Tissue storage composition, techniques, and methods of use

Abstract

An interoperative solution is provided for use as a temporary storage medium, at cryogenic or non-cryogenic temperatures, such as during a surgical procedure. The interoperative solution may also be used as a rinse agent, such as to rinse off or remove non-biocompatible preservatives or treatments, and / or as a transportation medium for maintaining the integrity of a tissue undergoing transport. In an embodiment, the interoperative solution comprises glycerol.

Description

TISSUE STORAGE COMPOSITION, TECHNIQUES, AND METHODSOF USECROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority and benefit of U.S. Provisional Patent Application No. 63 / 557,092, filed on 23 February 2024, both of which are incorporated herein by reference in their entirety.BACKGROUND

[0002] The present disclosure relates generally to bone growth compositions, such as cellular bone matrix compositions, and techniques associated with bone growth compositions.

[0003] This section is intended to introduce the reader to various aspects of art that may be related to various aspects of the present disclosure, which are described and / or claimed below. This discussion is believed to be helpful in providing the reader with background information to facilitate a better understanding of the various aspects of the present disclosure. Accordingly, it should be understood that these statements are to be read in this light, and not as admissions of prior art.

[0004] In clinical use, a variety of conditions may warrant repair and / or replacement of an internal body part, such as bone. For example, to repair a bone fracture, an adhesive agent may be applied to adhere sections of the separated bone together. A bone filler material may be applied to a bone in a subj ect to replace degenerated tissue and / or to provide a supportive matrix to support or reinforce the bone and promote bone growth.

[0005] Since the early 1990s, carefully processed allograft bone from donated human tissues have been routinely transformed into various type of bone grafting materials with many derivations in shape, form, and purpose. The first standards for tissue banking published by the American Association of Tissue Banks (AATB) in 1984. Due to an increasing medical and market demand for orthopaedic implants, a selection of grafting options has been developed. Fresh frozen allograft bone (derived from either deceased donors or surgical discards) has been used to supplement the available autograft bone recovered from patients undergoing surgery and in need of bone grafting. Osteotech launched the first formulated demineralized bone matrix product for bone grafting called Grafton™ Gel in 1991.

[0006] Commercial products intended as an alternative to autologous grafts have included growth factors, or a combination of natural or synthetic scaffold materials. While the use of demineralized bone fibers (DBF) creates structural matrices and promotes bone growth, the use of DBF alone lacks the viability of fresh tissue. Certain techniques may use cell-containing bone materials. However, clinical use of cell bone matrices has been associated with significant issues, such as high cost, burdensome and nonoverlapping regulatory matters globally, storage and transport challenges, and may infer complications associated with immunological response.

[0007] Cellular matrix products manufactured in the United States are regulated by section 361 of the Public Health Service Act and Code of Federal Regulation (CFR) title 21 section 1271, which does not require Food and Drug Administration (FDA) premarket review and approval. As such, the products will be considered human cells, tissues, or cellular or tissuebased products (HCT / Ps) so long as the products satisfy the following criteria: minimal manipulation; homologous use only; systemic effect absence; the primary function not dependent on the metabolic activity of viable cells, unless the product is intended for autologous use or use by a first-or second-degree blood relative.

[0008] Certain existing products on the market are also failing in forming stable spinal fusion, for example, in posterolateral fusion. Efficacy and safety studies, large randomized clinical trials, and additional research are needed to solidify the role of allograft with viable cells, especially in current studies on animal models that show controversial data. Cellular autograft, cellular bone matrix (CBM) / viable bone matrix (VBM)s, also need better understanding as to which product best aligns with a specific indication and cost-effective solution.

[0009] For each CBM, however, several intrinsic biological characteristics, such as viable cell sources, the donor age at the time of graft harvest, or cell survival after transplantation, cause variations among different lots of the same product in terms of expected outcomes. Processing may also introduce variability in product; such variability may include cell type, cell amount, cell viability dependent on donor material, cell viability after thawing, bone tissue processing, subsequent formulation, cryoprotectant agents, cry opreservation or freezing, and storage.

[0010] Further issues in regards to maintaining viability of cells remain. Cryoprotectant agents utilized to maintain cell viability at extremely low temperatures (e.g., less than about - 80°C) and for long-term storage and transport, e.g., dimethyl sulfoxide (DMSO), have an intrinsic cytotoxicity that requires rapid removal from grafts before implant. In fact, cryoprotectants during thaw cause cell death, therefore requiring additional rinsing, decanting, and handling that may be detrimental to the viable cells. Use of other cryoagents, or storing at less extreme cold temperatures has caused shrinking of the cells and / or caused formation of intracellular ice (which causes the cell to burst upon thaw).

[0011] A need exists to develop a CBM / VBM product that maintains a cellular viability similar to the live cell count of the extracted bone particles, granules, or fibers prior to processing. A CBM product is needed that will address the issues as described above.SUMMARY

[0012] Certain embodiments are summarized below. These embodiments are not intended to limit the scope of the disclosure. Indeed, the present disclosure may encompass a variety of forms that may be similar to or different from the embodiments set forth below. The bone growth composition of the current application will address the issues discussed prior, and further include an improved capability for use, manipulation, preservation, thaw and post-thaw applications. Such improvements are described herein.

[0013] In one embodiment, a bone growth composition is provided. The composition includes demineralized bone fibers generated from a donor bone tissue; cancellous chips and / or corti cocancell ous chips generated from the donor bone tissue; and an interoperative solution comprising glycerol in Lactated Ringer’s, wherein a glycerol content of the interoperative solution is equal to or less than 20% by volume.

[0014] In one embodiment a frozen bone growth composition product is provided. The product includes a sealed flexible pouch enclosing a bone growth composition: demineralized bone fibers generated from a donor bone tissue; cancellous chips and / or corticocancellous chips generated from the donor bone tissue; and an interoperative solution comprising glycerol in Lactated Ringer’s, wherein a glycerol content of the interoperative solution is equal to or less than 20% by volume, wherein the demineralized bone fibers, the cancellous chips, and theinteroperative solution are all frozen such that the bone growth composition is solid.

[0015] In one embodiment a thawed bone growth composition product is provided. The product includes a sealed flexible pouch enclosing a bone growth composition: demineralized bone fibers generated from a donor bone tissue; cancellous chips and / or corticocancellous chips generated from the donor bone tissue; and an interoperative solution comprising glycerol in Lactated Ringer’s, wherein a glycerol content of the interoperative solution is equal to or less than 20% by volume, wherein the demineralized bone fibers, the cancellous chips, and the interoperative solution are all thawed such that the bone growth composition is flowable within the sealed flexible pouch.

[0016] In one embodiment, a storage method for a fresh autograft tissue is provided. The method includes extracting the fresh autograft tissue from a patient, wherein the fresh autograft tissue includes bone, cartilage, or tissue. The fresh autograft tissue is placed in a receptacle containing an interoperative solution and, in some implementations, transferred to a larger secondary receptacle containing a cooling medium.

[0017] In one embodiment, a processing method for a fresh autograft tissue is provided. The method includes removing a receptacle containing a chilled, fresh autograft tissue in interoperative solution from a secondary receptacle containing a cooling medium. The temperature of the fresh autograft tissue is elevated (such as to at or near body temperature) for a period of time. The fresh autograft tissue is removed from the interoperative solution and implanted in a surgical or clinical procedure.

[0018] In one embodiment, a kit for preparation or preservation of a fresh autograft tissue is provided. The kit composition includes a receptacle for storing the fresh autograft tissue in an interoperative solution, wherein the interoperative solution includes glycerol in Lactated Ringer’s, a measuring glassware (in certain embodiments), wherein the measuring glassware is for measuring the interoperative solution, a mesh, and a bottle, wherein the bottle may include a premixed interoperative solution, glycerol, or Lactated Ringer’s.

[0019] In one embodiment, a composition of an interoperative solution is provided. The composition includes a glycerol fraction, wherein the glycerol fraction is less than or equal to 25% by volume (e.g., less than or equal to 20% by volume) and a Lactated Ringer’s fraction, whereinthe Lactated Ringer’s fraction is more than or equal to 75% by volume (e.g., more than or equal to 80% by volume).

[0020] In one embodiment, a method of rinsing and storing an autograft tissue is provided. The method includes retrieving the autograft tissue. The autograft tissue is rinsed with an interoperative solution to remove residue of a non-biocompatible solution used to store or transport the autograft tissue. The interoperative solution includes a glycerol fraction, wherein the glycerol fraction is less than or equal to 25% by volume (e.g., less than or equal to 20% by volume) and a Lactated Ringer’s fraction, wherein the Lactated Ringer’s fraction is more than or equal to 75% by volume (e.g., more than or equal to 80% by volume). The autograft tissue is stored in the interoperative solution.

[0021] In one embodiment, a composition comprising a storage mixture is provided. The composition includes an autograft tissue and an interoperative solution, wherein the interoperative solution includes a glycerol fraction and a Lactated Ringer’s fraction.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Advantages of the disclosed techniques may become apparent upon reading the following detailed description and upon reference to the drawings in which:

[0023] FIG. 1 is a flow diagram of a processing method to produce a cellular bone matrix from donor bone tissue, in accordance with an embodiment of the present disclosure;

[0024] FIG. 2 shows an example bone region including sources for cancellous, cortical, and corticocancellous chips, in accordance with an embodiment of the present disclosure;

[0025] FIG. 3A shows an example bone region including sources for cancellous, cortical, and corticocancellous chips, in accordance with an embodiment of the present disclosure;

[0026] FIG. 3B shows an example bone region including sources for cancellous, cortical, and corticocancellous chips, in accordance with an embodiment of the present disclosure;

[0027] FIG. 4 is a flow diagram of a fiber processing method that may be used in conjunction with FIG. 1, in accordance with an embodiment of the present disclosure;

[0028] FIG. 5 shows example fibers, in accordance with an embodiment of the present disclosure;

[0029] FIG. 6 is a flow diagram of a two-step chip washing method, in accordance with an embodiment of the present disclosure;

[0030] FIG. 7 is a flow diagram of a two-step chip washing method, in accordance with anembodiment of the present disclosure;

[0031] FIG. 8 shows effects of the two-step chip washing method on a tissue sample relative to a control;

[0032] FIG. 9 is a flow diagram of chip washing, in accordance with an embodiment of the present disclosure;

[0033] FIG. 10 shows a comparison of cell viability for different washing techniques;

[0034] FIG. 11 shows a comparison of cell viability for different washing techniques;

[0035] FIG. 12 shows a comparison of cell viability for different interoperative solutions;

[0036] FIG. 13 shows a comparison of cell viability for different interoperative solutions;

[0037] FIG. 14 is a flow diagram of a method of contacting chips with an interoperative solution for freezing, in accordance with an embodiment of the present disclosure;

[0038] FIG. 15 shows example components of a bone growth composition, in accordance with an embodiment of the present disclosure;

[0039] FIG. 16 shows an example bone growth composition packaged in a pouch and removed from an outer packaging, in accordance with an embodiment of the present disclosure;

[0040] FIG. 17 shows an example bone growth composition packaged in a pouch, in accordance with an embodiment of the present disclosure;

[0041] FIG. 18 shows an example bone growth composition packaged in a pouch and in a water bath for thawing, in accordance with an embodiment of the present disclosure;

[0042] FIG. 19 shows an example bone growth composition packaged in a pouch and being opened, in accordance with an embodiment of the present disclosure;

[0043] FIG. 20 depicts an example of a three-pouch embodiment for packaging, storing, and transporting bone growth composition, in accordance with an embodiment of the present disclosure;

[0044] FIG. 21 depicts the example of FIG. 20 in a use-configuration in which the inner pouch and pouch are nested within an outer pouch, in accordance with an embodiment of the present disclosure;

[0045] FIG. 22 shows an example of different conditions for a bone growth composition packaged in a pouch, in accordance with an embodiment of the present disclosure;

[0046] FIG. 23 shows an example bone growth composition packaging system including a release liner that is removed to permit draining, in accordance with an embodiment of the present disclosure;

[0047] FIG. 24 is a flow diagram of assessing cell viability, in accordance with anembodiment of the present disclosure;

[0048] FIG. 25 shows an example radiograph of a rat spine taken post-operatively;

[0049] FIG. 26 shows an example radiograph of the rat spine taken at eight weeks after explantation;

[0050] FIG. 27 shows an example histology of the rat spine taken at eight weeks after explantation;

[0051]

[0052] FIG. 28 shows a comparison of cell viability for different freezing techniques;

[0053] FIG. 29 shows a comparison of cell viability for different antimicrobial agents;

[0054] FIG. 30 shows a comparison of cell viability for different antimicrobial agents with respect to soak temperature;

[0055] FIG. 31 shows a comparison of cell viability for different antimicrobial agent concentrations;

[0056] FIG. 32 shows a comparison of cell viability for different antimicrobial agent rinse conditions;

[0057] FIG. 33 shows a comparison of post-thaw cell viabilities;

[0058] FIG. 34 shows a comparison of cell stability for different storage time conditions;

[0059] FIG. 35 is a flow diagram of a storage method for storing a fresh autograft tissue (e.g., bone or other tissue) in a secondary container with a cooling medium, in accordance with an embodiment of the present disclosure;

[0060] FIG. 36 is a flow diagram of a processing method for a fresh autograft tissue (e.g., bone or other tissue) for use in surgical applications, in accordance with an embodiment of the present disclosure;

[0061] FIG. 37 shows an example of a pouch having integrated drainage features, in accordance with an embodiment of the present disclosure;

[0062] FIG. 38 shows a further example of a pouch having integrated drainage features, in accordance with an embodiment of the present disclosure;

[0063] FIG. 39 shows an example of a sterilized kit that can be implemented in a surgical setting for the preparation and use of a fresh autograft tissue, in accordance with an embodiment of the present disclosure;

[0064] FIG. 40 is a flow diagram of a method for processing an autograft tissue for implantation, in accordance with an embodiment of the present disclosure;

[0065] FIG. 41 is a flow diagram of a method for storing, transporting, and implanting anautograft tissue, in accordance with an embodiment of the present disclosure; and

[0066] FIG. 42 shows an example of a medical grade intravenous (IV) type bag with a port that can contain the interoperative solution, transport solution, or cryosolution, in accordance with an embodiment of the present disclosure.DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

[0067] One or more specific embodiments will be described below. In an effort to provide a concise description of these embodiments, not all features of an actual implementation are described in the specification. It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementationspecific decisions must be made to achieve the developers’ specific goals, such as compliance with system-related and business-related constraints, which may vary from one implementation to another. Moreover, it should be appreciated that such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.

[0068] The methods discussed herein include various steps represented by blocks in flow diagrams. It should be noted that at least some steps may be performed as an automated procedure by one or more components of a system. Although the flow diagrams may illustrate the steps in a certain sequence, it should be understood that the steps may be performed in any suitable order and certain steps may be carried out simultaneously, where appropriate. Additionally, steps may be added to or omitted from of the methods.

[0069] When introducing elements of various embodiments of the present disclosure, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more of the elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. One or more specific embodiments of the present embodiments described herein will be described below. In an effort to provide a concise description of these embodiments, all features of an actual implementation may not be described in the specification. It should be noted that in the development of any such actual implementation, as in any engineering or design project, numerous implementationspecific decisions must be made to achieve the developers’ specific goals, such as compliance with system-related and business-related constraints, which may vary from one implementation to another. Moreover, it should be noted that such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, andmanufacture for those of ordinary skill having the benefit of this disclosure.

[0070] Certain embodiments of the present disclosure are discussed in the context of an “interoperative solution”, “transport solution”, or “cryosolution”. It should be noted that such solutions as disclosed herein may be used “interoperatively,” wherein the solutions are not directly in contact with the patient and are instead used for the processing or handling of transplantable or other anatomic materials away from the patient (i.e., at a different workspace or location or otherwise not directly part of a procedure being performed on the patient, such as in the back of an operating room while a procedure is performed elsewhere in the operating room. For example, rinsing, storing, or treating transplantable materials with the interoperative solution, transport solution, or cryosolution may be considered an interoperative use of the solutions as these may occur away from the surgical area and / or the vicinity of the patient (i.e., no contacting the patient), even when otherwise in the same operating room.

[0071] Certain embodiments of the present disclosure are discussed in the context of glycerol amount and mixing solution amount in the “interoperative solution”, “transport solution”, or “cryosolution”. Accordingly terms such as “by volume,” “content,” and “fraction,” may be used interchangeably to describe an amount of glycerol or mixing solution in the “interoperative solution”, “transport solution”, or “cryosolution”.

[0072] The disclosed bone graft compositions include demineralized allograft bone fibers from cortical bone, and cancellous and corticocancellous bone chips, and glycerol. The chips, being fully mineralized, retain their inherent radiopacity and enhance detection of the implant when using x-ray. The mineralized chips and demineralized fibers also impart desirable handling characteristics to the graft.

[0073] New bone formation in patients undergoing bone graft requires three elements; local signaling to drive new bone formation, a scaffolding material to template the shape of the future bone, and bone forming cells like osteoblasts and / or bone progenitor cells. The demineralized bone fibers expose local anabolic growth factors inherent in the tissue matrix. The demineralized fibers and cancellous bone chips also offer a scaffold material. In contrast to products in which the cells were provided by the recipient, the disclosed products provide allograft bone-forming cells.

[0074] The disclosed embodiments relate to bone growth compositions, such as bone matrix compositions. The bone matrix compositions may include cellular bone matrix compositions and techniques, also referred to as cellular bone matrices (CBMs) or viable bone matrices (VBMs). As provided herein, cellular bone matrixes provide a platform for new bone formation in a subject. A mix of bone elements and bone elements that have live bone cellsprovides an osteoconductive scaffold and viable cells with osteogenic potential. Specifically, the CBMs provide various components for new formation, namely, an osteoconductive scaffold, extracellular growth factors for cell proliferation and differentiation, and viable cells with osteogenic potential. The growth factors and proteins in the demineralized fibers provide an active signal (i.e., osteoinductivity) for bone healing. Both the chips and fibers act as scaffolds for bone regeneration (i.e. osteoconductivity). Cellular bone matrices may be used for bone grafts, e.g., allogenic bone grafts. Cellular bone matrices include live bone cells, such as live mesenchymal stem cells (MSCs).

[0075] The CBM product is an autograft extender having osteoconductive, osteoinductive, and osteogenic characteristics. The interoperative solution, as used herein (e.g., a glycerol and Lactated Ringer’s solution), has been formulated to retain cellular viability and, in some implementations, may comprise, incorporate, or correspond to a cryosolution or cryopreservative solution that in other embodiments is employed for tissue preservation at cryogenic temperatures. As used herein, however, an interoperative solution may be suitable for use with tissue at non-cryogenic temperatures as well, such as for temporary tissue storage in a clinical or operative setting, as a rinse for rinsing tissues stored in non-biologically safe media, such as cryoprotectants comprising DMSO, and / or as a transportation medium for storing or moving tissues at non-cryogenic temperatures. Also provided herein are effective manufacturing methods to optimize the manufacturing of this formulation in a way that maintains high cell viability, enhances microbiological safety, and enables high osteoinductive performance. These are described in more detail herein.

[0076] By design, the CBM is stored frozen using agents to maintain the viability of cells contained within the tissue. The tissue is prepared and packaged in a way that makes it ready- to-use following a short duration thawing process. The CBM can be utilized alone or mixed with autograft to enhance osteogenic characteristics and encourage bone fusion. The CBM may be moldable for insertion into a spinal implant cage or conforming to the contours of bone or other spinal region. Where the CBM is utilized for packing in a cage implant, the product may be compacted and malleable to conform to the designated space. In some instances, the composition can be dried by squeezing to alter its handling characteristics to the preference of the user. The composition alternatively can be easily hydrated with fluid, water, blood, marrow, or otherwise to alter handling and increase malleability. Further hydration can enhance the compositions flowability for use with a graft insertion gun or funnel and enable it to be easily pressed through long cannulas less than about 8mm in diameter. Once compacted or injected at the site for spinal repair, the graft is stable and unlikely to migrate due to theinherent cohesivity of the formulation. In some cases, the formulation is resistant to gentle irrigation.

[0077] Regarding appearance, the CBM has a fibrous appearance with particulate heterogeneity. In comparison with prior technology utilizing terminal sterilization, the embodiments herein comprise one or more steps of aseptic processing, variable according to batched donor supply and / or standardized for consistency across product line.

[0078] The improved thawing characteristics of the CBM include flexible packaging to permit an expanded surface area exposed to thawing temperatures and minimization of air pockets that inhibit thawing.

[0079] The bone repair donor materials disclosed herein may be autologous, allogenic, or xenogenic. For example, an autograft, as used herein, may refer to bone and / or tissue that is extracted from the intended recipient of an implant. An allograft refers to bone and / or tissue that is from a donor that is different than the intended recipient. A xenograft refers to bone and / or tissue that is from a donor that is a different species than the intended recipient. The disclosed compositions and techniques may be used in the context of autologous, allogenic, or xenogenic compositions or mixtures thereof. Further, the disclosed products may be formed from one or more donors, e.g., a single product may be formed from a single donor or from donor materials from multiple donors. For example, in certain cases, an allogenic material may be mixed with an autograft material to form a final product that is provided to the patient. The donor bone tissue may be cadaveric or from a living donor. In an embodiment, the bone growth composition uses tissue originating from a recently deceased human and is processed within a sufficiently short timeframe to enable retention of cellular components. The donor bone tissue may be a bone end of a long bone, from bone locations proximal to joints, or within the interior of vertebrae. By way of example, the donor bone tissue may be a distal end radius, proximal humerus, or proximal femur.

[0080] In embodiments, tissue recovered for use in the CBM product will be from donors screened, recovered, and tested in compliance with current AATB and FDA guidance documents. In an example, the source bone will originate anatomically from the extremities and from portions of the pelvis. In an embodiment, bone originating from the spine, ribs, cranial region, hand, or foot anatomy is not used. The disclosed embodiments may include testing and exclusion of any donor that tests positive for HTLV VII. Donors testing positive for certain markers, such as Cytomegalovirus IgG and IgM antibodies by ELISA, may also be excluded. Donor tissue which tests positive for Streptococcus pyogenes, Clostridium sp, or Mycobacterium tuberculosis organisms (by PCR and / or culture) may also be excluded.

[0081] In an embodiment, the donor tissue is in compliance with AATB Bulletin 22-2 and AATB Bulletin 23-6. These documents makes additional recommendations for tissue banks to consider to limit the risk of Mycobacterium tuberculosis transmission. The CBM products may exclude tissue from donors with a history of dialysis outside of an ICU setting or donor with a history of long-term steroid use prior to death.

[0082] Donor tissue is recovered within the time period defined by AATB guidelines. Following recovery, tissue is stored and transported to on wet ice or other appropriate conditions per AATB guidelines. In an embodiment, donor tissue may not be frozen prior to processing into the bone composition product. In an embodiment, donor tissue must complete its processing into the finished product within 96-120 hours of donor time of death or, in an embodiment, within 96 hours of donor time of death.

[0083] For clarification, viable cells within CBMs are cell populations capable of promoting synthesis of new bone, such as multipotent adult progenitor cells (MAPC), mesenchymal stem cells (MSCs), osteoprogenitor cells (OPCs), osteocytes, and / or osteoblasts (OBs). MAPCs and MSCs are both non-hematopoietic cells found in the bone marrow stroma; these cells retain the ability to self-replicate and differentiate into a specific phonotype by intrinsic and local environmental cues (e.g., spatial organization, mechanical forces, growth factors). Many other cells common to bone tissue may also be present in CBMs. Those could include fibroblasts, adipocytes, vascular endothelial cells, hematopoietic cells, lymphocytes, and other cells found commonly in bone.

[0084] In certain embodiments, techniques for preparing, manufacturing, and / or storing cellular bone matrix compositions are provided. In certain embodiments of the invention the disclosed techniques permit improved biological activity (e.g., osteoinductivity and / or osteoconductivity) of cellular bone matrices at one or more preparation stages. The cellular allografts, CBM / VBMs, are obtained by processing to largely remove immune-responsive signals generated by bone marrow components. Such components could include hematopoietic cells, retaining bone-forming cells within the bone matrix.

[0085] Embodiments of the disclosed techniques relate to cellular bone matrix processing methods that include an antimicrobial agent contact step. Use of an antimicrobial agent during processing may improve clinical results and end product characteristics by reducing potential for spoilage and improving shelf life of the cellular bone matrix. This contact step may also be used to reduce or eliminate microbiological contamination originating from the starting tissue or manufacturing process itself. However, the antimicrobial agent contact during processing may reduce cell viability for active cells of the chips. Certain antimicrobial contactconditions as disclosed herein may be associated with relatively higher cell viability. In addition, the disclosed techniques permit inclusion of an antimicrobial contact step in cancellous chip processing by maintaining or improving cell viability at other steps in chip processing, such that cell loss or reduced viability as a result of the antimicrobial contact step is compensated for by improved cell viability at other stages. These high viabilities in the manufacturing work flow result in a finished product with high cellular viability even while including aggressive antimicrobial steps to minimize the potential for bacterial contamination in the finished product while maintaining high performance.

[0086] In particular, cryopreservatives that permit storage of the cellular bone matrix products may nonetheless have harmful effects on cell viability. Embodiments of the disclosed techniques include cold preservation and cryopreservation in cryopreservative formulations and conditions with associated improved cell viability characteristics. The disclosed techniques also include cellular bone matrix processing with wash or rinsing step conditions with unexpected cell viability benefits relative to conventional processing steps. In embodiments, the disclosed techniques include contacting a cancellous portion with glycerol combined with Lactated Ringer’s at glycerol concentrations that, for example, provide enhanced cell viability at one or more freezing temperature conditions. In certain cases, the disclosed product can be maintained for two or more months (e.g., six months or more) at -70 to -80°C storage conditions.

[0087] Although popular among surgeons, many cellular bone matrices have complicated thawing and preparation steps. For example, thawing in a jar or rigid container can take up to 30 minutes. In contrast, in an embodiment, the disclosed cellular bone matrix compositions are frozen in flexible bags, flexible pouches, vessels, or receptacles such as a pouch. The CBM is frozen in pouches as the primary packaging which permits much faster thawing (<15 minutes). This facilitates access and quick access in a surgical setting. In one aspect, the malleable pouch allows for external manipulation of the CBM as it thaws. In addition, many cellular bone matrices require that the interoperative solution be poured / decanted off after thawing because of dimethyl sulfoxide (DMSO) toxicity. Sometimes, the tissue has to be rinsed with saline. In contrast, the disclosed cellular bone matrix compositions do not require decanting or washing to maintain high cell viability after thawing. If however, the user decants off any volume of liquid or storage solution, the pouch can be pinched off at the end to facilitate decanting. As well, a modified end portion of the pouch may incorporate a perforated sheet integrated with the sidewalls of the pouch to contain the CBM while allowing the extra volume of liquid to be easily extracted or poured off. As well, CBM packaged within individual cannulas internal tothe pouch will be capable of being frozen and thawed from a freezing state in reduced times compared to that of a jar. In further embodiments, the disclosed techniques describe methods and kit compositions containing materials relating to CBM packaging and / or processing such that CBMs can be prepared and implemented in a surgical or other clinical setting. These features enhance end user workflow relative to products that involve more complex handling.

[0088] A cellular bone matrix composition may be formed from a cancellous portion of a bone (e.g., cancellous chips) and a noncancellous, e.g., cortical or compact, portion of the bone (e.g., cortical fibers). One embodiment of the noncancellous portion of bone includes cortical bone particles, pieces, segments, and / or fibers. When harvesting or grating fibers from the donor tissue, the fibers are produced in a ribbon-like configuration, the fibers of which are irregular in shape with expansive surface areas. For exemplary purposes, and not limitation, the ribbon-like structures maintain the ribbon-like configuration that is a columnar-like sheet that wraps around itself. The ribbon-like configuration has increased surface area via the curled portion and, in embodiments, surface nanostructures. In some embodiments, nanostructures are imparted to bone matrix compositions wherein the nanofibrous properties on the surface may be compromised. In other embodiments, the bone is processed in a manner so as to expose and / or maintain the nanofibrous structure of the bone. The nanostructures may be as disclosed in U.S. Patent No. 10,220,115, the disclosure of which is hereby incorporated by reference in its entirety herein.

[0089] The particulates or fibers harvested range in size with variations from millimeters to centimeters, as described herein. At the end stage of milling, the particulates and fibers may range in size to form a non-homogenous fiber mix. In an embodiment, the fibers may range in micron sizes from about 50 microns to 5 cm or 10 cm. In an embodiment, the noncancellous portion contains less than 10% soft tissue. Both the chips and fibers, when provided as a cellular bone matrix, act as a scaffold for bone formation to a subject in need of bone repair. The cancellous portion, which contains viable bone cells, is processed separately from the noncancellous portion during cellular bone matrix manufacturing to retain the viability of the bone cells during processing. That is, processing steps applicable to the cortical fibers may be unsuitably harsh for the cancellous chips. However, certain processing steps to clean, disinfect, or cryopreserve the cancellous portion may nonetheless reduce bone cell viability and osteoinductivity. In an embodiment, the disclosed techniques permit processing of the cancellous portion to clean, disinfect, and / or cryopreserve while maintaining adequate bone cell viability. Thus, the cellular bone matrix end product produced by the disclosed techniqueshas desirable clinical characteristics.

[0090] The mix of chips and demineralized fibers in cellular bone matrix compositions as provided herein provide excellent handling. The fibers yield a cohesive putty -like implant, and the mineralized chips provide some grittiness. Internal testing has shown that the disclosed cellular bone matrix compositions are osteoinductive in the gold standard athymic rat muscle pouch model.

[0091] FIG. l is a processing method 10 to produce a cellular bone matrix from donor bone tissue. At step 20, the donor bone tissue is separated into a cancellous portion and a noncancellous portion, as shown by way of example in FIG. 2. In an embodiment, the donor bone tissue exterior is cleaned. Donor bone treatment may also include a debridement step to remove non-bone tissue content prior to separation. In one aspect, a high-pressure water cleaning process is used to clean the bone. In embodiments, the donor tissue is provided without having been previously frozen to maintain cell viability. Thus, the disclosed bone growth compositions, after freezing, may be considered fresh-frozen. The separation into the cancellous portion and the noncancellous portion may be via cutting or sawing techniques to remove cancellous tissue from the distal and proximal ends of a bone shaft. The cancellous portion and the noncancellous portion may be cut or segmented during or after separation into blocks that are suitably-sized for downstream steps of the process 10.

[0092] As is often the case, noncancellous bone is attached to or encasing or surrounding the cancellous bone elements of the tissue being segmented from the native anatomy. Thus, as provided herein, the cancellous portion may also include noncancellous elements in some nonzero percentage that are incorporated and retained within the subsequent chip form. Further, as provided herein, the noncancellous portion and the cancellous portion may not be achieved with 100% homogeneity. In fact, this imprecise segmentation is desirable as it minimizes donor material loss by eliminating unnecessary cutting and trimming of the tissue during segmentation. It should be understood that the cancellous and noncancellous portions of bone as described herein are predominately cancellous bone or noncancellous bone, but comprise some elements of both. In an example, the cancellous portion and the noncancellous portion may be cut into blocks that are between 3-10 cm in one dimension. Processing may be modified here to cut at a desired size. As well, the tools utilized for creating the particulates or fibers may determine the portion sizes created.

[0093] Turning to the processing of the noncancellous portion as discussed in FIG. 1, the noncancellous portion, at step 22, is milled or otherwise processed (e.g., cut, grated, shredded) into fibers. The fibers may be any suitable size or shape. In an embodiment, the fibers have alength dimension that is at least twice a width and height dimension. In an embodiment, the fibers have an aspect ratio of at least 50(length): 1 (height and / or width), and an average length greater than 0.5 cm. In an embodiment, the width and height dimension of the fiber have about a 1 : 1 aspect ratio while the length dimension is elongated. In an embodiment, a fiber length dimension is at least 5, 100, or 1000 times a width and height dimension. Example ranges of a length dimension relative to other dimensions are between 5-50 times, 5-100 times, or 5-1000 times. In an embodiment, the fibers may be uniform or nonuniform, e.g., may have a uniform or nonuniform length, width, and or surface area. In an embodiment, the fibers may be curled or ribbon-like. In an embodiment, the fibers are less than 10 or 5 mm in any dimension. In some embodiments, the bone fibers have a diameter from about 100 pm to about 2 mm. In some embodiments, the bone fibers have a length from about 0.5 mm to about 50 mm. In some embodiments, the bone fibers have an average length from about 0.5 cm to about 10 cm. In an embodiment, the average bone fiber thickness is between 0.05 to 0.5mm.

[0094] In one embodiment, the bone fibers are milled using an osteobiologic milling machine as disclosed in U.S. Patent No. 9,004,384, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. The osteobiologic milling machine may be computer-controlled, with instructions that are programmed, stored, and / or executed by a programmable logic controller. The instruction may control milling parameters such as fiber length, thickness, and / or surface area. In an embodiment, the output of the osteobiologic milling machine may include material that is outside of tolerance. Thus, in an embodiment, the output may undergo a sorting or sieving step to remove bone components that are too long, too short, too thin, or too thick.

[0095] At step 24, all or some of the fibers are demineralized, which may refer to a process of removing inorganic content, such as minerals, from cortical fibers. The fiber end product after demineralization may have at least 50% or at least 90% or at least 99% of minerals (e.g., calcium) removed. In an embodiment, the fibers may be demineralized such that the fiber end product after demineralization has less than 1% residual calcium. Demineralization may occur via an acid soak (e.g., HC1, 0.6N HC1). At step 26, the demineralized fibers are disinfected with an alcohol solution (70% alcohol in water by volume) and at step 28 rinsed / transferred to a solution, such as Lactated Ringer’s, to soak before being combined with the processed cancellous portion at step 40. An embodiment of cortical fiber processing is discussed in more detail in FIG. 4.

[0096] Demineralized bone matrix (DBM) has been shown to exhibit the ability to induce and / or conduct the formation of bone. Demineralization improves flexibility and handlingcharacteristics. Also, calcium / mineral phase of bone can cover the collagen phase of bone. Demineralization exposes the bone growth factors to facilitate bone growth. It is therefore desirable to implant and maintain demineralized bone matrix at a site which bone growth is desired. Bone fiber based-demineralized bone matrices for implantation exhibit improvements in mechanical properties, including cohesiveness, fiber length, fiber diameter or width, fiber aspect ratio, or a combination of multiple variables.

[0097] Turning to the processing of the cancellous portion, at step 30, the cancellous portion removed from the bone tissue is milled into chips. As disclosed, the cancellous portion may refer to a bone tissue portion that is majority or predominantly cancellous tissue (e.g., greater than 50%, greater than 75%, or greater than 90% by volume) but that nonetheless includes other elements, such as some encasing cortical tissue as well as marrow elements, blood, fat that remain before cleaning. Indeed, a benefit of the disclosed technique is that milling the cancellous bone with the marrow elements present protects the cells within the tissue to improve viability and / or cell retention at this stage.

[0098] While fibers may be characterized by an elongated form, a chip in certain embodiments may have a planar form having a width dimension that is greater than a width of a fiber and a length dimension that is less than a fiber length while having a similar height dimension. In an embodiment, a chip aspect ratio is at least 50: 1, and the chip an average length greater than 0.1cm (e.g., 0.5 mm-2 mm). In an embodiment, the chips may be uniform or nonuniform. In an embodiment, the chips are less than 5mm in any dimension.

[0099] FIGS. 3A-B show an example cancellous portion of a bone tissue (see FIG. 2), e.g., a femur end, that is processed to form chips and that is predominantly cancellous tissue but that includes example cancellous (A), corticocancellous (B), and cortical (C) bone regions. The cancellous, cortical, and corticocancellous bone regions are respective sources for cancellous, cortical, and corticocancellous chips. As discussed, the population of chips is predominantly cancellous (e.g., greater than 50%, greater than 75%, greater than 95%) by percentage. However, a certain percentage of chips include cortical or noncancellous tissue. Further, an individual chip of the chips may be comprised of 100% cancellous tissue, 100% cortical tissue, or may be a corticocancellous chip generated from a corticocancellous region, e.g., a heterogenous mixed chip that includes both cancellous tissue and cortical tissue present on a single chip. In one example, cancellous chips may include a population of chips are 50-99% cancellous with the remaining chips being one or more of noncancellous or mixed cancellous / noncancellous. In one example, the chips are about 80-90% cancellous and about 10-20% mixed cancellous / noncancellous. In one example, the chips are about 90-99% cancellous andabout 1-9% mixed cancellous / noncancellous. The cancellous chips as provided herein may refer to a set or population of chips that is at least 50% cancellous. Cortical chips may be less porous and more dense than cancellous chips. Thus, while the chips may be sized and shaped similarly, their structural qualities may be different. In an embodiment, cancellous chips have at least 1% noncancellous materials. Further, demineralized bone fibers formed from the cortical portion may also have a heterogenous composition that includes at least 1% noncortical materials.

[0100] The chips are washed at step 32 to begin removal of non-bone tissue elements and contacted with an antimicrobial agent at step 34. The chips may be soaked in the antimicrobial agent for a period of time, e.g., a predetermined period of time. In an embodiment, the antimicrobial soak is conducted in a temperature range of 25-37°C and for at least 1 hr. or in a range of 3 hrs-12 hrs. In an embodiment, the antimicrobial soak is conducted at 35-37°C or 37°C and for at least 5 hours but no more than 10 hours. The antimicrobial solution is selected based on characteristics of the antimicrobial agent and at a concentration sufficiently high to kill undesirable microorganisms. In an embodiment, the antimicrobial agent is mixed with water (e.g., filtered water). In an embodiment, the antimicrobial agent is mixed with an isotonic solution (e.g., lactated Ringer’s solution, saline, etc.). In an embodiment, the soak is conducted with agitation and / or stirring.

[0101] After the antimicrobial contact, the chips are washed to remove the antimicrobial agent (or to leave only residual amounts of the antimicrobial agent) using one or more washes at step 36. The wash step can use Lactated Ringer’s (e.g., Lactated Ringer’s solution or Ringer’s lactate) to facilitate transition to the cryopreservative contact at step 38. In certain embodiments, the wash or rinse is conducted multiple times, e.g., three times, with removal of rinse solution between the rinses. In certain embodiments, the wash or rinse is timed (e.g., 5- 15 minutes), and includes agitation (e.g. light stirring, etc.). After the antimicrobial contact, the chips are antimicrobial -treated chips. At step 38, the antimicrobial -treated chips are soaked in an interoperative solution (e.g., a cryopreservative solution). In an embodiment, the interoperative solution is 10-25% glycerol by volume and 75-90% mixing solution. In an embodiment, the interoperative solution is 15% glycerol by volume, 85% mixing solution. In an embodiment, the mixing solution is Lactated Ringer’ s. In an embodiment, the interoperative solution is 20% glycerol (e.g., has a glycerol content of 20% by volume), 80% Lactated Ringer’s. In an embodiment, the chips are soaked in the interoperative solution for a period of time, e.g., a predetermined period of time. In an embodiment, the chips are soaked in the interoperative solution for at least 30 minutes, at least 60 minutes, at least 120 minutes, or atleast 180 minutes. In an embodiment, the chips are soaked in the interoperative solution for 30 minutes to 240 minutes. In an embodiment, the chips are soaked in the interoperative solution for 60 minutes to 180 minutes. The interoperative solution can be provided in sufficient volume to cover the chips. In certain embodiments, after completion of the soak, at least some of the interoperative solution can be decanted or removed in preparation for freezing.

[0102] In an embodiment the Lactated Ringer’s as provided herein may be Lactated Ringer’s (e.g., Baxter Lactated Ringer’s from Baxter, B. Braun Lactated Ringer’s from B. Braun Medical Inc.) having the following composition: each 100 mL of Lactated Ringer's contains: Sodium chloride 600 mg; sodium lactate, anhydrous 310 mg; potassium chloride 30 mg; calcium chloride, dihydrate 20 mg. The pH is 6.6 (6.0 — 7.5). One liter has an ionic concentration of 130 mEq sodium, 4 mEq potassium, 2.7 mEq calcium, 109 mEq chloride and 28 mEq lactate. The osmolarity is 525 mOsmol / L (calc).

[0103] In certain embodiments, the cryopreservative is glycerol, and the interoperative solution does not include DMSO. In an embodiment, cryopreservative is glycerol, and the interoperative solution does not include any additional cryopreservative agents. This interoperative solution in combination with the viable bone cells and / or demineralized fibers may be referred to in embodiments as a cryo-ready product or bone growth composition.

[0104] At step 40, the chips and fibers are combined for storage. As discussed above, the chips are processed prior to freezing and complete processing wetted with interoperative solution. The fibers are processed and may complete processing with a soak or wash in the mixing solution without any cryopreservative, e.g., in a Lactated Ringer’s solution. Thus, mixing of the chips and fibers dilutes the existing cryopreservative present with the chips. The amount of dilution depends on 1) the volume of Lactated Ringer’s solution carried together with the fibers relative to the volume of interoperative solution carried together with the chips at combination and 2) the ratio of chips to fibers.

[0105] In an embodiment, the ratio of chips to fibers is 1 : 1. In some embodiments, the chips to fibers ratio is about 90: 10, 80:20, 75:25, 70:30, 60:40, 50:50, 40:60, 30:70, 25:75, 20:80 and / or 10:90. In some embodiments, the chips to fibers ratio is in a range between 70:30 and 30:70, between 60:40 and 40:60, between 55:45 and 45:55, or between 51 :49 and 49:51. The ratio can be assessed as a volume:volume or a weightweight ratio.

[0106] After combination, the combined chips and fibers that form the cryo-ready product are sealed in an appropriate freezing container, e.g., a pouch, and exposed to a chilled environment, a cold environment, or a freezing environment at step 42. In embodiments, the cold environment is a refrigerator temperature that is 5°C or less and / or between 0°C and 5°C.In embodiments, the freezing environment is a cryogenic environment. In an embodiment, the freezing environment is 0°C or less. In an embodiment, the freezing environment is 0°C to - 200°C. In an embodiment, the freezing environment is 0°C to -20°C. In an embodiment, the freezing environment is -15°C to -20°C. In an embodiment, the freezing environment is -15°C to -100°C. In an embodiment, the freezing environment is -15°C to -80°C. In an embodiment, the freezing environment is -40°C to -80°C. In an embodiment, the freezing environment is - 65°C to -80°C. In an embodiment, the freezing environment is -70°C + / - 15°C. In an embodiment, the freezing environment is -80°C + / - 15°C.

[0107] In certain embodiment, the disclosed cryo-ready product provides improved stability and viability even if certain steps in the freezing, storage, transport, and / or thawing process are not performed according to recommended guidelines. Further, the disclosed cryo- ready product, when frozen or chilled, may maintain viability even during conditions in which enzymatic or other chemical processes of the viable cells are still active at a lower level based on the storage temperature. In an embodiment, the disclosed cryo-ready product may be stored at temperatures at which certain cell processes, such as enzymatic reactions, are not completely shut down.

[0108] FIG. 4 is an example fiber processing method 50 that may be used in conjunction with FIG. 1. At step 52, bone shafts separated from bone ends are provided, and soft tissue and marrow is removed at step 56. At step 58, bone shafts, which may be provided as segments (e.g., 50-150 mm long segments) are milled into fibers. The milling may be performed using a cartridge mill. The fibers are sieved and shard material can be removed during the process at step 60. Post-milling. The fibers undergo at least one and, in embodiments at least two demineralizations at step 62. The demineralization may be an acid soak, such as a 0.6N HC1 soak. In an embodiment, the first demineralization is a 15-150-minute acid soak with occasional stirring. In an embodiment, the second demineralization is a 45-260-minute acid soak with occasional stirring. During acid soaks, pH may be monitored with a target pH of less than 1. Soak time or other conditions can be adjusted to achieve the target pH.

[0109] The fibers are rinsed in water (e.g., filtered water) at step 64. In an embodiment, the rinse is conducted at least two or three times (e.g., ten-minute soaks with soak solution replacement), and the fibers are subsequently soaked in alcohol at step 68. The alcohol soak may be in 70% ethanol (e.g., 20-200 minutes). The alcohol soak is followed by a soak in the mixing solution used for the cryopreservative, e.g., Lactated Ringer’s at step 72 until the measured pH is at least 3 (e.g., 5-800 minutes). The demineralized fibers are then combinedwith chips and cryopreservative at step 74. FIG. 5 shows example fibers.

[0110] In some embodiments, the bone fibers have an average length to average thickness ratio or aspect ratio of the fibers from about 50: 1 to about 1000: 1. In overall appearance the bone fibers can be in the form of ribbons, threads, narrow strips, and / or thin sheets. The elongated bone fibers can be substantially linear in appearance or they can be coiled to resemble springs. In some embodiments, the bone fibers have linear portions and coiled portions. In some embodiments, the bone fibers are of irregular shapes including, for example, linear, serpentine and / or curved shapes. In some embodiments, the fibers can be curled at the edges to have a substantially hemicircular cross-sections. In some embodiments, the fibers may be entirely or partially helical, circumvoluted or in the shape of a corkscrew. The elongated bone fibers can be demineralized however some of the original mineral content may be retained when desirable for a particular embodiment. The bone graft fiber may further comprise mineralized bone material.

[0111] The bone fibers may be elongated and curled to increase the surface area of the strips. The curled fibers may include frayed portions along the edges to facilitate interactions with other bone fibers. In some embodiments, the curled fibers are milled to have hooked portions along the edges of the fibers configured to engage with other fibers. The hooked portions may engage other hooked portions, frayed portions, straightened portions or curled portions of other fibers. The hooked and frayed portions and the curled shape of the fibers provide for entanglement between fibers such that the fibers may form a coherent mass without the need for a carrier or binding agent, as generally disclosed in U.S. Patent Publication No. US20210402060A1, the disclosure of which is incorporated by reference in its entirety herein. The fibers may include nanofibers of a submicron level or any fibers having at least one side or dimension at or below 100 nanometers. In specific embodiments, the fibers may have at least one side or dimension at or below 100 nanometers. The fibers may include nanostructures, such as nanofibers, nanoparticles, nanospheres, nanopores, nanomicelles, and nano-roughness on surfaces. Nanostructures include structures ranging from approximately 1 nm to approximately 100 nm in at least one dimension. The nanostructures may be part of a nanotextured surface.

[0112] In an embodiment, the bone growth composition materials, such as the fibers or chips, may include nanoscale textured surfaces attractive to cells. Nanoscale textured surfaces provided on a bone matrix aid in growth factor retention, remodeling, cell attachment, and osteoconductivity of the bone matrix. In embodiments wherein the nanostructures of the textured surface comprise nanofibers, the nanofibers may be oriented. In some embodiments,the nanostructures of the textured surface may be biologically active. For example, the nanostructure may comprise biologically active biomolecules or incorporated with other biological factors such as peptides, growth factors, cytokines, DNA, RNA, siRNA etc.

[0113] In an embodiment, the nanoscale textured surface of bone is retained during processing steps to prepare the bone growth composition. In an embodiment, the nanoscale textured surface is imparted to bone or enhanced via one or more processing steps. In certain cases, the fibers are prepared without lyophilization or drying. Because removal of moisture can deteriorate surface nanostructures, maintaining wetted fibers as provided herein may be associated with improved nanoscale feature retention. However, in embodiments, even in cases where the fibers and / or chips remain wet through the process, the bone growth composition may be subjected to steps that improve nanoscale features on the bone surface, such as providing hydrogel carriers (e.g., dextran, pluronics, N,O-carboxymethylchitosan glucosamine) and / or adding nanoscale features as a surface coating or treatment via electrospinning nanofibers (e.g., collagen, polylactide, polycaprolactone, polyglycolide, chitosan, gelatin, or other nanofibrous material) onto bone surface. The nanofibers may include a pharmaceutical agent or bioactive material as provided herein.

[0114] FIG. 6 is a two-step chip washing process 32 that may be used in conjunction with FIG. 1 or other bone handling processes. In an embodiment, the chip washing process 32 may be performed after milling a cancellous portion into chips. At step 122, cancellous bone chips are provided (e.g., from milling as in FIG. 1), and the chips are washed at a first temperature at step 124 and subsequently washed at step 126 at a second temperature lower than the first temperature. The first wash may be in a same or different solution than the second wash.

[0115] FIG. 7 is an embodiment of the two-step chip washing process 32 of FIG. 4. At step 132, cancellous bone chips are provided (e.g., from milling as in FIG. 1), and the chips are washed in a first washing solution that is a nonisotonic solution (e.g., water, filtered water) at a first temperature for a first time period at step 134 and subsequently at step 136 washed in a second washing solution that is Lactated Ringer’s at a second temperature lower than the first temperature and for a second time period longer than the first time period (e.g., the first time period is shorter than the second time period). The illustrated process 32 is by way of example, and the time of washes may, in embodiments, be the same (e.g., 5-15 minutes), or the second wash may be shorter in time than the first wash in another embodiment.

[0116] In an embodiment, the first wash step is conducted at 40-50°C (e.g., 42-44°C) for 5-15 minutes in a closed vessel with agitation, spinning, and / or a motorized impeller. In an embodiment, the first wash step is conducted for a minimum of 5 minutes and a maximum of15 minutes. The second wash may be conducted at 22-35°C (e.g., 25°C) for 5-20 minutes or at least 20 minutes in a closed vessel with agitation, spinning, and / or a motorized impeller.

[0117] The water used in the first wash is nonisotonic relative to the bone cells of the chips. The two-step wash maintains cell viability relative to a wash in an isotonic solution, which is an unexpected benefit.

[0118] FIG. 8 shows a side-by-side comparison of a cancellous tissue portion sample 150 and associated histology image 160 washed with the two-step wash as discussed in FIG. 5 relative to a control cancellous tissue portion sample 152 and associated histology image 162 processed in a one-step wash. As shown in the image 160, the sample 150 has limited marrow and soft tissue attachment to chips while the image 162 is indicative of marrow and / or soft tissue attached to most chips in the control sample 152. The effectiveness of the two-step chip washing is demonstrated. Using this technique, there is very little marrow and soft tissue still attached to the bone chips after cleaning (left image 160). Very small infrequent pockets of non-bone tissue (circled in left image 160) can only be found at high magnification. When a single-step wash technique is used, considerable bone marrow and soft tissue still remain (circled in right image 162). Large pockets of non-bone tissue are evident throughout even at low magnification

[0119] FIG. 9 is a method 200 of monitoring viability of osteogenic cells after a two-step washing process, as discussed with respect to FIGS. 4-5 and with reference to FIGS 1-3. At step 202, milled cancellous bone chips are provided. The chips are washed using a two-step wash (see FIGS. 4-5) at step 204 as provided herein, and the washed chips are provided to additional steps of the bone growth composition processing at step 206.

[0120] FIGS. 10-11 show example cell viability data for different washing conditions. FIG. 10 is a comparison of cell viability, as assessed by PrestoBlue, of washed non-frozen tissue using saline to wash non-frozen tissue using Lactated Ringer’s from a single donor. Lactated Ringer’s as a wash solution demonstrated improved viability relative to saline. The cell viability is shown as a percentage comparison to Lactated Ringer’s of 0.9% saline for washed, non-frozen chips.

[0121] FIG. 11 is a comparison of cell viability, as assessed by PrestoBlue, of different washing procedures assessed for a single donor. A single-step Lactated Ringer’s wash by stirring or a single-step MicroAire lavage with Lactated Ringer’s, did not perform as well as a two-step water (CPW denoting a clean room water source or filtered water) and Lactated Ringer’s wash. The cell viability is shown as a cell viability percentage compared to a two- step water and Lactated Ringer’s wash for a single-step Lactated Ringer’s wash or single-stepMicroAire lavage with Lacatated Ringer’s in which the two-step wash showed improved viability.

[0122] The disclosed techniques may use a glycerol cryopreservative that can be used with direct freezing in certain embodiments. That is, packaged and sealed cellular bone matrix products may be placed directly in a conventional freezer, and do not necessarily require a controlled freezing process to maintain sufficient cell viability. In addition, in certain embodiments, the cellular bone matrix products as provided herein may be used without decanting liquid cryopreservative from the thawed product and / or without rinsing or centrifugation. In certain embodiments, the product is thawed to 37°C in a solution bath, and the packaged and sealed cellular bone matrix products can remain in the cryopreservative at the thawing conditions for at least 2 hours or 4-6 hours after thawing without significant cell viability impacts.

[0123] In an embodiment, the frozen bone growth composition is in a solid state and transitions to a partially liquid state after thawing. That is, certain portions of the bone growth composition may remain solid while certain portions may be liquid. In an embodiment, the bone growth composition, after thawing, may have a flowable, slurry, or putty -like consistency. The term “flowable” may refer to compositions whose consistencies range from those which can be described as shape-sustaining but readily deformable, e.g., those which behave like putty, to those which are runny. Specific forms of flowable bone powder compositions include cakes, pastes, creams and fillers. Reference is made to U.S. Pat. No. 5,290,558, herein incorporated by reference in its entirety, for discussion of flowable materials. The bone growth composition may be a gel, putty, paste, cake, or solid in embodiments. The disclosed bone growth composition provides a product with sufficient cohesion for efficient manipulation of the product interoperatively and, in embodiments, improved mixing with other materials (autograft, other bone graft materials, etc.), and improved bone defect filling and shaping to optimize bone defect repair.

[0124] FIG. 12 shows a comparison of cell viability for a glycerol cryopreservative in Lactated Ringer’s (e.g., 80% Lactated Ringer’s) relative to certain commercial interoperative solutions demonstrating that 20% glycerol by volume outperforms multiple commercial cryopreservatives under the same conditions. The cell viability is shown as a cell viability comparison normalized to the 20% glycerol by volume results for various other interoperative solutions.

[0125] FIG. 13 shows cell viability for various glycerol concentrations in Lactated Ringer’s. The cell viability is shown as a cell viability comparison for various glycerolconcentrations in Lactated Ringer’s normalized to 20% glycerol by volume.

[0126] FIG. 14 is a method 220 of combining chips and fibers during cellular bone matrix production and with reference to FIGS. 1-5. At step 222, milled cancellous bone chips are provided. At step 224, chips are contacted with an interoperative solution or cryopreservative as provided herein. In certain embodiments, the cryopreservative contact occurs subsequent to the antimicrobial contact and rinse. In certain embodiments, the rinse is Lactated Ringer’s, and the rinse solution is removed to facilitate the cryopreservative soak in the rinse solution with 10-20% glycerol by volume. In other embodiments, the glycerol may be directly added to the rinse solution. The soak may be conducted at 22-30°C in an embodiment.

[0127] After the appropriate amount of time has elapsed (e.g., a minimum of 60 minutes to a maximum of 180 minutes in an embodiment), the excess interoperative solution of the chips can be decanted, leaving enough solution behind to keep the combined cellular bone matrix product wetted. At step 226, the chips and fibers are combined in an approximate 1 : 1 ratio. By way of example, chips and fibers can be aliquoted using a same-sized volume (e.g., cubic centimeters) measure. The combined material can be filled into various pouches according to desired size and sealed. The chips and fibers, together with the interoperative solution, may be measured according to total volume, such as cubic centimeters. In an embodiment, the liquid of the interoperative solution may be measured in mL. For example, a bone growth composition may be aliquoted using 1-15 cubic centimeters of fibers and 1-15 cubic centimeters of chips. The chips and fibers may carry liquid that, in the packaging, forms the interoperative solution. This solution may be present in liquid volume amounts of 1-10 mL (e.g., 1-50 cc), in an embodiment. In an embodiment, the excess (e.g., free or decantable) total liquid amount in the packaging is 0-2 mL and a total amount of the fibers or chips is 1-10 cubic centimeters or 6-8 cubic centimeters.

[0128] It should be understood that the disclosed compositions are wetted and, therefore, the interoperative solution that is present may be associated with, absorbed by, or otherwise coupled together with the bone tissue. Thus, more liquid may be present than is decantable within the package. In contrast to less viable compositions, the disclosed wetted bone growth compositions are exposed to associated interoperative solution, even after thawing, and nonetheless maintain significant viability.

[0129] In an embodiment, any remaining solution in the combined chips and fibers composition has a cryopreservative, e.g., glycerol, present in a range of 1-25%. In certain embodiments, the fibers, stored in Lactated Ringer’s, are drained prior to combination. Thus,little or no Lactated Ringer’s from the stored fibers may be carried over into the combined composition, and a majority of the solution present is the interoperative solution. Accordingly, chips in an interoperative solution having 20% glycerol may yield a cellular bone matrix composition having 1-20% glycerol in an embodiment. In certain embodiments, the Lactated Ringer’s solution is present in about equal volume as the interoperative solution. By way of example, in a 1 : 1 ratio combination, the existing cryopreservative is halved in percentage after combination. Accordingly, an interoperative solution having 20% glycerol has about 10% glycerol after combination in such an embodiment. In certain embodiments, the fibers are retained in Lactated Ringer’s prior to combination while the chips have been drained or partially drained. Accordingly, an interoperative solution having 20% glycerol has less than 10% glycerol after combination in such an embodiment.

[0130] The disclosed cellular bone matrix production techniques incorporate a cryopreservative contact step to maintain cell viability at cryogenic temperatures. However, cryopreservative agents, while protective at cryogenic temperatures, may be cytotoxic at temperatures higher than the cryogenic temperatures. Thus, cryopreservatives have potential to reduce cell viability both during the freezing process as well as during the thawing process. In addition, the time spent in contact with the cryopreservative after thawing is dependent on the end user preparation conditions and preferences. Certain techniques may involve a decanting and / or centrifugation step post-thawing to remove the cryopreservative present in the thawed sample. However, these steps may also be associated with loss of viable cells to handling. In addition, certain techniques may also involve a controlled freezing process that involves a specialized freezing container with a linear temperature decrease per minute to reduce cell loss as a result of cryopreservative contact during the freezing process.

[0131] FIG. 15 shows an example bone growth composition product in which wetted demineralized bone matrix fibers 290 are combined with a cancellous chip mixture in interoperative solution 292 having viable cells that are in interoperative solution. As discussed herein, the cancellous chips in interoperative solution 292 may include a mix of cancellous chips 294, cortical chips 296, and corticocancellous chips 298. The chips 292 may be predominantly cancellous chips 294 (e.g., 50-90% or greater) with the remainder being cortical chips 296 and / or corticocancellous chips 298.

[0132] The fibers 290 and cancellous chips in interoperative solution 292 are packaged in a container, shown as a pouch 304, to form a cryo-ready bone growth composition 306. The pouch 304 is sealed, and the bone growth composition 306 is frozen, stored, and / or transported for use. Thus, the bone growth composition product may, in embodiments, include both thepackaging, such as the pouch 304, and the bone growth composition 306.

[0133] FIG. 16 is an image of an example packaging including a sealed interior pouch with bone growth composition being aseptically removed from a protective outer packaging. FIG. 17 shows the sealed interior pouch with bone growth composition. FIG. 18 shows thawing of bone growth composition in the sealed interior pouch. FIG. 19 shows an end user opening the interior pouch after thawing to access the bone growth composition.

[0134] In certain embodiments, and as illustrated in FIGS. 20 and 21, the bone growth composition 306 may be “triple-pouched”, with the pouch 304 (e.g., main or intermediary pouch) containing a sterile sealed inner pouch 704 in which the bone growth composition 306 is contained. An outer or protective pouch 706 may in turn contain the pouch 304. In certain embodiments the pouch 304 and / or the outer or protective pouch 706 may be ClearFoil pouches or other suitable composition. In one embodiment the inner pouch 704 may be a nylon pouch, but in practice the inner pouch 704 may be fabricated using any medically suitable polymer or composition. In one such example, using suitable sterile techniques, the pouch 304 may be opened (e.g., peeled open using unsealed flaps at an end, cut open at a visually marked indicator, tom open along a perforation, and so forth) and the sterile inner pouch 704 containing the bone growth composition 306 may be aseptically accessed and transferred to a sterile field. By way of example, FIG. 20 depicts the three pouches of such an embodiment separate and relative to one another while FIG. 21 depicts the three pouches of such an embodiment in a practical implementation for storage and transport, with the pouch 304 placed within the outer pouch 706, the inner pouch 704 placed within the pouch 304, and the bone growth composition 306 placed within the inner pouch 704. The pouches may also have an integrated sealing feature 714 (e.g., a user-activated complementary sealing feature, such as an interlocking groove and ridge seal mechanism), such that tissues may remain stored within the pouch.

[0135] FIG. 22 shows the bone growth composition packaging in different conditions. At the end of processing of the donor tissue, as shown in FIG. 15, the fibers 290 and chips 292 (e.g., chip mixture) are combined to form the cryo-ready bone growth composition 306 that is packaged and sealed in the pouch 304. The processing conditions may be room temperature conditions, e.g., 20-25°C. After packaging, the pouch is placed in a freezing environment, e.g., a cryogenic environment, and the bone-growth composition 306 in the pouch transitions from a room temperature or nonfrozen state to a frozen bone growth composition 306. When the bone growth composition 306 is ready for use in a patient, the pouch 304 is removed from the freezing environment and thawed, and the frozen bone growth composition 306 transitions to a thawed bone growth composition 306.

[0136] In an embodiment, the number of viable cells of the bone growth composition 306 present at the time of packaging is reduced by less than 30% or less than 50% after thawing.

[0137] FIG. 23 shows a bone growth composition packaging system in which the pouch 304 includes features to permit decanting of liquid via a port 309 that, when opened, breaks the pouch seal. Additionally, or alternatively, the pouch 304 may include a release liner 310 that can be peeled away or removed to reveal a perforated wall 312 including perforations that extend from an exterior of the pouch 304 through to an interior space 310 to permit draining of cryopreservative liquid via the perforations. The perforations may be sized to retain chips and fibers in the pouch 304 while permitting liquid drainage.

[0138] As provided herein, osteogenic may refer to the ability of a material to enhance or accelerate the growth of new bone tissue by one or more mechanisms such as osteogenesis, osteoconduction, and / or osteoinduction. FIG. 24 is a method 800 of monitoring viability of osteogenic cells after chip processing. At step 802, milled cancellous bone chips are provided. The chips are processed at step 804 as provided herein, and the viability of the osteogenic cells is assessed at step 806. In the illustrated embodiment, the viability is assessed after processing, e.g., after the chips are contacted with the cryopreservative. Viability may additionally be assessed following contact with the antimicrobial agent and / or after thawing.

[0139] In an embodiment, the viability is assessed using a modified PrestoBlue assay. Viable cells are able to convert resazurin (blue) to resorufin (pink). The absorbance indicative of pink color conversion can be compared against a standard curve based on known numbers of viable cells to estimate a viable cell count in a sample.

[0140] As provided herein, the cancellous chips may have an estimated viable cell concentration of at least 50,000 viable cells / cc.

[0141] Osteoinductivity, may refer to the quality of being able to recruit cells from the host that have the potential to stimulate new bone formation. Any material that can induce the formation of ectopic bone in the soft tissue of an animal is considered osteoinductive. For example, most osteoinductive materials induce bone formation in athymic rats when assayed according to the method of Edwards et al., “Osteoinduction of Human Demineralized Bone: Characterization in a Rat Model,” Clinical Orthopaedics & Rel. Res., 357:219-228, December 1998. In other instances, osteoinduction is considered to occur through cellular recruitment and induction of the recruited cells to an osteogenic phenotype. Osteoinductivity score refers to a score ranging from 0 to 4 as determined according to the method of Edwards et al. (1998) or an equivalent calibrated test. In the method of Edwards et al., a score of “0” represents no new bone formation; “1” represents l%-25% of implant involved in new bone formation; “2”represents 26-50% of implant involved in new bone formation; “3” represents 51%-75% of implant involved in new bone formation; and “4” represents >75% of implant involved in new bone formation. In most instances, the score is assessed 28 days after implantation. However, the osteoinductivity score may be obtained at earlier time points such as 7, 14, or 21 days following implantation. Percentage of osteoinductivity refers to an osteoinductivity score at a given time point expressed as a percentage of activity, of a specified reference score.

[0142] As provided herein, the cellular bone matrix thawed from frozen may have an osteoinductivity (01) score of at least 1 or at least 2 based on a 50 / 50 chip vs. fiber formulation. Results of osteoinductivity tests for cellular bone matrix products prepared according to the disclosed techniques are shown below in Table 1. The osteoinductivity tests were performed as in Edwards et al. and using a rat muscle pouch assay. Table 2 demonstrates that the osteoinductivity of the tissue was maintained during extended storage at -70°C. In particular, Table 2 illustrates that osteoinductivity is maintained following storage at -70C. In the testing conditions, samples were stored in the freezer at -70C for an extended amount of time (shown in table 2) and tested again in the rats. The results confirm that the tissue retained its osteoinductivity. On average, the osteoinductive score for the disclosed cellular bone matrix compositions is higher than that of tested competitive products. By way of example, OI scores were assessed for competitive products as follow: OI for Osteocel Pro (n=3 lots) was 0.9 ± 0.1. For OsteoCel Plus (n=5 lots), it was 0.2 ± 0.3. Map3 (no longer on market) was 0.25 ± 0.0 (n=3 lots).TABLE 1TABLE 2

[0143] The disclosed cellular bone matrix compositions are associated with relatively higher viable cell counts and longer shelf lives and / or longer time windows for use after thawing as a result of improved processing. In one example, disclosed cellular bone matrix products maintain viability for at least 2 hours or at least 4-6 hours after thawing and without separation from the interoperative solution. In an embodiment, the total thaw time for a 15 cubic centimeter (as packaged) size is 6 minutes or less.

[0144] The ability of the cellular bone matrix to promote bone healing in vivo was demonstrated in an athymic rat spine model. Each rat (n=8 animals) received 0.6 cubic centimeter (cc) of test article on each side of the spine (a total of 1.2 cc implant per animal) that was in contact with the bleeding bone of the decorticated L3-L5 transverse processes. The animals were sacrificed at 8 weeks post-operatively and were assessed for fusion by manual palpation, radiography, and histology. Table 3 demonstrates the number of sites evaluated via manual palpation and radiographic fusion assessment performed on explants during two-level rat spine fusion study. Four sites were targeted per each rat on the left and right side of the L3- L4 and L4-L5 (total of 32 sites). In general, all operative segments (32 / 32) had no motion with manual palpation, indicating that all eight rats were fused. FIGS. 25-27 show data (e.g., radiographs, histology) of a rat spine (e.g., Rat 1243).TABLE 3

[0145] FIG. 25 shows an example radiograph of the rat spine taken post-operatively (e.g.,post-delivery of the test article). In contrast, FIG. 26 shows an example radiograph of the rat spine taken at eight weeks after explantation of the spine. In general, FIG. 26 displayed new mineralization and complete bridging bone, which is representative for all 32 / 32 operative segments for all eight rats graded as fused. FIG. 27 shows an example histology of the rat spine taken at eight weeks after explantation (scale bar = 5 millimeters). Histopathology confirmed each implant site contained extensive new bone formation and marrow elements that span the transverse processes. In summary, the disclosed cellular bone matrix compositions demonstrated 100% fusion by manual palpation and radiography with bridging bone confirmed by histology in this model.

[0146] FIG. 28 is a percentage comparison between the direct and controlled freezing showing very similar viability results. As shown, the use of a controlled freezing device provided no cell viability benefit under the assessed conditions. Accordingly, in certain embodiments, the pouches or other containers may be placed directly in cryogenic conditions, such as a -70°C freezer, without use of the controlled freezing device. A benefit of the disclosed production techniques for cellular bone matrix compositions is a less complex and more user- friendly freezing process.

[0147] FIGS. 29-31 show results from different antimicrobial agent soaks and contact conditions. FIG. 29 shows results of different soak times and antimicrobial agents, such as a gentamicin and vancomycin or 1% povidone-iodine. The cell viability is shown as with results normalized to a 2-hour gentamicin and vancomycin soak. The presence of povidone iodine was associated with decreased cell viability.

[0148] FIG. 30 shows benefits of performing the antimicrobial contact step at temperatures above room temperature. A 12-hour antimicrobial soak at 37°C demonstrated elevated results relative to room temperature soaks at various lengths. The cell viability is shown normalized to a no antimicrobial contact control.

[0149] FIG. 31 shows results of different soak concentrations. A concentration-dependent decrease in bone cell viability was observed. The cell viability is shown normalized to a no antimicrobial contact control.

[0150] FIG. 32 shows effects on cell viability using different post-antimicrobial soak rinse conditions results relative to a no rinse control. Including at least one rinse step did not reduce cell viability.

[0151] FIG. 33 shows post-thaw viability data for the bone growth composition of approximately, 100% at 1 hour and 90% at 5 hours normalized to the number of cells viable immediately after thawing (time zero or T=0). FIG. 34 shows post-thaw viability data for thebone growth composition of approximately 90% after 3 months, 6 months, 9 months, 12, 15, and 18 months of storage at -70°C normalized to the number of cells when the tissue was first placed into the freezer at the end of processing (time zero or T=0).

[0152] In further embodiments, the interoperative solution as discussed herein may be more generally used as a temporary, non-cryogenic storage or preservation medium for tissue prepared and / or used in a medical procedure, such as a surgical procedure. By way of example, FIG. 35 illustrates a method 810 in which a fresh or locally harvested and prepared tissue, such as a fresh autograft tissue or mixture, as discussed herein, is extracted from a subject, such as an intended recipient of an implant or autograft procedure and stored temporarily for subsequent transplantation into the intended recipient. At block 812, a fresh autograft tissue is extracted from an intended recipient during a surgical procedure with the expectation that the autograft will be transplanted into the recipient, such as during the same surgical procedure. As such, the autograft is considered “fresh” in this context. Further, the autograft material will typically be chilled, but not frozen (e.g., 0° - 12° C), during the procedure until it is needed for implantation. It should be noted that the fresh autograft tissue may include, but is not limited to, bone, tissue, or cartilage, etc. After the fresh autograft tissue is extracted from the intended recipient, the autograft tissue may be transferred into a receptacle or other suitable biosafe vessel containing the interoperative solution, as described herein (block 814) or to which the interoperative solution is subsequently added. The interoperative solution, as described herein, may contain a glycerol -based solution containing glycerol and a Lactated Ringer’s solution, wherein in certain embodiments the glycerol content (e.g., fraction) of the interoperative solution is equal to or less than 50% by volume, equal to or less than 45% by volume, equal to or less than 40% by volume, equal to or less than 35% by volume, equal to or less than 30% by volume, equal to or less than 25% by volume, equal to or less than 20% by volume, equal to or less than 15% by volume, equal to or less than 10% by volume, and so forth. In certain embodiments, the Lactated Ringer’s fraction of the interoperative solution is equal to or more than 50% by volume, equal to more than 55% by volume, equal to or more than 60% by volume, equal to or more than 65% by volume, equal to or more than 70% by volume, equal to more than 75% by volume, equal to more than 80% by volume, equal to or more than 85% by volume, equal to or more than 90% by volume, and so forth. For example, the interoperative solution may contain 10-25% glycerol by volume and 75-90% by volume mixing solution, 20% glycerol by volume and 80% mixing solution by volume, 15% glycerol by volume and 85% mixing solution by volume, or 10% glycerol by volume and 90% mixing solution by volume. In other embodiments, the interoperative solution may contain a combination of glycerol, LactatedRinger’s, and an antimicrobial agent (e.g., antibiotics, antiviral agents, or antifungal). It should be noted that the mixing solution described herein may contain Lactated Ringer’s and an antimicrobial agent. The interoperative solution can maintain cell viability of the autograft tissue, as the autograft tissue is fresh and will be transplanted into the intended recipient during the same procedure.

[0153] Further, at block 816, in certain embodiments the receptacle containing the fresh autograft tissue in interoperative solution is transferred or otherwise placed within a larger, secondary receptacle or receptacle containing a cooling medium that helps maintain a consistent, chilled temperature of the interoperative solution, upon where the autograft will remain chilled until it is needed for implantation. In some embodiments, the cooling medium represents a cold but non-cryogenic temperature and is at a temperature that is between -70°C and 15°C. For example, the temperature of the cooling medium may range between -50 and 15°C, -50 and 5°C, between -10°C and 0°C, or between 0°C and 5°C. Further, the cooling medium may consist of ice, a mixture of ice and water, a mixture of ice, water, and salt, or dry ice. It should be noted that while temperatures of the cooling medium may range between - 20°C and 10°C, the fresh autograft tissue in interoperative solution may exhibit chilled temperatures ranging between 0°C and 5°C. The processing conditions for harvesting and preparing the fresh autograft tissue to be placed in the interoperative solution may be room temperature conditions, e.g., 20-25°C.

[0154] In another embodiment, a bone growth composition as discussed herein may be combined with a fresh autograft tissue prior to placement in the receptacle containing the interoperative solution. The interoperative solution can maintain cell viability of the autograft, as the autograft is fresh and will be transplanted into the intended recipient during the same procedure. In such an embodiment, the receptacle including the bone growth composition and fresh autograft tissue mixture in interoperative solution may be placed into a larger, secondary container with a cooling medium as described above, upon where the autograft and bone growth composition mixture will remain chilled until the mixture is needed for implantation. Furthermore, the fresh autograft tissue in interoperative solution or mixture of fresh autograft tissue and bone graft composition in interoperative solution may be chilled for approximately less than or equal to 6 hours (e.g., less than 6 hours, about 4 hours, or about 2 hours) without substantially impacting cell viability. Further, the examples provided herein are meant to be non-limiting and may be utilized with other grafts, including allogenic grafts or xenografts.

[0155] With the preceding in mind, FIG. 36 illustrates a method 820 for processing the chilled, fresh autograft tissue in interoperative solution for use in surgical implantation. Asdescribed with respect to FIG. 35, the fresh autograft tissue in interoperative solution is placed in a secondary container with a cooling medium to maintain a desired (i.e., chilled) temperature. In another example, the tissue may be an allogeneic graft or a xenograft. In certain implementations the chilled, fresh autograft tissue in interoperative solution is removed from the secondary container containing the cooling medium, as described at block 822 prior to use, such as to allow the tissue to increase in temperature prior to use.

[0156] In one such embodiment, the chilled sample may include a mixture of fresh autograft tissue and bone growth composition. The fresh autograft tissue in interoperative solution may be cold (e.g., between 0°C and 5°C). To this end, the temperature of the chilled fresh autograft tissue may be elevated, as described in block 824. The temperature of the fresh autograft tissue can be elevated to either room temperature, or optionally, recipient body temperature immediately prior to implantation. The temperature of the fresh autograft tissue may be elevated to a desired temperature required for transplanting the fresh autograft tissue into the body of a suitable recipient using a heated water bath or such like device, submerging the cold or chilled packaged into a sample of growth medium, biological buffer, or tissue / organ storage solution (e.g., pre-warmed to the desired temperature). The techniques disclosed herein can bring the temperature of a cold or chilled tissue sample to the desired temperature required for transplanting the fresh autograft tissue or mixture of fresh autograft tissue and bone growth composition into the body of a suitable recipient.

[0157] Furthermore, at block 826, the fresh autograft tissue in interoperative solution undergoes a separation step, wherein the interoperative solution is removed through a decanting process, as described in FIG. 23 or alternatively by manually lifting or straining the autograft tissue from the interoperative solution. In another example, a sterile mesh may be used to help decant or pour out excess interoperative solution and retain the fresh autograft tissue with no or minimal loss. In certain embodiments, the samples as provided herein may be used without decanting liquid interoperative solution from the thawed product and / or without rinsing. That is, the interoperative solution is non-toxic and may be primarily removed by decanting or draining, without having to rinse the interoperative solution completely from the tissue prior to use (e.g., reimplantation).

[0158] After the interoperative solution is removed from the fresh autograft tissue and receptacle or, alternatively, the tissue is removed from the interoperative solution, the fresh autograft tissue or mixture of fresh autograft tissue and bone growth composition can be utilized in a surgical transplantation, as described in block 828. In another embodiment, the fresh autograft tissue or mixture of fresh autograft tissue and bone growth composition can betransferred to an application device for transplantation into the intended recipient. As such, the interoperative solution may be utilized in settings where autografts are prepared for surgical procedures such as cranioplasty, sinus lift, mandibular bone augmentation, cleft palate, cosmetic procedures (e.g., rhinoplasty, etc.), bone, spine, foot & ankle and synovial joint fusion procedures, vertebroplasty, long-bone trauma, revision joint, revision fusion, revision nonunion, foot and ankle fusion, and procedures involving “free-flap” bone. It should be noted that the specific examples described herein are meant to be non-limiting and are intended merely to provide useful real-world context and examples.

[0159] By way of further example, FIGS. 37 and 38 depict an example of a packaging and transportation approach incorporating integrated rinsing and drainage solution for the interoperative solution, transport solution, or cryosolution as described herein. By way of example, and turning to FIG. 37, a pouch or bag (e.g., pouch 304 or inner pouch 704 is depicted that incorporates a plurality of perforations or drain features through which interoperative solution, transport solution, or cryosolution may be drained without loss of bone growth composition 306 (e.g., microsize) or autograft tissue. In another embodiment, the perforations or drain features 710 allow for the bone growth composition 306 or autograft material to be rinsed within the pouch. In such an embodiment the rinsing or drainage features may have a uniform, staggered, or random placement pattern and may positioned on one or more sidewalls of the pouch. Such a pouch or package having integrated rinsing or drainage features would be sealed or otherwise stored in a larger pouch or bag (e.g., pouch 304 or outer pouch 706) through which the interoperative solution, transport solution, or cryosolution may flow during storage and into which the interoperative solution, transport solution, or cryosolution may during preparation. By way of example, in such an embodiment a user may open the outer pouch or bag and, in the process of lifting or removing the inner pouch or bag having the bone growth composition or autograft tissue, the interoperative solution, transport solution, or cryosolution may be drained from the inner pouch simply by the act of lifting or partially removing the inner, perforated pouch from the outer pouch. As discussed in other embodiments, such a pouch that incorporates integrated drainage or rinsing features may include an integrated sealing feature 714 (e.g., a user-activated complementary sealing feature, such as an interlocking groove and ridge seal mechanism) and / or a built-in release mechanism to access the bone growth composition or autograft tissue, such as a perforated tear-off region 718. In practice, the two pouches may be integrated as nested or attached pouches having integrated drainage or rinsing features (e.g., as a bag-in-a-bag solution). In such an integratedapproach, the two pouches may be in fluid communication with respect to the interoperative solution, transport solution, or cryosolution when the drainage or rinsing features are exposed.

[0160] Turning to FIG. 38, a partially transparent view of one embodiment is depicted. In the depicted example, the pouch incorporating the integrated drainage or rising features may incorporate such features (e.g., perforations) in a diaphragm region 726 (e.g., an inner wall or inner membrane), such perforations may be sealed prior to use for draining, such as by a removeable seal or layer that may be chemically adhered and / or structurally attached until removed, at which point the drainage features may be exposed and used. In this manner, pouch may be fluid tight up until a seal is removed to expose the drainage or rinsing features 710, at which point the interoperative solution, transport solution, or cryosolution may be drained or rinsed prior to use. In the depicted example, the pouch may include an integrated sealing feature 714 and / or a built-in release mechanism, such as a perforated tear-off region 718, as discussed herein. In such an example, a user may open the pouch may tearing or opening along the tear-off region 718, may insert autograft material 716 into the pouch along with the bone growth composition 306, and may reseal the pouch using integrated sealing feature 714. Once the autograft material 716 is added and the pouch is resealed, a user may knead or manipulate the sealed pouch so as to mix the bone growth composition 306 and the autograft material 716. Once mixed, the user may unseal or expose the integrated drainage features 710 and pour off the cryosolution. Alternatively, the user may rinse the pouch and the materials within using interoperative solution, transport solution, or cryosolution. It should be noted that other materials (e.g., allograft materials, xenograft materials) may be added to the pouch as well.

[0161] As such, FIG. 39 describes a composition 830 of a kit 832 that can be implemented in a surgical setting for the preparation and use of a fresh autograft tissue or a mixture of fresh autograft tissue and bone growth composition, as described in FIGS. 35 and 36. Several nonlimiting examples of the composition of the kit 832 are described here with respect to FIGS. 35 and 36. The kit 832 contains materials that may be used in the preparation and use of a fresh autograft tissue. The sterilized kit 832 includes sterilized receptacles 834 and 836 sterilized bottles 834 and 836, a sterilized graduated cylinder 838 or other marked measurement device, sterilized bottles 840a-b, wherein the bottles 840a-b may include a pre-mixed solution of glycerol in Lactated Ringer’s, and a sterile mesh 442 (e.g., a sieve). In certain embodiments the sterile mesh 442 may be provided in the form of a pouch or bag as described above that includes perforations of drain features 710 and to which material or tissue to be rinsed, stored,or transported using interoperative solution may be added. In one embodiment, sterilized receptacles 834 and 836 may be marked with “fill here” lines to facilitate mixing of the interoperative solution at various pre-defined levels of the constituents (e.g., glycerol and Lactated Ringers). In another embodiment, the receptacles may be unmarked. Further, bottles 840a-b may contain a pre-mixed interoperative solution including glycerol in Lactated Ringer’s solutions. For example, bottles 840a-b may include an interoperative solution containing a glycerol fraction the glycerol content (e.g., fraction) of the interoperative solution is equal to or less than 50% by volume, equal to or less than 45% by volume, equal to or less than 40% by volume, equal to or less than 35% by volume, equal to or less than 30% by volume, equal to or less than 25% by volume, equal to or less than 20% by volume, equal to or less than 15% by volume, equal to or less than 10% by volume, and so forth. Bottles 840a-b may also include an interoperative solution containing a Lactated Ringer’s fraction equal to or more than 50% by volume, equal to more than 55% by volume, equal to or more than 60% by volume, equal to or more than 65% by volume, equal to or more than 70% by volume, equal to more than 75% by volume, equal to more than 80% by volume, equal to or more than 85% by volume, equal to or more than 90% by volume, and so forth. For example, bottle 440 may contain pre-mixed interoperative solutions including 10-25% glycerol fraction by volume and 75-90% mixing solution fraction. In another example, bottles 840a-b may contain pre-mixed interoperative solutions including 20% glycerol by volume and 80% mixing solution by volume, 15% glycerol by volume and 85% mixing solution by volume, or 10% glycerol by volume and 90% mixing solution by volume. In other embodiments, the interoperative solution may contain a combination of glycerol, Lactated Ringer’s, and an antimicrobial agent (e.g., antibiotics, antiviral agents, or antifungal). It should be noted that the mixing solution described herein may contain Lactated Ringer’s and an antimicrobial agent.

[0162] Pre-mixed interoperative solutions can be measured in the graduated cylinder 838 prior to pouring into receptacle 834 or added directly to receptacle 834. Once the interoperative solution is added to the receptacle 834, the fresh autograft tissue can be placed into the receptacle 834. Receptacle 834 containing the interoperative solution and fresh autograft tissue can subsequently be placed in the larger receptacle 836, which may contain a cooling medium to chill the fresh autograft tissue, as described in FIG. 30. Prior to implantation, a sterile mesh 442 may be used to help decant or pour out excess interoperative solution and retain the tissue. The sterile mesh 442 may be fitted to the dimensions of the receptacle 834, or alternatively, the sterile mesh 442 can be held while pouring out the excess interoperative solution. In practice, the sterile mesh may be provided as a rigid structure or device, such as a screen, sieve, orcolander. Alternatively, the sterile mesh may be provided as a flexible or other non-rigid structure, such as a net or “cheese cloth” type fabric or structure. In a further embodiment, antimicrobial agent (e.g., antibiotics, antiviral agents, or antifungal) may be added to the interoperative solution. In general, it should be noted that the specific examples relating to the materials described in the kit 832 or interoperative solution compositions described herein are meant to be non-limiting and can be utilized for other grafts including allogeneic grafts or xenografts.

[0163] In another embodiment, a person preparing the fresh autograft tissue can prepare an interoperative solution containing glycerol in Lactated Ringer’s with respect to a specific procedure. A graduated cylinder 838 can be utilized to measure and pour a specified volume of glycerol using bottle 840a and Lactated Ringer’s using bottle 840b, respectively. For example, an interoperative solution containing 20% by volume of glycerol fraction and 80% by volume of Lactated Ringer’s fraction can be prepared by measuring 2 mL of glycerol in bottle 840a into the graduated cylinder 838, followed by pouring it into the receptacle 834, measuring 8 mL of Lactated Ringer’s in bottle 840b, and pouring the mixing solution into the receptacle 834. In another embodiment, the receptacle 834 may be pre-marked with lines designating the volumes of glycerol and Lactated Ringer’s, respectively.

[0164] For example, receptacle 834 may be pre-marked with lines for preparing an interoperative solution containing a glycerol fraction equal to or less than 50% by volume, equal to or less than 45% by volume, equal to or less than 40% by volume, equal to or less than 35% by volume, equal to or less than 30% by volume, equal to or less than 25% by volume, equal to or less than 20% by volume, equal to or less than 15% by volume, equal to or less than 10% by volume, and so forth. In a further example, receptacle 834 may be pre-marked with lines for preparing an interoperative solution containing a Lactated Ringer’s fraction equal to or more than 50% by volume, equal to more than 55% by volume, equal to or more than 60% by volume, equal to or more than 65% by volume, equal to or more than 70% by volume, equal to more than 75% by volume, equal to more than 80% by volume, equal to or more than 85% by volume, equal to or more than 90% by volume, and so forth. Accordingly, interoperative solutions with varying amounts of glycerol and Lactated Ringer’s can be prepared (e.g., 10-25% glycerol fraction by volume and 75-90% mixing solution by volume, 15% glycerol by volume and 85% mixing solution by volume, or 10% glycerol by volume and 90% mixing solution by volume. In a further embodiment, an antimicrobial agent (e.g., antibiotics, antiviral agents, or antifungal) may be added to the interoperative solution. Once the interoperative solution is added to the receptacle 440, the fresh autograft tissue can be placed into the receptacle 834.Receptacle 834 containing the interoperative solution and fresh autograft tissue can subsequently be placed in the larger receptacle 836, to which a cooling medium is added to chill the fresh autograft tissue, as described in FIG. 35. Prior to implantation, a sterile mesh 442 may be used to help decant or pour out excess interoperative solution and retain the issue. The sterile mesh 442 may be fitted to the dimensions of the receptacle 834, or alternatively, the sterile mesh 442 can be held while pouring out the excess interoperative solution.

[0165] With the preceding in mind, FIG. 40 illustrates a method 850 for processing an autograft tissue for transplantation. An autograft tissue may be processed and temporarily stored in a non-biocompatible solution. It should be noted that the fresh autograft tissue or autograft tissue may include, but is not limited to, bone, tissue, or cartilage, etc. A nonbiocompatible solution, as described herein, is a solution that may reduce cell viability of an autograft tissue until the autograft tissue is no longer functional and / or which is not suitable for introduction into the human body, such as due to tissue toxicity. Thus, the non-biocompatible solution must be removed or rinsed off from the autograft tissue prior to transplantation into a patient. As described herein, at block 852, an autograft tissue is removed from a nonbiocompatible solution. Because the autograft tissue was stored in a non-biocompatible solution, the autograft tissue is rinsed (e.g., irrigated) with an interoperative solution, as described in block 854. In general, the interoperative solution is non-bio toxic and may be used at room temperature (about 20°C to about 25°C). The interoperative solution, as described herein, may contain a glycerol -based solution containing glycerol and a Lactated Ringer’s solution, wherein in certain embodiments the glycerol content (e.g., fraction) of the interoperative solution is equal to or less than 50% by volume, equal to or less than 45% by volume, equal to or less than 40% by volume, equal to or less than 35% by volume, equal to or less than 30% by volume, equal to or less than 25% by volume, equal to or less than 20% by volume, equal to or less than 15% by volume, equal to or less than 10% by volume, and so forth.. In certain embodiments, the Lactated Ringer’s fraction of the interoperative solution is equal to or more than 50% by volume, equal to more than 55% by volume, equal to or more than 60% by volume, equal to or more than 65% by volume, equal to or more than 70% by volume, equal to more than 75% by volume, equal to more than 80% by volume, equal to or more than 85% by volume, equal to or more than 90% by volume, and so forth. For example, the interoperative solution may contain 10-25% glycerol by volume and 75-90% by volume mixing solution, 20% glycerol by volume and 80% mixing solution by volume, 15% glycerol by volume and 85% mixing solution by volume, or 10% glycerol by volume and 90% mixing solution by volume. In other embodiments, the interoperative solution may contain acombination of glycerol, Lactated Ringer’s, and an antimicrobial agent (e.g., antibiotics, antiviral agents, or antifungal). For example, a medical grade intravenous (IV) polyvinyl chloride (PVC) type bag with a port may be used to rinse or irrigate the autograft tissue. In further embodiments, a containment system such as a packaging or pouch containing a CBM product or other bone product / tissues may have a perforated membrane integrated with the packing or pouch. As such, the perforations may allow for rinsing or irrigation. For example, the perforations or other drainage features will allow for the interoperative solution to flow out the pores during rinsing. In another embodiment, the autograft tissue may be placed in a receptacle containing the interoperative solution to rinse off the non-biocompatible solution. Once the autograft tissue is thoroughly rinsed with the interoperative solution, the autograft tissue may be implanted into a recipient during surgery or other clinical procedure, as described in block 856. In other embodiments, the autograft tissue may be temporarily (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, and so forth) stored in a container with the interoperative solution during a surgical procedure until it is ready to be implanted into a recipient. In further embodiments, the autograft tissue may be further combined or mixed with other bone materials (e.g., bone growth composition, allograft materials, synthetic bone product, cellular bone matrix, bone growth protein, xenograft materials, or other autograft materials) prior to implantation into a recipient during surgery.

[0166] By way of example, FIG. 41 illustrates a method 860 for storing, transporting, and implanting an autograft tissue. An autograft tissue or sample may be retrieved for a surgical procedure, as described in block 862. For example, the autograft tissue may be rinsed (e.g., cleaned or irrigated) with the interoperative solution and subsequently placed in a container containing a transport solution, as described at block 864. The container holding the tissue may be a resealable pouch, a cooler, or a combination thereof, to transport the tissue accordingly. As described herein, the interoperative solution may be used as a transport solution to transport autograft tissues or alternative tissues (e.g., allograft materials, xenograft materials, bone growth compositions, or combination of materials), or the interoperative solution may be used as a cryosolution to store or hydrate autograft tissues or alternative tissues and placed in storage for use at a later time.

[0167] The transport solution or cryosolution, as described herein, may contain a glycerolbased solution containing glycerol and a Lactated Ringer’s solution, wherein in certain embodiments the glycerol content (e.g., fraction) of the transport solution or cryosolution is equal to or less than 50% by volume, equal to or less than 45% by volume, equal to or less than 40% by volume, equal to or less than 35% by volume, equal to or less than 30% by volume,equal to or less than 25% by volume, equal to or less than 20% by volume, equal to or less than 15% by volume, equal to or less than 10% by volume, and so forth. In certain embodiments, the Lactated Ringer’s fraction of the transport solution or cryosolution is equal to or more than 50% by volume, equal to more than 55% by volume, equal to or more than 60% by volume, equal to or more than 65% by volume, equal to or more than 70% by volume, equal to more than 75% by volume, equal to more than 80% by volume, equal to or more than 85% by volume, equal to or more than 90% by volume, and so forth. For example, the transport solution or cryosolution may contain 10-25% glycerol by volume and 75-90% by volume mixing solution, 20% glycerol by volume and 80% mixing solution by volume, 15% glycerol by volume and 85% mixing solution by volume, or 10% glycerol by volume and 90% mixing solution by volume. In other embodiments, the transport solution or cryosolution may contain a combination of glycerol, Lactated Ringer’s, and an antimicrobial agent (e.g., antibiotics, antiviral agents, or antifungal). It should be noted that the transport solution or cryosolution can maintain cell viability of tissues. After the autograft tissue is placed in a container with the transport solution, the autograft tissue can be transported to its intended location, as described in block 866. For example, the autograft tissue may be stored in the container with the transport solution for up to approximately 24 hours to approximately 48 hours until it reaches the intended location for transplantation. In certain embodiments, it may be stored less than 24 hours (e.g., 18 hours, 12 hours, 4 hours). In other embodiments, the autograft tissue or other tissues in the cryosolution in the container may be placed in long-term storage for use at a later time. For example, the autograft tissue or other tissues may be stored up to one month, two months, three months, four months, five months, or up to six months in storage between approximately -60°C and approximately -80°C. In other embodiments, the autograft tissue or other tissues may be stored in long-term storage at room temperature (e.g., 25°C) or alternatively, 0°C for up to one month, two months, three months, four months, five months, or up to six months in storage. Once the autograft tissue is transported to its intended location, the autograft tissue can be removed from the container, as described in block 868. At block 870, the autograft tissue is implanted into the recipient directly from the container. In other embodiments, the autograft tissue may be rinsed with an interoperative solution prior to implantation into the intended recipient. In further embodiments, the autograft tissue may be combined or mixed with other materials (e.g., bone growth composition, allograft materials, xenograft materials, or other autograft materials) prior to implantation during surgery. In even further embodiments, the autograft tissue may be stored in the interoperative solution duringthe duration of a surgery prior to implantation into a recipient.

[0168] By way of example, FIG. 42 shows an example of a medical grade intravenous (IV) polyvinyl chloride (PVC) type bag with a port that can contain the interoperative solution, transport solution, or cryosolution. The port may allow for fluid to be poured out of the bag. Further, the port may be adapted such that it can connect to tubes. It should be noted that alternative materials or containers may be used to contain or hold the interoperative solution, transport solution, or cryosolution.

[0169] The disclosed techniques provide improvements in maintaining the cells, tissues, or other biologically active components of a bone growth composition from harvesting through processing and until the composition is readied for transplantation into a recipient. During the interval between harvest and implantation, it is also desirable to monitor and control the environmental conditions, and storage parameters to maintain the integrity, viability, and biochemical activity of the harvested and processed biological material from which the bone growth composition is formed.

[0170] Thus, the disclosed bone growth compositions have improved viability and / or biologic activity. While it is contemplated that the parameters of storage temperature, atmospheric pressure, ambient environmental conditions, and such like will provided to users of the bone growth composition, the bone growth compositions have a wider tolerance of storage and handling conditions to reduce the effects of end user variability and preferences on the therapeutic effectiveness of the disclosed bone growth compositions. For example, not all end users have access to stringent storage conditions, such as a -70° C freezer. However, the disclosed bone growth compositions maintain suitable activity in a conventional -20° C freezer. The bone growth composition has been demonstrated to be viable (e.g., greater than 80% viability) measured after 5 days at -20°C. In particular, viability was observed to be 100% after 5 days at -20°C. and 96% after 15 days at -20°C. In addition, viability was observed to be 95% after 4 weeks at -40°C and 71% after 8 weeks at -40°C. Further, because freezers may be subject to variability in operating conditions, the disclosed bone growth compositions are able to maintain sufficient viability through fluctuations in freezer operating conditions, such that intermittent changes of temperature within a tolerance band (e.g., -15° C to -25° C) will not significantly alter the therapeutic effectiveness of the disclosed bone growth compositions.

[0171] The disclosed techniques are directed to bone growth compositions and methods. In certain embodiments, the bone growth compositions and methods may be cryogenically preserved. In an embodiment, cryogenic preservation, e.g., freezing, refers to exposing a population of cells, tissues, or organs to a cryogenic environment. A cryogenic environmentmay refer to temperatures below 0°C, below -20°C, below -70°C. In an embodiment, a cryogenic environment may be between about 0°C to about -200°C.

[0172] A cryoprotectant or cryopreservative may refer to agents or materials that prevents or reduces undesirable damage to a biological material caused by lowering the temperature of a biological material or any substance or material which enhances, strengthens or otherwise increases the ability of the biological material to withstand lowered temperatures. As disclosed herein, the bone growth compositions and associated cryopreservation methods may include the addition of one or more cryoprotectants or cryopreservative compounds to permit freezing of the sample, and / or maintenance of the sample at temperatures generally below 0° C. Exemplary cryoprotectants and / or cryopreservative compounds, as used in the context of the disclosed techniques may include, but are not limited to, ice-suppressing cryoprotectants (e.g., non-colligative agents such as Supercool X-1000™ and Supercool Z-1000™, 21stCentury Medicine, Rancho Cucamonga, Calif.) glycerol, dimethylsulfoxide (DMSO), ethylene glycol, propylene glycol, polyethylene oxide (PEO), acetamide, ethanol, methanol, butanediol, carbohydrates (including sugars such as glucose, fructose, dextrans, sucrose, lactose, and trehalose), polyvinyl alcohols, hydroxyethyl starch, serum albumin. With CBMs, cry opreservation ensures the osteogenic potential of allogeneic cells providing benefits for the bone grafting site.

[0173] In certain embodiments, steps that involve freezing and / or thawing of a tissue sample or cell population may be achieved by, e.g., bringing the temperature of a refrigerated tissue or cell sample down to a suitable sub-zero temperature, or alternatively, bringing the temperature of a sub-zero stored sample up to refrigerated (and, optionally, to either room or recipient body temperature immediately prior to implantation). Freezing may include direct freezing a room temperature composition. Such steps in the disclosed methods may employ submersion vessels or frozen storage means to prepare the frozen tissue or cell sample, while conventional means such as a heated water bath or such like device, submerging the packaged frozen sample into a sample of growth medium, biological buffer, or tissue / organ storage solution (e.g., pre-warmed to the desired temperature), may be employed to bring the temperature of a frozen tissue sample to the desired temperature required for transplanting the bone growth compositions into the body of a suitable recipient.

[0174] It should be understood that exposure to a cryogenic environment may result in the freezing of liquid portions of a bone growth composition. However, the resultant bone growth composition may include composite structures that do not change in state between freezing and thawing and / or that change state at different temperatures relative to water in the sample. Thus,in an embodiment, a bone growth composition as provided herein may be referred to as frozen or may freeze at temperatures that cause liquid portions of the composition to transition to a solid state.

[0175] As provided herein, antimicrobial includes, for example, antibiotics, antifungal, antiviral agents or the like. Antimicrobial agents to treat infection include by way of example and not limitation, antiseptic agents, antibacterial agents; polyene antifungals (e.g., amphotericin B), quinolones and in particular fluoroquinolones (e.g., norfloxacin, ciprofloxacin, lomefloxacin, ofloxacin, etc.), aminoglycosides (e.g., gentamicin, tobramycin, etc.), glycopeptides (e.g., vancomycin, etc.), lincosamides (e.g., clindamycin), cephalosporins (e.g., first, second, third generation) and related beta-lactams, macrolides (e.g., azithromycin, erythromycin, etc.), nitroimidazoles (e.g., metronidazole), penicillins, polymyxins, tetracyclines, or combinations thereof. Some exemplary antimicrobial agents include, by way of illustration and not limitation, acedapsone; acetosulfone sodium; alamecin; alexidine; amdinocillin; amdinocillin pivoxil; amicycline; amifloxacin; amifloxacin mesylate; amikacin; amikacin sulfate; aminosalicylic acid; aminosalicylate sodium; amoxicillin; amphomycin; ampicillin; ampicillin sodium; apalcillin sodium; apramycin; aspartocin; astromicin sulfate; avilamycin; avoparcin; azithromycin; azlocillin; azlocillin sodium; bacampicillin hydrochloride; bacitracin; bacitracin methylene disalicylate; bacitracin zinc; bambermycins; benzoylpas calcium; berythromycin; betamicin sulfate; biapenem; biniramycin; biphenamine hydrochloride; bispyrithione magsulfex; butikacin; butirosin sulfate; capreomycin sulfate; carbadox; carbenicillin disodium; carbenicillin indanyl sodium; carbenicillin phenyl sodium; carbenicillin potassium; carumonam sodium; cefaclor; cefadroxil; cefamandole; cefamandole nafate; cefamandole sodium; cefaparole; cefatrizine; cefazaflur sodium; cefazolin; cefazolin sodium; cefbuperazone; cefdinir; cefepime; cefepime hydrochloride; cefetecol; cefixime; cefmenoxime hydrochloride; cefmetazole; cefmetazole sodium; cefonicid monosodium; cefonicid sodium; cefoperazone sodium; ceforanide; cefotaxime sodium; cefotetan; cefotetan disodium; cefotiam hydrochloride; cefoxitin; cefoxitin sodium; cefpimizole; cefpimizole sodium; cefpiramide; cefpiramide sodium; cefpirome sulfate; cefpodoxime proxetil; cefprozil; cefroxadine; cefsulodin sodium; ceftazidime; ceftibuten; ceftizoxime sodium; ceftriaxone sodium; cefuroxime; cefuroxime axetil; cefuroxime pivoxetil; cefuroxime sodium; cephacetrile sodium; cephalexin; cephalexin hydrochloride; cephaloglycin; cephaloridine; cephalothin sodium; cephapirin sodium; cephradine; cetocycline hydrochloride; cetophenicol; chloramphenicol; chloramphenicol palmitate; chloramphenicol pantothenate complex; chloramphenicol sodium succinate; chlorhexidine phosphanilate; chloroxylenol;chlortetracycline bisulfate; chlortetracycline hydrochloride; cinoxacin; ciprofloxacin; ciprofloxacin hydrochloride; cirolemycin; clarithromycin; clinafloxacin hydrochloride; clindamycin; clindamycin hydrochloride; clindamycin palmitate hydrochloride; clindamycin phosphate; clofazimine; cioxacillin benzathine; cioxacillin sodium; chlorhexidine, cloxyquin; colistimethate sodium; colistin sulfate; coumermycin; coumermycin sodium; cycloserine; dalfopristin; dapsone; daptomycin; demeclocycline; demeclocycline hydrochloride; demecycline; denofungin; diaveridine; dicloxacillin; dicloxacillin sodium; dihydrostreptomycin sulfate; dipyrithione; dirithromycin; doxycycline; doxycycline calcium; doxycycline fosfatex; doxycycline hyclate; droxacin sodium; enoxacin; epicillin; epitetracycline hydrochloride; erythromycin; erythromycin acistrate; erythromycin estolate; erythromycin ethyl succinate; erythromycin gluceptate; erythromycin lactobionate; erythromycin propionate; erythromycin stearate; ethambutol hydrochloride; ethionamide; fleroxacin; floxacillin; fludalanine; flumequine; tromethamine; fumoxicillin; furazolium chloride; furazolium tartrate; fusidate sodium; fusidic acid; ganciclovir and ganciclovir sodium; gentamicin sulfate; gloximonam; gramicidin; haloprogin; hetacillin; hetacillin potassium; hexedine; ibafloxacin; imipenem; isoconazole; isepamicin; isoniazid; josamycin; kanamycin sulfate; kitasamycin; levofuraltadone; levopropylcillin potassium; lexithromycin; lincomycin; lincomycin hydrochloride; lomefloxacin; lomefloxacin hydrochloride; lomefloxacin mesylate; loracarbef; mafenide; meclocycline; meclocycline sulfosalicylate; megalomicin potassium phosphate; mequidox; meropenem; methacycline; methacycline hydrochloride; methenamine; methenamine sodium; methenamine mandelate; methicillin sodium; metioprim; metronidazole hydrochloride; metronidazole phosphate; mezlocillin; mezlocillin sodium; minocycline; minocycline hydrochloride; mirincamycin hydrochloride; monensin; monensin sodium; nafcillin sodium; nalidixate sodium; nalidixic acid; natainycin; nebramycin; neomycin palmitate; neomycin sulfate; neomycin undecylenate; netilmicin sulfate; neutramycin; nifuiradene; nifuraldezone; nifuratel; nifuratrone; nifurdazil; nifurimide; nifiupirinol; nifurquinazol; nifurthiazole; nitrocycline; nitrofurantoin; nitromide; norfloxacin; novobiocin sodium; ofloxacin; onnetoprim; oxacillin and oxacillin sodium; oximonam; oximonam sodium; oxolinic acid; oxytetracycline; oxytetracycline calcium; oxytetracycline hydrochloride; paldimycin; parachlorophenol; paulomycin; pefloxacin; pefloxacin mesylate; penamecillin; penicillins such as penicillin g benzathine, penicillin g potassium, penicillin g procaine, penicillin g sodium, penicillin v, penicillin v benzathine, penicillin v hydrabamine, and penicillin v potassium; pentizidone sodium; phenyl aminosalicylate; piperacillin sodium; pirbenicillin sodium; piridicillin sodium; pirlimycin hydrochloride; pivampicillinhydrochloride; pivampicillin pamoate; pivampicillin probenate; polymyxin b sulfate; porfiromycin; propikacin; pyrazinamide; pyrithione zinc; quindecamine acetate; quinupristin; racephenicol; ramoplanin; ranimycin; relomycin; repromicin; rifabutin; rifametane; rifamexil; rifamide; rifampin; rifapentine; rifaximin; rolitetracycline; rolitetracy cline nitrate; rosaramicin; rosaramicin butyrate; rosaramicin propionate; rosaramicin sodium phosphate; rosaramicin stearate; rosoxacin; roxarsone; roxithromycin; sancycline; sanfetrinem sodium; sarmoxicillin; sarpicillin; scopafungin; sisomicin; sisomicin sulfate; sparfloxacin; spectinomycin hydrochloride; spiramycin; stallimycin hydrochloride; steffimycin; streptomycin sulfate; streptonicozid; sulfabenz; sulfabenzamide; sulfacetamide; sulfacetamide sodium; sulfacytine; sulfadiazine; sulfadiazine sodium; sulfadoxine; sulfalene; sulfamerazine; sulfameter; sulfamethazine; sulfamethizole; sulfamethoxazole; sulfamonomethoxine; sulfamoxole; sulfanilate zinc; sulfanitran; sulfasalazine; sulfasomizole; sulfathiazole; sulfazamet; sulfisoxazole; sulfisoxazole acetyl; sulfisboxazole diolamine; sulfomyxin; sulopenem; sultamricillin; suncillin sodium; talampicillin hydrochloride; teicoplanin; temafloxacin hydrochloride; temocillin; tetracycline; tetracycline hydrochloride; tetracycline phosphate complex; tetroxoprim; thi amphenicol; thiphencillin potassium; ticarcillin cresyl sodium; ticarcillin disodium; ticarcillin monosodium; ticlatone; tiodonium chloride; tobramycin; tobramycin sulfate; tosufloxacin; trimethoprim; trimethoprim sulfate; trisulfapyrimidines; troleandomycin; trospectomycin sulfate; tyrothricin; vancomycin; vancomycin hydrochloride; virginiamycin; zorbamycin; or combinations thereof.

[0176] Certain embodiments of the disclosure are discussed in the context of cellular bone matrices or bone matrices. However, it should be understood that one or more compositions or processing steps may be used for other bone growth compositions. For example, the disclosed process steps may be used alone or in combination with other type of bone growth processing that includes one or both of chips or demineralized fibers.

[0177] In other embodiments, interoperative solution, transport solution, or cryosolution may be utilized “intraoperatively” by which it is meant that such solutions may incidentally come into contact with the patient during a procedure. For example, as used herein such solutions are non-toxic and bio-safe and may, therefore, be present as a residue on tissues or materials held in such solution over the course of a procedure and then introduced into the patient at some point in the procedure. In such a context, though the primary characterization of the solution may be considered interoperative, the residual contact with the patient during the procedure may itself be considered an intraoperative contact. That is, residual amounts ofinteroperative solution, transport solution, or cryosolution on a material to be transplanted into a patient may be considered intraoperative once contact is made with the patient.

[0178] The disclosed bone growth compositions may include a bioactive or pharmaceutical agent, which may be disposed in, packaged with, coated on or combined with the bone growth composition. The term “bioactive agent” as used herein is generally meant to refer to any substance that alters the physiology of a patient. The term “bioactive agent” may be used interchangeably herein with the terms “therapeutic agent,” “therapeutically effective amount,” and “active pharmaceutical ingredient”, “API” or “drug”.

[0179] Bioactive agent or bioactive compound is used herein to refer to a compound or entity that alters, inhibits, activates, or otherwise affects biological or chemical events. For example, bioactive agents may include, but are not limited to, osteogenic or chondrogenic proteins or peptides, anti-AIDS substances, anti-cancer substances, antibiotics, immunosuppressants, anti-viral substances, enzyme inhibitors, hormones, neurotoxins, opioids, hypnotics, anti-histamines, lubricants, tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinson substances, anti-spasmodics and muscle contractants including channel blockers, miotics and anti-cholinergics, anti-glaucoma compounds, anti-parasite and / or anti -protozoal compounds, modulators of cell-extracellular matrix interactions including cell growth inhibitors and antiadhesion molecules, vasodilating agents, inhibitors of DNA, RNA or protein synthesis, anti-hypertensives, analgesics, anti-pyretics, steroidal and non-steroidal anti-inflammatory agents, anti-angiogenic factors, angiogenic factors, anti- secretory factors, anticoagulants and / or antithrombotic agents, local anesthetics, prostaglandins, anti-depressants, anti-emetics, and imaging agents. In certain embodiments, the bioactive agent is a drug. Bioactive agents further include RNAs, such as siRNA, and osteoclast stimulating factors. In some embodiments, the bioactive agent may be a factor that stops, removes, or reduces the activity of bone growth inhibitors. In some embodiments, the bioactive agent is a growth factor, cytokine, extracellular matrix molecule or a fragment or derivative thereof, for example, a cell attachment sequence such as RGD.

[0180] In some embodiments, the pharmaceutical agent may include one or a plurality of therapeutic agents and / or pharmacological agents for release, including sustained release, to treat, for example, pain, inflammation, degeneration, or infection. The agent may be an analgesic agent including but are not limited to acetaminophen, a local anesthetic, such as for example, lidocaine, bupivicaine, ropivacaine, opioid analgesics such as buprenorphine, butorphanol, dextromoramide, dezocine, dextropropoxyphene, diamorphine, fentanyl, alfentanil, sufentanil, hydrocodone, hydromorphone, ketobemidone, levomethadyl,levorphanol, mepiridine, methadone, morphine, nalbuphine, opium, oxycodone, papaveretum, pentazocine, pethidine, phenoperidine, piritramide, dextropropoxyphene, remifentanil, sufentanil, tilidine, tramadol, codeine, dihydrocodeine, meptazinol, dezocine, eptazocine, flupirtine or a combination thereof.

[0181] The agent may be an anti-inflammatory agent including, but are not limited to, a statin, sulindac, sulfasalazine, naroxyn, diclofenac, indomethacin, ibuprofen, flurbiprofen, ketoprofen, aclofenac, aloxiprin, aproxen, aspirin, diflunisal, fenoprofen, mefenamic acid, naproxen, phenylbutazone, piroxicam, meloxicam, salicylamide, salicylic acid, desoxysulindac, tenoxicam, ketoralac, flufenisal, salsalate, triethanolamine salicylate, aminopyrine, antipyrine, oxyphenbutazone, apazone, cintazone, flufenamic acid, clonixeril, clonixin, meclofenamic acid, flunixin, colchicine, demecolcine, allopurinol, oxypurinol, benzydamine hydrochloride, dimefadane, indoxole, intrazole, mimbane hydrochloride, paranylene hydrochloride, tetrydamine, benzindopyrine hydrochloride, fluprofen, ibufenac, naproxol, fenbufen, cinchophen, diflumidone sodium, fenamole, flutiazin, metazamide, letimide hydrochloride, nexeridine hydrochloride, octazamide, molinazole, neocinchophen, nimazole, proxazole citrate, tesicam, tesimide, tolmetin, triflumidate, fenamates (mefenamic acid, meclofenamic acid), nabumetone, celecoxib, etodolac, nimesulide, apazone, gold, tepoxalin; dithiocarbamate, or a combination thereof. Anti-inflammatory agents also include other compounds such as steroids, such as for example, fluocinolone, cortisol, cortisone, hydrocortisone, fludrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, dexamethasone, beclomethasone, fluticasone interleukin- 1 receptor antagonists, thalidomide (a TNF-a release inhibitor), thalidomide analogues (which reduce TNF-a production by macrophages), bone morphogenetic protein (BMP) type 2 or BMP-4 (inhibitors of caspase 8, a TNF-a activator), quinapril (an inhibitor of angiotensin II, which upregulates TNF-a), interferons such as IL- 11 (which modulate TNF-a receptor expression), and aurin-tricarboxylic acid (which inhibits TNF-a), guanidinoethyldisulfide, or a combination thereof. Exemplary anti-inflammatory agents include, for example, naproxen; diclofenac; celecoxib; sulindac; diflunisal; piroxicam; indomethacin; etodolac; meloxicam; ibuprofen; ketoprofen; r-flurbiprofen; mefenamic; nabumetone; tolmetin, and sodium salts of each of the foregoing; ketorolac bromethamine; ketorolac tromethamine; ketorolac acid; choline magnesium trisalicylate; rofecoxib; valdecoxib; lumiracoxib; etoricoxib; aspirin; salicylic acid and its sodium salt; salicylate esters of alpha, beta, gamma-tocopherols and tocotrienols (and all their d, 1, and racemic isomers); methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, t-butyl,esters of acetylsalicylic acid; tenoxicam; aceclofenac; nimesulide; nepafenac; amfenac; bromfenac; flufenamate; phenylbutazone, or a combination thereof.

[0182] An anti-inflammatory agent can be a steroid. Exemplary steroids include, for example, 21 -acetoxy pregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, dexamethasone 21 -acetate, dexamethasone 21 -phosphate di -Na salt, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluoromethoIone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinonide, halobetasol propionate, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, predni carb ate, prednisolone, prednisolone 25-diethylamino-acetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide or a combination thereof.

[0183] Anti-inflammatory agents also include those with anti-inflammatory properties, such as, for example, amitriptyline, carbamazepine, gabapentin, pregabalin, clonidine, or a combination thereof. For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities of ingredients, percentages or proportions of materials, reaction conditions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” For example, “about” may refer to ±0.1%, ±0.25%, ±0.5%, ±0.75%, ±1%, ±1.5%, ±2, ±5%, ±10%, or ±15%.

[0184] Examples:

[0185] Example 1 : An interoperative solution to prepare an autograft bone material includes a glycerol fraction, wherein the glycerol fraction is less than or equal to 25% byvolume and a Lactated Ringer’s fraction, wherein the Lactated Ringer’s fraction is more than or equal to 75% by volume.

[0186] Example 2: The interoperative solution of the preceding example, wherein the interoperative solution is used as a cryosolution.

[0187] Example 3: The interoperative solution of any preceding example, wherein the interoperative solution is used as a rinse or transport solution.

[0188] Example 4: The interoperative solution of any preceding example, wherein the interoperative solution includes an antimicrobial agent.

[0189] Example 5: The interoperative solution of any preceding example, wherein the antimicrobial agent includes antibiotics or antiviral agents.

[0190] Example 6: The interoperative solution of any preceding example, wherein the interoperative solution preserves viable bone at an ambient room temperature.

[0191] Example 7: The interoperative solution of any preceding example, wherein the interoperative solution is packaged in a medical grade PVC IV bag having a port for dispensing.

[0192] Example 8: The interoperative solution of any preceding example, further including an autograft bone material stored in the interoperative solution.

[0193] Example 9: The interoperative solution of any preceding example, further including a second bone material to create a mixture, wherein the second bone material includes an allograft, synthetic bone product, cellular bone matrix, and bone growth protein, or a combination of such materials.

[0194] Example 10: The interoperative solution of any preceding example, further including a containment system having a perforated wall to rinse the composition prior to interoperative use.

[0195] Example 11 : The interoperative solution of any preceding example, wherein a sterile mesh is used to decant excess interoperative solution and retain the mixture.

[0196] Example 12: A method of rinsing and storing an autograft tissue includes retrieving the autograft tissue. The method includes rinsing the autograft tissue with an interoperative solution to remove residue of a non-biocompatible solution used to store or transport the autograft tissue, wherein the interoperative solution includes a glycerol fraction, wherein the glycerol fraction is less than or equal to 25% by volume and a Lactated Ringer’s fraction,wherein the Lactated Ringer’s fraction is more than or equal to 75% by volume. The method further includes storing the autograft tissue in the interoperative solution.

[0197] Example 13 : The method of example 12, wherein the interoperative solution is used as cryosolution.

[0198] Example 14: The method of examples 12 or 13, wherein the autograft tissue is stored within the interoperative solution for all or part of a duration of a surgical procedure.

[0199] Example 15: The method of examples 12, 13, or 14, wherein the autograft tissue is stored in the interoperative solution for up to six months.

[0200] Example 16: The method of examples 12, 13, 14, or 15 further including transporting the stored autograft tissue to a different operation or clinical site where the autograft tissue will be implanted.

[0201] Example 17: The method of examples 12, 13, 14, 15, or 16, wherein the autograft tissue is combined with a bone growth composition, allograft materials, autograft materials, or a combination of such materials.

[0202] Example 18: A composition includes an autograft tissue and an interoperative solution, the interoperative solution including a glycerol fraction and a Lactated Ringer’s fraction.

[0203] Example 19: The composition of example 18, wherein the glycerol fraction is less than or equal to 25% by volume.

[0204] Example 20: The composition of examples 18 or 19, wherein the autograft tissue is combined with a bone growth composition, allograft materials, autograft materials, or a combination of such materials.

[0205] While the disclosure may be susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and have been described in detail herein. However, it should be understood that the embodiments provided herein are not intended to be limited to the particular forms disclosed. Rather, the various embodiments may cover all modifications, equivalents, and alternatives falling within the spirit and scope of the disclosure as defined by the following appended claims. Further, it should be understood that certain elements of the disclosed embodiments may be combined or exchanged with one another.

Claims

CLAIMSWhat is claimed is:

1. An interoperative solution to prepare an autograft bone material, comprising: a glycerol fraction, wherein the glycerol fraction is less than or equal to 25% by volume; and a Lactated Ringer’s fraction, wherein the Lactated Ringer’s fraction is more than or equal to 75% by volume.

2. The interoperative solution of claim 1, wherein the interoperative solution is used as a cryosolution.

3. The interoperative solution of claim 1 or claim 2, wherein the interoperative solution is used as a rinse or transport solution.

4. The interoperative solution of any one of claims 1-3, wherein the interoperative solution comprises an antimicrobial agent, wherein the antimicrobial agent comprises antibiotics or antiviral agents.

5. The interoperative solution of any one of claims 1-4, wherein the interoperative solution preserves viable bone at an ambient room temperature.

6. The interoperative solution of any one of claims 1-5, wherein the interoperative solution is packaged in a medical grade PVC IV bag having a port for dispensing.

7. The interoperative solution of any one of claims 1-6, further comprising an autograft bone material stored in the interoperative solution.

8. The interoperative solution of any one of claims 1-7, further comprising: a second bone material to create a mixture, wherein the second bone material comprises an allograft, synthetic bone product, cellular bone matrix, and bone growth protein, or a combination of such materials; and wherein a sterile mesh is used to decant excess interoperative solution and retain the mixture.

9. The interoperative solution of any one of claims 1-8, further comprising a containment system having a perforated wall to rinse the composition prior to interoperative use.

10. A method of rinsing and storing an autograft tissue, comprising: retrieving the autograft tissue; rinsing the autograft tissue with an interoperative solution to remove residue of a nonbiocompatible solution used to store or transport the autograft tissue, wherein the interoperative solution comprises: a glycerol fraction, wherein the glycerol fraction is less than or equal to 25% by volume; and a Lactated Ringer’s fraction, wherein the Lactated Ringer’s fraction is more than or equal to 75% by volume; and storing the autograft tissue in the interoperative solution.

11. The method of claim 10, wherein the interoperative solution is used as a cryosolution.

12. The method of claim 10 or claim 11, wherein the autograft tissue is stored within the interoperative solution for all or part of a duration of a surgical procedure.

13. The method of any one of claims 10-12, wherein the autograft tissue is stored in the interoperative solution for up to six months.

14. The method of any one of claims 10-13, further comprising: transporting the stored autograft tissue to a different operation or clinical site where the autograft tissue will be implanted.

15. The method of any one of claims 10-14, wherein the autograft tissue is combined with a bone growth composition, allograft materials, autograft materials, or a combination of such materials.

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