeneration: Ten Years of Research and a Literature Review”, J Nippon Med Sch., 2009, vol. 76, No. 2, pp. 56-66.Applicant
Mulliken, et al., “Induced Osteogenesis—The Biological Principle and Clinical Applications”, Journal of Surgical Research, 1984, vol. 37, pp. 487-496.Applicant
Mulliken, et al., “Use of Demineralized Allogeneic Bone Implants for the Correction of Maxillocraniofacial Deformities”, Ann Surg., 1981, vol. 194, No. 3, pp. 366-372.Applicant
Niemeyer, et al., “Comparison of Immunological Properties of Bone Marrow Stromal Cells and Adipose Tissue-Derived Stem Cells Before and After Osteogenic Differentiation in Vitro”, Tissue Eng., 2007, vol. 13, No. 1, pp. 111-121.Applicant
Nih et al., “Stem Cell Basics”, National Institute of Health, [accessed on Oct. 20, 2015, retrieved from the Internet: <http://stemcells.nih.gov/info/basics/pages/basics4.aspx>].Applicant
Noel, et al., “Cell Specific Differences Between Human Adipose-Derived and Mesenchymal-Stromal Cells Despite Similar Differentiation Potentials”, Exp Cell Res., 2008, vol. 314, No. 7, pp. 1575-1584.Applicant
Office Action dated Apr. 10, 2013 in U.S. Appl. No. 12/965,335, 41 pages.Applicant
Pate, D.W. et al., Isolation and differentiation of mesenchymal stem cells from rabbit muscle, 44 Surg Forum 587 (1993).Applicant
Prockop, O.J., et al., “Isolation and Characterization of Rapidly Self-Renewing Stem Cells from Cultures of Human Marrow Stromal Cells”, Cytotherapy, 2001, vol. 3, No. 5, pp. 393-396.Applicant
Qian, et al., “Reconstruction of Bone Using Tissue Engineering and Nanoscale Technology”, Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 2006, vol. 20, No. 5, pp. 560-564.Applicant
Rada, et al., “Adipose Tissue-Derived Stem Cells and Their Application in Bone and Cartilage Tissue Engineering”, Tissue Engineering: Part B, 2009, vol. 15, No. 2, pp. 113-125.Applicant
Reddi, “Morphogenesis and Tissue Engineering of Bone and Cartilage: Inductive Signals, Stem Cells, and Biomimetic Biomaterials”, Tissue Eng., 2000, vol. 6, No. 4, pp. 351-359.Applicant
Reddi, “Role of Morphogenetic Proteins in Skeletal Tissue Engineering and Regeneration”, Nat Biotechnol., 1998, vol. 16, No. 3, pp. 247-252.Applicant
Rhie, J-W. et al., “Chondrogenic differentiation of human adipose-derived stem cells in PLGA (Poly(Lactide-co-Glycolide Acid)) Scaffold,” Key Engineering Materials, 342, 345-348 (2007).Applicant
Rickard, D.J., Induction of rapid osteoblast differentiation in rat bone marrow stromal cell cultures by dexamethasone and BMP-2, 161 Dev. Biol. 218 (1994).Applicant
Rigotti, et al., “Clinical Treatment of Radiotherapy Tissue Damage by Lipoaspirate Transplant: A Healing Process Mediated by Adipose-Derived Adult Stem Cells”, Plast Reconstr Surg., 2007, vol. 119, No. 5, pp. 1409-1422.Applicant
Riordan, NH et al., “Non-expanded adipose stromal vascular fraction cell therapy for multiple sclerosis,” Journal of Translational Medicine 2009, 7:29, 9 pages.Applicant
Sakaguchi et al., Arthritis & Rheumatism, vol. 52, No. 8, Aug. 2005, pp. 2521-2529.Applicant
Salgado, A.J. et al., “Adult Stem Cells in Bone and Cartilage Tissue Engineering,”.Applicant
Singh, S. et al., “Leukocyte depletion for safe blood transfusion,” Biotechnol J (2009) 4:1140-1151.Applicant
Strem, et al., “Multipotential Differentiation of Adipose Tissue-Derived Stem Cells”, Keio J Med., 2005, vol. 54, No. 3, pp. 132-141.Applicant
Substrate. (n.d.). The American Heritage® Stedman's Medical Dictionary, [accessed on Oct. 20, 2015, retrieved from the Internet: <http://dictionary.reference.com/browse/substrate>.Applicant
Tapp, et al., “Adipose-Derived Stem Cells: Characterization and Current Application in Orthopaedic Tissue Repair”, Exp Bioi Med (Maywood), 2008.Applicant
Tapp, et al., “Adipose-Derived Stem Cells: Characterization and Current Application in Orthpaedic Tissue Repair”, Exp Bioi Med (Maywood), 2009, vol. 234, No. 1, pp. 1-9.Applicant
Tsiridis, et al., “In Vitro and In Vivo Optimization of Impaction Allografting by Demineralization and Addition of rh-OP-1”, J Orthop Res, 2007, vol. 25, No. 11, pp. 1425-1437.Applicant
Van Dijk, et al., “Differentiation of Human Adipose-Derived Stem Cells Towards Cardiomyocytes is Facilitated by Laminin”, Cell Tissue Res, 2008, vol. 334, No. 3, pp. 457-467.Applicant
Van, R.L. et al., Cytological and enzymological characterization of adult human adipocyte precursors in culture, 58 J. Clin. Invest. 699 (1976).Applicant
Varma, Mjo et al., “Phenotypical and Functional Characterization of Freshly Isolated Adipose Tissue-Derived Stem Cells,” Stem Cells and Development, 16:91-104 (2007).Applicant
Wang, et al., “Characterization of Demineralized Bone Matrix-Induced Osteogenesis in Rat Calvarial Bone Defects: III. Gene and Protein Expression”, Calcif Tissue Int., 2000, vol. 67, No. 4, pp. 314-320.Applicant
Wang, J., et al., “Characterization of Matrix-Induced Osteogenesis in Rat Calvarial Bone Defects: I. Differences in the Cellular Response to Demineralized Bone Matrix Implanted in Calvarial Defects and in Subcutaneous Sites”, Calcif Tissue Int., 1999, vol. 65, No. 2, pp. 156-165.Applicant
Wang, J., et al., “Characterization of Matrix-Induced Osteogenesis in Rat Calvarial Bone Defects: II. Origins of Bone-Forming Cells”, CalcifTissue Int., 1999, vol. 65, No. 6, pp. 486-493.Applicant
Wei, et al., “A Novel Injectable Scaffold for Cartilage Tissue Engineering Using Adipose-Derived Adult Stem Cells”, J Orthop Res., 2008, vol. 26, No. 1, pp. 27-33.Applicant
Wei, et al., “Adipose-Derived Stem Cells and Chondrogenesis”, Cytotherapy, 2007, vol. 9, No. 8, pp. 712-716.Applicant
Xie, et al., “The Performance of a Bone-Derived Scaffold Material in the Repair of Critical Bone Defects in a D Rhesus Monkey Model”, Biomaterials, 2007, vol. 28, No. 22, pp. 3314-3324.Applicant
Yang, Q. et al., “A cartilage ECM-derived 3-D porous acellular matrix scaffold for in vivo cartilage tissue engineering with PKH26-labeled chondrogenic bone marrow-derived mesenchymal stem cells,” Biomaterials, 29, 2378-2387 (2008).Applicant
Yoo, et al., “Comparison of Immunomodulatory Properties of Mesenchymal Stem Cells Derived from Adult Human Tissues”, Cell Immunol., 2009, vol. 259, No. 2, pp. 150-156.Applicant
Yoon, et al., “In Vivo Osteogenic Potential of Human Adipose-Derived Stem Cells/Poly Lactide-co-Giycolic Acid Constructs for Bone Regeneration in a Rat Critical-Sized Calvarial Defect Model”, Tissue Eng., 2007, vol. 13, No. 3, pp. 619-627.Applicant
Yoshimura, et al., “Comparison of Rat Mesenchymal Stem Cells Derived from Bone Marrow, Synovium, Periosteum, Adipose Tissue, and Muscle”, Cell Tissue Res., 2007, vol. 327, No. 3, pp. 449-462.Applicant
Young, H.E. et al, Pluripotent mesenchymal Stem Cells Reside within Avian Connective Tissue Matrices, 29A, In Vitro Cell. Div. Biol. 723-736 (1993).Applicant
Zhang, et al., “Adipose Tissue Engineering with Human Adipose-Derived Stem Cells and Fibrin Glue Injectable Scaffold”, Zhonghua Yi Zue Za Zhi, 2008, vol. 88, No. 38, pp. 2705-2709.Applicant
Zhao, L. et al., “The study of the feasibility of segmental bone defect repair with tissue-engineered bone membrane: a qualitative observation,” Strat. Traum. Limb Recon. 3:57-64 (2008).Applicant
Liu, G., et al., “Evaluation of Partially Demineralized Osteoporotic Cancellous Bone Matrix Combined with Human Bone Marrow Stromal Cells for Tissue Engineering: An In Vitro and In Vivo Study.” Calcif. Tissue Int. 83(3):176-185 (Sep. 2008).Applicant
Extended European Search Report for European Patent Application No. 16183850.3, dated Sep. 27, 2016, all pages.Applicant
Finkemeier, “Bone Grafting and Bone-Graft Substitutes”; The Journal of Cone and Joint Surgery; Mar. 2002; pp. 454-464; vol. 84-A, No. 3.Applicant
Gepstein, et al. “Bridging Large Defects in Bone by Demineralized Bone Matrix in the Form of a Powder”; The Journal of Bone and Joint Surgery; Sep. 1987; pp. 984-992; vol. 69-A, No. 7.Applicant
Jeon, et al., “In Vivo Bone Formation Following Transplantation of Human Adipose-Derived Stromal Cells That Are Not Differentiated Osteogenically”, Tissue Engineering Part A, vol. 14. No. 8., Aug. 2008, pp. 1285-1294.Applicant
Lovric et al., “The Effect of Processing Conditions of the Osteoinductivity of Sheep Demineralized Bone Matrix in an Ectopic Nude Rat Model: A Pilot Study”, ORS 2012 Annualk Meeting, Poster No. 0629.Applicant
Mauney. et al., “Osteogenic Differentiation of Human Bone Marrow Stromal Cells on Partially Demineralized Bone Scaffolds in Vitro”, Tissue Engineering, vol. 10. No. 1-2, Jan. 2004, pp. 81-92.Applicant
Pietrzak, et al., “Assay of Bone Morphogenetic Protein-2, -4, and -7 in Human Demineralized Bone Matrix”, Scientific Foundation, pp. 84-90, Jun. 1972.Applicant
Reddi, et al., “Biochemical Sequences in the Transformation of Normal Fibroblasts in Adolescent Rats”, Proc. Nat. Acad. Sci. USA vol. 69, No. 6, pp. 1601-1605, Jun. 1972.Applicant
Schwartz, et al. “Osteoinductivity of Demineralized Bone Matrix is Independent of Donoe Bisphosphonate Use”; The Journal of Bone and Joint Surgery; Dec. 21, 2011; pp. 2278-2286; vol. 93-A, No. 24.Applicant
Shi, et al., “Adipose-Derived Stem Cells Combined with a Demineralized Cancellous Bone Substrate for Bone Regeneration” Tissue Engineering Part A, Jun. 2012.Applicant
Shih, et al., “Restoration of bone defect and enhancement of bone ingrowth using partially demineralized bone matrix and marrow stromal cells”, Journal of Orthopaedic Research, vol. 23., No. 6., Nov. 2005, pp. 1293-1299.Applicant
Urist, M., “Bone Formation by Autoinduction”, Science, vol. 150, No. 3698, pp. 893-899. Nov. 12, 1965.Applicant
Aubin, et al., “Isolation of Bone Cell Clones with Differences in Growth, Hormone Responses, and Extracellular Matrix Production,” 92 J. Cell. Biol. 452-61 (1982).Applicant
Bennett, et al., “Adipocytic cells cultured from marrow have osteogenic potential,” 99 J. Cell. Sci. 131 (1991).Applicant
Notice of Allowance dated Sep. 25, 2015 for U.S. Appl. No. 12/612,583, filed Nov. 4, 2009, all pages.Applicant
International Preliminary Report on Patentability for International Application No. PCT/US2013/063674 filed Oct. 7, 2013, dated Apr. 21, 2016, 10 pages.Applicant
“Preparation of the stromal vascular fraction (SVF) from human lipaspirate” Miltenyi Biotec, 2008.Applicant
Boquest, A., et al. Neclear Reprogramming: Methods and Protocols, Chapter 4, “Isolation of Stromal Stem Cells From Human Adipose Tissue,” 35-46 Humana Press, Neew Jersey, 2006.Applicant
Kroeze, R.J.; “Adipose stem cells on a biodegradable polymer for spinal fusion” Vrije Universiteit, 2014.Applicant
Zhao, et al. “The study of the feasibility of segmental bone defect repair with tissue-engineered bone membrane: a qualitative observation,” Strat. Traum, Limb Recon. (2008) 3:57-64.Applicant
Lin et al.,“Commonly used mesenchymal stem cell markers and tracking labels: Limitations and challenges,” Histol. Histopathol. 28:1109-1116 (2013).Applicant
Medtronic Product Brochure: Infuse Bone Graft, retrieved from the internet Mar. 5, 2017: (www.infusebonegraft.com/healthcare-providers/bone-grafting-options/categorization-of-bone-grafts/allograft-tissue/index.htm).Applicant
Sakaguchi et al., Comparison of human stem cells derived from various mesenchymal tissues, Arthritis & Rheumatism, vol. 52, No. 8, Aug. 2005, pp. 2521-2529.Applicant
Simper, et al., “Smooth Muscle Progenitor Cells in Human Blood”, Circulation (by the American Heart Association), vol. 106, pp. 1199-1204, Aug. 19, 2002.Applicant

Abstract

Description

CROSS-REFERENCES TO RELATED APPLICATIONS

BACKGROUND OF THE INVENTION

[0002] Regenerative medicine requires an abundant source of human adult stem cells that can be readily available at the point of care.

[0003] Adipose-derived stem cells (ASCs), which can be obtained in large quantities, have been utilized as cellular therapy for the induction of bone formation in tissue engineering strategies.

[0004] Allografts may be combined with stem cells. This requires a significant amount of tissue processing and cellular processing prior to seeding the allograft substrate.

[0005] Allografts seeded with living cells generally provide better surgical results.

BRIEF SUMMARY OF THE INVENTION

[0016] Other embodiments are also disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Illustrative embodiments of the invention are illustrated in the drawings, in which:

[0018] FIG. 1 illustrates a flow chart of the combination of mesenchymal stem cells with a bone substrate;

[0019] FIG. 2 illustrates a prior art example of a pellet of a stromal vascular fraction containing the desired stem cells and unwanted cells;

[0021] FIG. 4 illustrates a standard curve of total live ASCs using the CCK-8 assay;

[0022] FIG. 5 illustrates mineral deposition by ASCs cultured in osteogenic medium (images shown at 20× magnification); and

[0023] FIG. 6 illustrates H&E staining showed that cells adhered to the bone surface.

DETAILED DESCRIPTION OF THE INVENTION

[0027] Rada, T., R. L. Reis, and M. E. Gomes, Adipose Tissue-Derived Stem Cells and Their Application in Bone and Cartilage Tissue Engineering. Tissue Eng Part B Rev, 2009.
[0028] Ahn, H. H., et al., In vivo osteogenic differentiation of human adipose-derived stem cells in an injectable in situ forming gel scaffold. Tissue Eng Part A, 2009. 15(7): p. 1821-32.
[0029] Anghileri, E., et al., Neuronal differentiation potential of human adipose-derived mesenchymal stem cells. Stem Cells Dev, 2008. 17(5): p. 909-16.
[0030] Arnalich-Montiel, F., et al., Adipose-derived stem cells are a source for cell therapy of the corneal stroma. Stem Cells, 2008. 26(2): p. 570-9.
[0031] Bunnell, B. A., et al., Adipose-derived stem cells: isolation, expansion and differentiation. Methods, 2008. 45(2): p. 115-20.
[0032] Chen, R. B., et al., [Differentiation of rat adipose-derived stem cells into smooth-muscle-like cells in vitro]. Zhonghua Nan Ke Xue, 2009. 15(5): p. 425-30.
[0033] Cheng, N. C., et al., Chondrogenic differentiation of adipose-derived adult stem cells by a porous scaffold derived from native articular cartilage extracellular matrix. Tissue Eng Part A, 2009. 15(2): p. 231-41.
[0034] Cui, L., et al., Repair of cranial bone defects with adipose derived stem cells and coral scaffold in a canine model. Biomaterials, 2007. 28(36): p. 5477-86.
[0035] de Girolamo, L., et al., Osteogenic differentiation of human adipose-derived stem cells: comparison of two different inductive media. J Tissue Eng Regen Med, 2007. 1(2): p. 154-7.
[0036] Elabd, C., et al., Human adipose tissue-derived multipotent stem cells differentiate in vitro and in vivo into osteocyte-like cells. Biochem Biophys Res Commun, 2007. 361(2): p. 342-8.
[0037] Flynn, L., et al., Adipose tissue engineering with naturally derived scaffolds and adipose-derived stem cells. Biomaterials, 2007. 28(26): p. 3834-42.
[0038] Flynn, L. E., et al., Proliferation and differentiation of adipose-derived stem cells on naturally derived scaffolds. Biomaterials, 2008. 29(12): p. 1862-71.
[0039] Fraser, J. K., et al., Adipose-derived stem cells. Methods Mol Biol, 2008. 449: p. 59-67.
[0040] Gimble, J. and F. Guilak, Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy, 2003. 5(5): p. 362-9.
[0041] Gimble, J. M. and F. Guilak, Differentiation potential of adipose derived adult stem (ADAS) cells. Curr Top Dev Biol, 2003. 58: p. 137-60.
[0042] Jin, X. B., et al., Tissue engineered cartilage from hTGF beta2 transduced human adipose derived stem cells seeded in PLGA/alginate compound in vitro and in vivo. J Biomed Mater Res A, 2008. 86(4): p. 1077-87.
[0043] Kakudo, N., et al., Bone tissue engineering using human adipose-derived stem cells and honeycomb collagen scaffold. J Biomed Mater Res A, 2008. 84(1): p. 191-7.
[0044] Kim, H. J. and G. I. Im, Chondrogenic differentiation of adipose tissue-derived mesenchymal stem cells: greater doses of growth factor are necessary. J Orthop Res, 2009. 27(5): p. 612-9.
[0045] Kingham, P. J., et al., Adipose-derived stem cells differentiate into a Schwann cell phenotype and promote neurite outgrowth in vitro. Exp Neural, 2007. 207(2): p. 267-74.
[0046] Mehlhorn, A. T., et al., Chondrogenesis of adipose-derived adult stem cells in a poly-lactide-co-glycolide scaffold. Tissue Eng Part A, 2009. 15(5): p. 1159-67.
[0047] Merceron, C., et al., Adipose-derived mesenchymal stem cells and biomaterials for cartilage tissue engineering. Joint Bone Spine, 2008. 75(6): p. 672-4.
[0048] Mischen, B. T., et al., Metabolic and functional characterization of human adipose-derived stem cells in tissue engineering. Plast Reconstr Surg, 2008. 122(3): p. 725-38.
[0049] Mizuno, H., Adipose-derived stem cells for tissue repair and regeneration: ten years of research and a literature review. J Nippon Med Sch, 2009. 76(2): p. 56-66.
[0050] Tapp, H., et al., Adipose-Derived Stem Cells: Characterization and Current Application in Orthopaedic Tissue Repair. Exp Biol Med (Maywood), 2008.
[0051] Tapp, H., et al., Adipose-derived stem cells: characterization and current application in orthopaedic tissue repair. Exp Biol Med (Maywood), 2009. 234(1): p. 1-9.
[0052] van Dijk, A., et al., Differentiation of human adipose-derived stem cells towards cardiomyocytes is facilitated by laminin. Cell Tissue Res, 2008. 334(3): p. 457-67.
[0053] Wei, Y., et al., A novel injectable scaffold for cartilage tissue engineering using adipose-derived adult stem cells. J Orthop Res, 2008. 26(1): p. 27-33.
[0054] Wei, Y., et al., Adipose-derived stem cells and chondrogenesis. Cytotherapy, 2007. 9(8): p. 712-6.
[0055] Zhang, Y. S., et al., [Adipose tissue engineering with human adipose-derived stem cells and fibrin glue injectable scaffold]. Zhonghua Yi Xue Za Zhi, 2008. 88(38): p. 2705-9.

[0057] Gimble, J. and F. Guilak, Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy, 2003. 5(5): p. 362-9.
[0058] Gimble, J. M. and F. Guilak, Differentiation potential of adipose derived adult stem (ADAS) cells. Curr Top Dev Bioi, 2003. 58: p. 137-60.
[0059] Strem, B. M., et al., Multipotential differentiation of adipose tissue-derived stem cells. Keio J Med, 2005. 54(3): p. 132-41.
[0060] De Ugarte, D. A., et al., Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells Tissues Organs, 2003. 174(3): p. 101-9.
[0061] Hayashi, O., et al., Comparison of osteogenic ability of rat mesenchymal stem cells from bone marrow, periosteum, and adipose tissue. Calcif Tissue Int. 2008. 82(3): p. 238-47.
[0062] Kim, Y., et al., Direct comparison of human mesenchymal stem cells derived from adipose tissues and bone marrow in mediating neovascularization in response to vascular ischemia. Cell Physiol Biochem, 2007. 20(6): p. 867-76.
[0063] Lin, L., et al., Comparison of osteogenic potentials of BMP4 transduced stem cells from autologous bone marrow and fat tissue in a rabbit model of calvarial defects. Calcif Tissue Int, 2009. 85(1): p. 55-65.
[0064] Niemeyer, P., et al., Comparison of immunological properties of bone marrow stromal cells and adipose tissue-derived stem cells before and after osteogenic differentiation in vitro. Tissue Eng, 2007. 13(1): p. 111-21.
[0065] Noel, D., et al., Cell specific differences between human adipose-derived and mesenchymal-stromal cells despite similar differentiation potentials. Exp Cell Res, 2008. 314(7): p. 1575-84.
[0066] Yoo, K. H., et al., Comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues. Cell Immunol, 2009.
[0067] Yoshimura, H., et al., Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res, 2007. 327(3): p. 449-62.

[0069] Rada, T., R. L. Reis, and M. E. Gomes, Adipose Tissue-Derived Stem Cells and Their Application in Bone and Cartilage Tissue Engineering. Tissue Eng Part B Rev, 2009.
[0070] Tapp, H., et al., Adipose-Derived Stem Cells: Characterization and Current Application in Orthopaedic Tissue Repair. Exp Bioi Med (Maywood), 2008.
[0071] Tapp, H., et al., Adipose-derived stem cells: characterization and current application in orthopaedic tissue repair. Exp Bioi Med (Maywood), 2009. 234(1): p. 1-9.
[0072] De Girolamo, L., et al., Human adipose-derived stem cells as future tools in tissue regeneration: osteogenic differentiation and cell-scaffold interaction. Int J Artif Organs, 2008. 31(6): p. 467-79.
[0073] Di Bella, C., P. Farlie, and A. J. Penington, Bone regeneration in a rabbit critical-sized skull defect using autologous adipose-derived cells. Tissue Eng Part A, 2008. 14(4): p. 483-90.
[0074] Grewal, N. S., et al., BMP-2 does not influence the osteogenic fate of human adipose-derived stem cells. Plast Reconstr Surg, 2009. 123(2 Suppl): p. 158S-65S.
[0075] Li, H., et al., Bone regeneration by implantation of adipose-derived stromal cells expressing BMP-2. Biochem Biophys Res Commun, 2007. 356(4): p. 836-42.
[0076] Yoon, E., et al., In vivo osteogenic potential of human adipose-derived stem cells/poly lactide-co-glycolic acid constructs for bone regeneration in a rat critical-sized calvarial defect model. Tissue Eng, 2007. 13(3): p. 619-27.

[0078] Butt, O. I., et al., Stimulation of peri-implant vascularization with bone marrow-derived progenitor cells: monitoring by in vivo EPRoximetry. TissueEng, 2007. 13(8): p. 2053-61.
[0079] Rigotti, G., et al., Clinical treatment of radiotherapy tissue damage by lipoaspirate transplant: a healing process mediated by adipose-derived adult stem cells. Plast Reconstr Surg, 2007. 119(5): p. 1409-22; discussion 1423-4.

[0081] Chen, L. Q., et al., [Study of MSCs in vitro cultured on demineralized bone matrix of mongrel]. Shanghai Kou Qiang Yi Xue, 2007. 16(3): p. 255-8.
[0082] Gamradt, S. C. and J. R. Lieberman, Bone graft for revision hip arthroplasty: biology and future applications. Clin Orthop Relat Res, 2003(417): p. 183-94.
[0083] Honsawek, S., D. Dhitiseith, and V. Phupong, Effects of demineralized bone matrix on proliferation and osteogenic differentiation of mesenchymal stem cells from human umbilical cord. J Med Assoc Thai, 2006. 89 Suppl 3: p. S189-95.
[0084] Kasten, P., et al., [Induction of bone tissue on different matrices: an in vitro and a in vivo pilot study in the SCID mouse]. Z Orthop Ihre Grenzgeb, 2004.142(4): p. 467-75.
[0085] Kasten, P., et al., Ectopic bone formation associated with mesenchymal stem cells in a resorbable calcium deficient hydroxyapatite carrier. Biomaterials, 2005. 26(29): p. 5879-89.
[0086] Qian, Y., Z. Shen, and Z. Zhang, [Reconstruction of bone using tissue engineering and nanoscale technology]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 2006. 20(5): p. 560-4.
[0087] Reddi, A. H., Role of morphogenetic proteins in skeletal tissue engineering and regeneration. Nat Biotechnol, 1998. 16(3): p. 247-52.
[0088] Reddi, A. H., Morphogenesis and tissue engineering of bone and cartilage: inductive signals, stem cells, and biomimetic biomaterials. Tissue Eng, 2000.6(4): p. 351-9.
[0089] Tsiridis, E., et al., In vitro and in vivo optimization of impaction allografting by demineralization and addition of rh-OP-1. J Orthop Res, 2007. 25(11): p. 1425-37.
[0090] Xie, H., et al., The performance of a bone-derived scaffold material in the repair of critical bone defects in a rhesus monkey model. Biomaterials, 2007.28(22): p. 3314-24.
[0091] Liu, G., et al., Tissue-engineered bone formation with cryopreserved human bone marrow mesenchymal stem cells. Cryobiology, 2008. 56(3): p. 209-15.
[0092] Liu, G., et al., Evaluation of partially demineralized osteoporotic cancellous bone matrix combined with human bone marrow stromal cells for tissue engineering: an in vitro and in vivo study. Calcif Tissue Int, 2008. 83(3): p. 176-85.
[0093] Liu, G., et al., Evaluation of the viability and osteogenic differentiation of cryopreserved human adipose-derived stem cells. Cryobiology, 2008. 57(1): p. 18-24.

[0098] In one embodiment, an allograft product may include a combination of mesenchymal stem cells with a bone substrate such that the combination is manufactured by the above exemplary embodiment.

[0104] In one embodiment, an allograft product may include a combination of mesenchymal stem cells with a bone substrate such that the combination is manufactured by the above exemplary embodiment.

[0107] In one embodiment, an allograft product may include combination of mesenchymal stem cells with a bone substrate such that the combination is manufactured by the above exemplary embodiment.

[0111] In one embodiment, an allograft product may include a combination of mesenchymal stem cells with a bone substrate such that the combination is manufactured by the above exemplary embodiment.

[0127] Arinzeh T L, Peter S J, Archambault M P, van den Bas C, Gordon S, Kraus K, Smith A, Kadiyala S. Allogeneic mesenchymal stem cells regenerate bone in a critical sized canine segmental defect. J Bone Joint Surg Am. 2003; 85-A:1927-35.
[0128] Bruder S P, Kurth A A, Shea M, Hayes W C, Jaiswal N, Kadiyala S. Bone regeneration by implantation of purified, culture-expanded human mesenchymal stem cells, J Orthop Res. 1998; 16:155-62.

Example 1

Adipose Recovery

[0129] Adipose was recovered from cadaveric donors. Adipose aspirate may be collected using liposuction machine and shipped on wet ice.

Washing

[0131] The solution was suspended in an equal volume of growth media (DMEM/F12, 10% FBS, 1% penicillin/streptomycin) and stored in a refrigerator at about 4° C.

Digestion and Combining of Cell Suspension with Allografts

[0132] Digestion of the adipose was undertaken to acquire a stromal vascular fraction (SVF) followed by combining the solution onto an allograft.

[0134] Next, take out the washed adipose and mix with collagenase I solution at 1:1 ratio. Mixture was added to a shaker flask.

[0135] The flask was placed in an incubating shaker at 37° C. with continuous agitation (at about RPM=75) for about 45 to 60 minutes until the tissue appeared smooth on visual inspection.

[0137] Growth media was added into every tube (i.e., 40 ml total was added into the 4 tubes) followed by gentle shaking.

[0139] Aliquot cell mixtures were measured into 2 centrifuge tubes (of 10 ml each) and centrifuged at about 300 g for 5 minutes. The supernatant was aspirated.

[0140] A cell pellet obtained from one tube was used for seeding onto allografts. The allografts may include cortical/cancellous or both which was subjected to a demineralization process.

Example 2

Adipose Recovery

[0143] Adipose was recovered from cadaveric donors. Adipose aspirate may be collected using liposuction machine and shipped on wet ice.

Washing

Acquire Ficoll Concentrated Stem Cells and Combine onto Allograft

[0147] Next, 2 volumes of 0-PBS were added. The tubes were capped and mix gently by inversion to wash the cells.

[0148] The tubes with the diluted cells were then subjected to centrifugation at 900 g for 5 minutes at room temperature to pellet the cells with the brake on during deceleration.

[0150] The remainder of the cell mixtures were centrifuged at about 300 g for about 5 minutes. The supernatant was aspirated.

Example 3

Bone Marrow Recovery

[0154] Adipose was recovered from cadaveric donors. Adipose aspirate may be collected using liposuction machine and shipped on wet ice.

Washing

Acquire Stem Cells and Combine onto Allograft

Example 4

Skeletal Muscle Recovery

[0157] Skeletal muscle may be recovered from cadaveric donors.

Washing

Acquire Stem Cells and Combine onto Allograft

Example 5

Adipose Recovery

[0160] Adipose was recovered from a cadaveric donor within 24 hours of death and shipped in equal volume of DMEM in wet ice.

Washing

Cortical Cancellous Bone Recovery

Digestion and Combining of Cell Suspension with Allograft

Example 6

Adipose-Derived Stem Cell Characterization

Flow Cytometry Analysis

In Vitro Tri-Lineage Differentiation

Final Product Characterization

Histology

Statistical Analysis

Results

Final Product Appearance

[0173] FIGS. 3A-3D illustrate an appearance of strips, dowels and disks. In these embodiments, all have a cortical bottom and cancellous top. Other embodiments may be used.

ASC Characterization

Flow Cytometry—Immunophenotype of SVF

Estimated Mean Total Percentage of MSC

In Vitro Tri-Lineage Differentiation

Final Product Characterization

Cell Count: CCK-8 Assay

[0179] 28 grafts were tested from 8 donors and had an average of 50,000 live cells/graft.

Histology

CONCLUSIONS

[0182] Liu, G., et al., Evaluation of partially demineralized osteoporotic cancellous bone matrix combined with human bone marrow stromal cells for tissue engineering: an in vitro and in vivo study. Calcif Tissue Int, 2008. 83(3): p. 176-85.
[0183] Wang, J. and M. J. Glimcher, Characterization of matrix-induced osteogenesis in rat calvarial bone defects: II. Origins of bone forming cells. Calcif Tissue Int, 1999. 65(6): p. 486-93.
[0184] Wang, J. and M. J. Glimcher, Characterization of matrix-induced osteogenesis in rat calvarial bone defects: I. Differences in the cellular response to demineralized bone matrix implanted in calvarial defects and insubcutaneous sites. Calcif Tissue Int, 1999. 65(2): p. 156-65.
[0185] Wang, J., et al., Characterization of demineralized bone matrix-induced osteogenesis in rat calvarial bone defects: III. Gene and protein expression. Calcif Tissue Int, 2000. 67(4): p. 314-20.
[0186] Bruder, S. P. and B. S. Fox, Tissue engineering of bone. Cell based strategies. Clin Orthop Relat Res, 1999(367 Suppl): p. S68-83.

[0188] Chen, L. Q., et al., [Study of MSCs in vitro cultured on demineralized bone matrix of mongrel]. Shanghai Kou Qiang Yi Xue, 2007. 16(3): p. 255-8.
[0189] Honsawek, S., D. Dhitiseith, and V. Phupong, Effects of demineralized bone matrix on proliferation and osteogenic differentiation of mesenchymal stem cells from human umbilical cord. J Med Assoc Thai, 2006. 89 Suppl 3: p. 8189-95.
[0190] Kasten, P., et al., Ectopic bone formation associated with mesenchymal stem cells in a resorbable calcium deficient hydroxyapatite carrier. Biomaterials, 2005. 26(29): p. 5879-89.
[0191] Qian, Y., Z. Shen, and Z. Zhang, [Reconstruction of bone using tissue engineering and nanoscale technology]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 2006. 20(5): p. 560-4.
[0192] Liu, G., et al., Tissue-engineered bone formation with cryopreserved human bone marrow mesenchymal stem cells. Cryobiology, 2008. 56(3): p. 209-15.
[0193] Ko, E. K., et al., In vitro osteogenic differentiation of human mesenchymal stem cells and in vivo bone formation in composite nanofiber meshes. Tissue Eng Part A, 2008. 14(12): p. 2105-19.