Method for preparing a composition for supporting the reconstitution or regeneration of bone substance

Abstract

Described is a method for preparing a composition for supporting the reconstitution or regeneration of bone substance in a patient in need thereof, wherein the method comprises the following steps: (i) decalcification / demineralization of decellularized bone material at a nearly physiological pH value; (ii) fragmentation of the decalcified / demineralized bone material of step (i); (iii) incubation of the fragmented bone material of step (ii) in an aqueous solution at a nearly physiological pH value; (iv) separation of the liquid supernatant from the fragmented bone material / debris; and (v) recovery of the liquid supernatant of step (iv), thereby obtaining a liquid composition for supporting reconstitution or regeneration of bone substance. Moreover, described is a composition for supporting reconstitution or regeneration of bone substance, obtainable by the method according to the present invention. Further, described is said composition for use in a method of supporting reconstitution or regeneration of bone substance in a patient.

Description

[0001] Method for preparing a composition for supporting the reconstitution or regeneration of bone substance

[0002] The present invention relates to a method for preparing a composition for supporting the reconstitution or regeneration of bone substance in a patient in need thereof, wherein the method comprises the following steps: (i) decalcification / demineralization of decellularized bone material at a nearly physiological pH value; (ii) fragmentation of the decalcified / demineralized bone material of step (i); (iii) incubation of the fragmented bone material of step (ii) in an aqueous solution at a nearly physiological pH value; (iv) separation of the liquid supernatant from the fragmented bone material / debris; and (v) recovery of the liquid supernatant of step (iv), thereby obtaining a liquid composition for supporting reconstitution or regeneration of bone substance. Moreover, the present invention relates to a composition for supporting reconstitution or regeneration of bone substance, obtainable by the method according to the present invention. Further, the present invention relates to said composition for use in a method of supporting reconstitution or regeneration of bone substance in a patient.

[0003] At present, it is standard to use human bone replacement material for the repair of defects of deceased and injured bone and has been subject to substantial research over decades. Moreover, human derived bone replacement material is used increasingly in many surgeries.

[0004] Using demineralized bone replacement material increased success rates in different surgical settings compared to using non demineralized bone replacement material.

[0005] The human bone extracellular matrix consists of proteins, lipids, sugar compounds and salts in macromolecular and molecular formations. The macromolecules / molecules are linked to each other by either peptide bonds or ester bonds. While breaking off peptide bonds requires specific enzymes, ester bond hydrolysis is regulated by enzymatic regulation of buffer systems regulating shifts of the pH of bone extracellular matrix.

[0006] Physiological buffer systems consist of a weak acid (HA) and the corresponding base A". In the blood serum, the main buffer system is the hydrogen carbon system, which keeps constantly a pH of 7.4. To lesser extent, protein buffer systems are involved in the regulation of blood pH, while the phosphate buffer system (H2PO4 / H-PO4) of the blood serum contributes about 2%. The pK of that system is around 6.8. The pK of H2PO4 is 7.2, the buffer capacity ranges from 6.2 - 8.2.

[0007] Bone extracellular matrix is the main storage place for the ubiquitously required phosphate. The exchange of serum phosphate and bone stored phosphate requires an equilibrium which is enzymatically regulated by shifts of the pH of the serum and the bone extracellular matrix as well.

[0008] The physiological range of pH required for the constant bone extracellular matrix / blood serum equilibrium is 6.8-7.4. The buffer capacity of H2PO4 ranges from 6.2 to 8.2. Any shift of the pH below 6.2 or beyond 8.2 exceeds the buffer capacity of phosphate.

[0009] It is standard that demineralization procedures currently used apply technically complex machines to expose the bone matrix to strong inorganic acids combined with alcoholic compounds. These strong acids are in use to solubilize hydroxyapatite, which is supposed to be the form of calcium-phosphatic deposition in bone. Therefore, efforts were undertaken to minimize hydroxyapatite deposits in the bone matrix to an extent favorable for wound healing. Yet, exposure of bone matrix to strong acids irreversibly drives ester bond hydrolysis, disabling molecular bonds essential for the functional integrity of the bone matrix.

[0010] Accordingly, there is a need to provide improved means and methods for preparing a composition for supporting the reconstitution or regeneration of bone substance.

[0011] The present invention provides a method for preparing a composition for supporting the reconstitution or regeneration of bone substance in a patient in need thereof, wherein the method comprises the following steps:

[0012] (i) decalcification / demineralization of decellularized bone material at a nearly physiological pH value;

[0013] (ii) fragmentation of the decalcified / demineralized bone material of step (i);

[0014] (iii) incubation of the fragmented bone material of step (ii) in an aqueous solution at a nearly physiological pH value;

[0015] (iv) separation of the liquid supernatant from the fragmented bone material / debris; and

[0016] (v) recovery of the liquid supernatant of step (iv), thereby obtaining a liquid composition for supporting reconstitution or regeneration of bone substance, preferably, wherein said liquid composition comprises electrolytes containing HPCh / PCh / Glucose and / or Ribose.

[0017] Surprisingly, the present invention demonstrates that the obtained liquid composition, i.e., a transudate (which is a liquid composition that is poor in proteinaceous compounds), polymerizes during condensation to an organelle-like structure and, accordingly, provides a suitable additive to, e.g., tissue transplants which unexpectedly achieves that the healing phase of tissue transplants is clinically and radiologically shortened to 9 to 17 days. Thus, the frequency of wound healing disorders can be significantly reduced, thereby beneficially providing a liquid composition for supporting reconstitution or regeneration of bone substance.

[0018] Indeed, as exemplified in more detail in the Example section below, patients were analysed in a follow up study who underwent bone augmentation of the upper and the lower jaw. This was done using x-ray diagnosis monthly over a period of 4 to 5 month postoperatively, thereby evaluating the bone density.

[0019] As a side remark, bone density is commonly evaluated with conventional x-ray techniques and digital volume tomography (DVT) in clinical settings to evaluate normal versus decreased bone mineral status (diagnosis of osteopetrosis, osteoporosis), diagnostic and follow up of bone fractures, follow up in kidney disease where bone density might be affected by secondary hyperparathyroidism, diagnosis and follow up of primary and secondary malignant bone lesions, diagnosis, planning and follow up of treatment regiments in head and neck / oral cavity and dental procedures. Thus, the determination of the bone density is the outstanding criterium for the evaluation of bone quality which translates into a brighter or darker appearance of bone structures on radiographs.

[0020] In the study as exemplified in the Example section below, autologous bone replacement material (also termed "bone material" in the present invention) used for the reconstruction of the jaws was demineralized with either HCI or EDTA.

[0021] 60 patients were included in this study that underwent augmentation of the jaws with allogens of demineralized bone replacement material. In these studies, these patients were followed up over a period of 4 months with monthly x-rays of the jaws .

[0022] Surprisingly, it was shown that the composition of the present invention obtainable by the above-claimed method of the present invention (i.e., the "supernatant" as it is termed in the appended Examples) is capable of rapidly and strongly reconstituting, regenerating and / or healing bone substance. The composition of the present invention obtained from bone material demineralized with EDTA turned out to have an even more pronounced effect in reconstituting, regenerating and / or healing bone substance compared to the composition of the present invention obtained from bone material (also termed bone replacement material above) demineralized with HCI.

[0023] This has been analysed by determining the bone density by radiography while "visibility" in the radiographs corresponds to a high bone density. Indeed, as shown in the appended Examples, the composition of the present invention (obtainable by the method of the present invention, when bone replacement material demineralized with HCI was used as a starting material for the claimed method), increased the bone density (at least to a certain extent). In these patients, a slight "visibility" was detectable on the 3-D x-rays, after 4 months.

[0024] In a different setting, the effect was more pronounced when the composition of the present invention (obtainable by the method of the present invention, when bone replacement material that has been decalcified with EDTA + / - was used as a starting material for the claimed method). In these patients, it has been shown that the visibility was high in all patients during the post operative radiological follow up. This indicates that in this setting, the bone density rapidly increased.

[0025] More specifically, as exemplified in more detail in the Example section below, it has been observed that demineralization of human bone matrix in non-toxic ethylene-diamine- tetraacetate (EDTA) solutions provided decalcified bone graft material for surgical use without side effects but with superior healing properties.

[0026] Moreover, as shown below, it has been observed that bone matrix decalcified in EDTA- solutions treated under cell culture like conditions, regarding pH and temperature, separates the matrix into a sediment fraction, a fraction solubilized in the medium, containing protein, sugar, and inorganic compounds, and a third fraction which appears at the surface of the medium over days, as did bone matrix decalcified with hydrochloric acid (HCI).

[0027] In studies of the interaction between the composition of the present invention and metals, functional properties of objects larger than 1 millimeter have been observed, which are reminiscent of the innate immune system. Metal objects smaller than 90 microns were absorbed by the objects, then led to vital staining in the case of Fe particles having a size of <90 microns, to organelle destruction in the case of CuCh, and to unresponsive incorporation in the case of zirconium oxide.

[0028] In studies on the interaction between bone fragments, analogous processes were seen, which led to the formation of mechanically resilient connections between the bone fragments in each examination.

[0029] In view of the prior art, taking into account these beneficial and surprising effects, the technical problem underlying the present invention is the provision of improved methods for preparing a composition for supporting the reconstitution or regeneration of bone substance and of the so obtained compositions.

[0030] The technical problem is solved by provision of the embodiments characterized in the claims.

[0031] The present invention relates to: a method for preparing a composition for supporting the reconstitution or regeneration of bone substance in a patient in need thereof, wherein the method comprises the following steps:

[0032] (i) decalcification / demineralization of decellularized bone material at a nearly physiological pH value;

[0033] (ii) fragmentation of the decalcified / demineralized bone material of step (i); (iii) incubation of the fragmented bone material of step (ii) in an aqueous solution at a nearly physiological pH value;

[0034] (iv) separation of the liquid supernatant from the fragmented bone material / debris; and

[0035] (v) recovery of the liquid supernatant of step (iv), thereby obtaining a liquid composition for supporting reconstitution or regeneration of bone substance, preferably, wherein said liquid composition comprises electrolytes containing HPCh / PCh / Glucose and / or Ribose.

[0036] The purpose of the method of the present invention is to prepare a composition for supporting the reconstitution or regeneration of bone substance in a patient in need thereof.

[0037] The term "bone substance" is not particularly limited to a specific kind of bone. "Bone" in accordance with the present invention is a rigid organ that constitutes part of the skeleton in most vertebrate animals. Without being bound by theory, bones have the function of protecting the various other organs of the body, produce red and white blood cells (as they contain the site of production of red and white blood cells), store minerals, provide structure and support for the body, and enable mobility. Bones come in a variety of shapes and sizes and have complex internal and external structures. They are lightweight, yet strong and hard and serve multiple functions.

[0038] Bone tissue (osseous tissue) is hard tissue, a type of specialised connective tissue. It has a honeycomb-like matrix internally, which helps to give the bone rigidity. Bone tissue is made up of different types of bone cells. Osteoblasts and osteocytes are involved in the formation and mineralisation of bone; osteoclasts are involved in the resorption of bone tissue. Osteoclasts are located within bone, express the parathyroid hormone receptor and are under the control of the parathyroid hormone secreted from the parathyroid glands into the circulation to regulate serum phosphate and Ca2+levels. Modified (flattened) osteoblasts become the lining cells that form a protective layer on the bone surface. The mineralised matrix of bone tissue has an organic component of mainly collagen, called ossein, and an inorganic component of bone mineral made up of various salts. Bone tissue is mineralized tissue of two types, cortical bone and cancellous bone. Other types of tissue found in bones include bone marrow, endosteum, periosteum, nerves, blood vessels and cartilage.

[0039] Bone is not uniformly solid, but is known to consist of a flexible matrix (about 30%) and bound minerals (about 70%) which are intricately woven and endlessly remodelled by a group of specialized bone cells. Their unique composition and design allow bones to be relatively hard and strong, while remaining lightweight.

[0040] Bone matrix is composed from 90 to 95% of elastic collagen fibers, also known as ossein, and the remainder is known as ground substance. The elasticity of collagen improves fracture resistance. The matrix is hardened by the binding of inorganic mineral salt, calcium phosphate, in a chemical arrangement known as bone mineral, a form of calcium hydroxylapatite. It is the mineralization that gives bones rigidity due to the minerals, phosphate and Ca2+stored in the bone.

[0041] Bone is actively constructed and remodelled throughout the entire lifetime by special bone cells known as osteoblasts and osteoclasts. Osteoclasts are controlled by the parathyroid hormone, osteoblasts are regulated by VEGF and are involved in bone remodelling, as well as ALCAM-positive and other immune competent cell-lines. Moreover, as exemplified in the appended Examples, autonomous processes are likely involved in the various remodelling processes, involving matrix intrinsic matrix-metalloprotease enzyme systems, alkaline phosphatase (ALPL), tissue-non-specific-alkaline-phosphatase (TNAP). Within any single bone, the tissue is woven into two main patterns, known as cortical and cancellous bone, each with a different appearance and different characteristics.

[0042] Bones are known to consist of living cells (osteoblasts and osteocytes) embedded in a mineralized organic matrix. The primary inorganic component of human bone is hydroxyapatite, the dominant bone mineral, having the nominal composition of Caio(P04)6(OH)2. The organic components of this matrix consist mainly of type I collagen and type II collagen - "organic" commonly referring to materials produced as a result of the human body - and inorganic components, which alongside the dominant hydroxyapatite phase, include other compounds of calcium and phosphate including salts. Approximately 30% of the (extracellular) acellular component of bone consists of organic matter, while roughly 70% by mass is attributed to the inorganic phase. The collagen fibers give bone its tensile strength, and the interspersed crystals of hydroxyapatite give bone its compressive strength. These effects are synergistic. The exact composition of the matrix may be subject to change over time due to nutrition and biomineralization, with the ratio of calcium to phosphate varying between 1.3 and 2.0 (per weight), and trace minerals such as magnesium, sodium, potassium and carbonate can also be found.

[0043] The term "bone matrix" or "organic matrix" collectively refers to the extracellular matrix (ECM) of the bone. These terms are used interchangeably in the present invention. The term "extracellular matrix", also called intercellular matrix, generally refers in biology and medicine to a network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite that provide structural and biochemical support to surrounding cells. Because m ulticell ularity evolved independently in different multicellular lineages, the composition of ECM varies between multicellular structures; however, cell adhesion, cel l-to-ce II communication and differentiation are common functions of the ECM.

[0044] In the bone, type I, II collagen is known to compose 90-95% of the organic matrix, with the remainder of the matrix being a homogenous liquid called ground substance consisting of proteoglycans such as hyaluronic acid and chondroitin sulfate, as well as non-collagenous proteins such as osteocalcin, osteopontin or bone sialoprotein. Collagen consists of strands of repeating units, which give bone its tensile strength, and they are arranged in an overlapping fashion that prevents shear stress. The function of ground substance is not fully known. Two types of bone can be identified microscopically according to the arrangement of collagen: woven and lamellar. Woven bone (also known as fibrous bone), which is characterized by a haphazard organization of collagen fibers and is mechanically weak. Lamellar bone, which has a regular parallel alignment of collagen into sheets ("lamellae") and is mechanically strong. Woven bone is produced when osteoblasts produce osteoid rapidly, which occurs initially in all fetal bones but is later replaced by more resilient lamellar bone. In adults, woven bone is created after, e.g., fractures. Woven bone is weaker, with a smaller number of randomly oriented collagen fibers, but forms quickly; it is for this appearance of the fibrous matrix that the bone is termed woven. It is soon replaced by lamellar bone, which is highly organized in concentric sheets with a much lower proportion of osteocytes to surrounding tissue. Lamellar bone, which makes its first appearance in humans in the fetus during the third trimester, is stronger and filled with many collagen fibers parallel to other fibers in the same layer (these parallel columns are called osteons). In cross-section, the fibers run in opposite directions in alternating layers, assisting in the bone's ability to resist torsion forces. After a fracture, woven bone forms initially and is gradually replaced by lamellar bone during a process known as "bony substitution". Compared to woven bone, lamellar bone formation takes place more slowly. The orderly deposition of collagen fibers restricts the formation of osteoid to about 1 to 2 pm per day. Lamellar bone also requires a relatively flat surface to lay the collagen fibers in parallel or concentric layers.

[0045] The extracellular matrix of bone is known to be established by osteoblasts, which secrete both collagen and ground substance. These synthesise collagen within the cell and then secrete collagen fibrils. The collagen fibers rapidly polymerise to form collagen strands. At this stage, they are not yet mineralised, and are called "osteoid". Around the strands calcium and phosphate precipitate on the surface of these strands, becoming crystals of hydroxyapatite within days to weeks.

[0046] In order to mineralise the bone, the osteoblasts secrete vesicles containing alkaline phosphatase. This cleaves the phosphate groups and acts as the foci for calcium and phosphate deposition. The vesicles then crack and act as a centre for crystals to grow on. More particularly, bone mineral is formed from globular and plate structures.

[0047] While, as mentioned, the bone is a rather rigid organ with a certain strength, there are conditions and / or disorders and / or diseases that lead to the destruction and / or degradation of the bone and / or the structure of the bone.

[0048] Without being bound by theory, conditions and / or disorders and / or diseases that lead to the destruction and / or degradation of the bone and / or the structure of the bone are known to the skilled person and can, e.g., be caused by the following: fractures, tumors, cancer and osteoporosis. Indeed, a number of conditions / diseases / disorders are known in the art to affect bone (structure), including arthritis, fractures, infections, osteoporosis and tumors. Conditions relating to bone can be managed by a variety of doctors, including rheumatologists for joints, and orthopaedic surgeons, who may conduct surgery to fix broken bones. Other doctors, such as rehabilitation specialists, may be involved in recovery, radiologists in interpreting the findings of imaging, pathologists in investigating the cause of the disease, and family doctors may play a role in preventing complications of bone disease, such as osteoporosis.

[0049] When a doctor sees a patient, a history and exam is usually taken. Bones are often imaged, called radiography. Ultrasound X-ray, CT scan, MRI scan and other imaging, such as a Bone scan, might be included to investigate cancer. Other tests such as a blood test for autoimmune markers may be taken, or a synovial fluid aspirate may be taken.

[0050] In normal bone, fractures occur when significant force is applied or when there is repetitive trauma over a long time. Fractures can also occur when a bone is weakened, such as by osteoporosis, or when there is a structural problem, such as when the bone remodels excessively (for instance by Paget's disease) or when the site is a site of cancer growth. Common fractures include wrist fractures and hip fractures, associated with osteoporosis, vertebral fractures associated with high-energy trauma and cancer, and fractures of long- bones. Compound fractures involve the bone's penetration through the skin. Some complex fractures can be treated by, e.g., bone grafting procedures that replace missing bone portions. Fractures and their underlying causes can be investigated by X-rays, CT scans and MRIs. Fractures are described by their location and shape. Several classification systems exist, depending on the location of the fracture. In addition, in general, surgical measures, such as internal fixation, may be used. Because of the immobilisation, people with fractures are often advised to undergo rehabilitation.

[0051] There are several types of tumors that can affect bone; examples of benign bone tumors include osteoma, osteoid osteoma, osteochondroma, osteoblastoma, enchondroma, giantcell bone tumors and aneurysmal bone cysts.

[0052] Cancer can arise in bone tissue, and bones are also a common site for other cancers to spread (metastasise) to. Cancers that arise in bone are called "primary" cancers, although such cancers are rare. Metastases within bone are "secondary" cancers, with the most common ones being breast cancer, lung cancer, prostate cancer, thyroid cancer and kidney cancer. Secondary cancers that affect bone can either destroy bone (called a "lytic" cancer) or create bone (a "sclerotic" cancer). Cancers of the bone marrow inside the bone can also affect bone tissue, examples include leukemia and multiple myeloma. Bone may also be affected by cancers in other parts of the body. Cancers in other parts of the body may release parathyroid hormone or parathyroid hormone-related peptide. This increases bone reabsorption and can lead to bone fractures. Bone tissue that is destroyed or altered as a result of cancers is distorted, weakened, and more prone to fracture. This may lead to compression of the spinal cord, destruction of the marrow resulting in bruising, bleeding and immunosuppression, and is a cause of bone pain. If the cancer is metastatic, then there might be other symptoms depending on the site of the original cancer. Some bone cancers can also be felt.

[0053] Other conditions that affect the bone are known in the art and include, inter alia, osteomyelitis (which is inflammation of the bone or bone marrow due to bacterial infection), osteomalacia (which is a painful softening of adult bone caused by severe vitamin D deficiency), osteogenesis imperfecta, osteochondritis dissecans, ankylosing spondylitis or skeletal fluorosis (which is a bone disease caused by an excessive accumulation of fluoride in the bones; in advanced cases, skeletal fluorosis damages bones and joints and is painful).

[0054] Osteoporosis is known as a disease of bone where there is reduced bone mineral density, increasing the likelihood of fractures. Osteoporosis is defined in women by the World Health Organization as a bone mineral density of 2.5 standard deviations below peak bone mass, relative to the age and sex-matched average. This density is measured using dual energy X-ray absorptiometry (DEXA), with the term "established osteoporosis" including the presence of a fragility fracture. Osteoporosis is most common in women after menopause, where it is called "postmenopausal osteoporosis", but may develop in men and premenopausal women as well in the presence of particular hormonal disorders and other chronic diseases or as a result of smoking and medications, specifically with glucocorticoids. Osteoporosis usually has no symptoms until a fracture occurs. For this reason, DEXA scans are often done in people with one or more risk factors, who have developed osteoporosis and are at risk of fracture.

[0055] Thus, in accordance with the present invention, "a patient in need of supporting the reconstitution or regeneration of bone substance" is, in its broadest sense, a patient that suffers from some kind of condition and / or disorder and / or disease that leads to the (at least partial) destruction and / or degradation of the bone and / or the structure of the bone. The above are only given as examples in order to illustrate that there are many conditions / disorders / diseases that lead to destruction and / or degradation of the bone and / or the structure of the bone.

[0056] The "destruction" and / or "degradation" may be gradual and may include all stages or grades that a given bone tissue may have in terms of an alteration and / or destruction and / or weakening and / or distortion of the present invention.

[0057] In certain embodiments, the destruction and / or degradation is a destruction and / or degradation of a part of the structure of a certain bone. In certain other embodiments, the destruction and / or degradation is a destruction and / or degradation and / or distortion of the entire or full structure of a certain bone.

[0058] Thus, bone tissue / bone structure that is destroyed, degraded, altered, distorted and / or weakened in terms of the present invention may be prone to the reconstitution or regeneration of said bone substance in a patient in need thereof in terms of the present invention.

[0059] The terms "reconstitution" and "regeneration" in the present invention have the same meaning as the terms have in the art. "Regeneration" in biology and in medicine generally refers to the process of renewal, restoration, and tissue growth that makes cells or tissues resilient to natural fluctuations or events that cause disturbance or damage. Every species is capable of regeneration. Regeneration can either be complete where the new tissue is the same as the lost tissue, or incomplete after which the necrotic tissue becomes fibrotic.

[0060] At its most elementary level, it is known that regeneration is mediated by the molecular processes of gene regulation and involves the cellular processes of cell proliferation, morphogenesis and cell differentiation. More specifically, regeneration is carried out by molecular processes within and by conformational changes of the damaged extracellular matrix of bone priorto receptor mediated gene regulation setting up cell proliferation and cell differentiation. Any cell involved in any regenerative activity in any tissue of an organism is regulated by incoming signals from another cell or the matrix environment. Phenotypic plasticity of ectodermal / mesenchymal embryonic stem cells translate into morphogenesis, yet are silenced down at adolescence, restricting restitutio ad integrum to tissue of endothermal origin, like blood vessels through CD14+ endothelial precursors organized by CD34+ cells. Ectodermal and mesodermal derived tissue / cells carry out defect repair. Any kind of morphologic information is no more available at a cellular level, yet it is within the chemically determined storehouse place for minerals in the extracellular matrix of bone.

[0061] Regeneration in biology and in medicine, however, mainly refers to the morphogenic processes that characterize the phenotypic plasticity of traits allowing multi-cellular organisms to repair and maintain the integrity of their physiological and morphological states.

[0062] The present invention is not particularly limited to a certain degree of reconstitution or regeneration of bone substance in a patient in need thereof. The composition prepared in accordance with the method of the present invention has the capability of "supporting" reconstitution or regeneration of bone substance in a patient in need thereof.

[0063] The term "supporting" not only includes a full reconstitution or regeneration of bone substance (in terms of a full recovery) but all stages or degrees of reconstitution or regeneration, even if there is only a gradual, but still significant, improvement into the direction of a regeneration or reconstitution in terms of the present invention. Thus, in its broadest sense, the term "supporting" means that the degree of reconstitution or regeneration is slightly, but still significantly, advanced compared to a patient in need thereof who has not been in contact with the composition prepared in accordance with the present invention. As will be outlined in more detail further below, therapeutic progress in terms of the degree of reconstitution or regeneration of bone substance in a patient in need thereof can be monitored by periodic assessment.

[0064] The nature of the bone or bone substance which can be reconstituted or regenerated with the composition of the present invention is not particularly limited and can be any bone or bone substance which is in need of a reconstitution or regeneration. Preferred bones are iliac crest, calcaneus, femur head, tibia, femur and rib.

[0065] In the present invention, the terms "patient", "subject" and "individuum" are used interchangeably.

[0066] In the present invention, the patient / subject / individuum is in a preferred embodiment a mammal such as a dog, cat, pig, cow, sheep, horse, rodent, e.g., rat, mouse, and guinea pig, or a primate, e.g., gorilla, chimpanzee and a human. In a most preferable embodiment, the subject is a human.

[0067] As mentioned above, the method for preparing a composition for supporting the reconstitution or regeneration of bone substance comprises five or six major steps preferably in the following subsequent order: step (i), step (ii), step (iii), step (iv) and step (v), and, optionally, step (vi). In the method of the present invention, (a) further step(s) may be performed before, between an / or after the above five or six major steps.

[0068] In another embodiment, the present invention relates to an alternative method wherein the order of step (i) and step (ii) can be exchanged, i.e., the fragmentation is performed before the decalcification / demineralization. In other words, in another preferred embodiment, the bone material can be fragmented prior to the decalcification / demineralization of the thus obtained fragmented bone material.

[0069] Accordingly, in this embodiment, the alternative method of the present invention comprises the following steps:

[0070] (i) fragmentation of decellularized bone material the decalcified / demineralized bone material;

[0071] (ii) decalcification / demineralization of fragmented bone material of step (i) at a nearly physiological pH value;

[0072] (iii) incubation of the decalcified / demineralized fragmented bone material of step (ii) in an aqueous solution at a nearly physiological pH value;

[0073] (iv) separation of the liquid supernatant from the fragmented bone material / debris; and

[0074] (v) recovery of the liquid supernatant of step (iv), thereby obtaining a liquid composition for supporting reconstitution or regeneration of bone substance. In the following, the five major steps are described in more detail (while the optional sixth major step is described in more detail further below).

[0075] With the exception of the exchange in the order of step (i) and step (ii) in the above-mentioned alternative method of the present invention wherein the fragmentation is performed before the decalcification / demineralization, the same applies, mutatis mutandis, to this alternative method of the present invention as described in the following for the method of the present invention (wherein step (i) is performed prior to step (ii)).

[0076] The first step (i) is the decalcification / demineralization of decellularized bone material at a nearly physiological pH value.

[0077] The starting material for the method for preparing a composition for supporting the reconstitution or regeneration of bone substance in a patient in need thereof is "bone material". Any bone material can be used as a starting material for the method of the present invention.

[0078] The nature of the bone material is not particularly limited. Any kind of osseous structure can be used that provides cortico-cancellous bone. Preferred bones / bone material as the starting material for the method of the present invention are iliac crest, calcaneus, femur head, tibia, femur and rib.

[0079] In preferred embodiments, the bone material may, e.g., be allogenic or autologous in relation to the patient / individuum in need of a reconstitution or regeneration of bone substance.

[0080] In short, allogeneic means "from another person" while autologous means "from oneself".

[0081] Thus, in a preferred embodiment, the bone material is allogenic in relation to the patient / individuum in need of a reconstitution or regeneration of bone substance.

[0082] The term "allogenic" is known in the art and relates to cells or tissues which are genetically different (and, hence, commonly immunologically incompatible) in relation to a certain patient / individuum, while belonging to the same species.

[0083] In another preferred embodiment, the bone material is autologous in relation to the patient / individuum in need of a reconstitution or regeneration of bone substance.

[0084] The term "autologous" is known in the art and relates to cells or tissues which are genetically identical (and, hence, commonly immunologically compatible) in relation to a certain patient / individuum.

[0085] In a different, yet, less preferred embodiment, the bone material may also be xenogeneic in relation to the patient / individuum in need of a reconstitution or regeneration of bone substance.

[0086] The term "xenogeneic" is known in the art and relates to cells or tissues which are genetically different (and, hence, commonly immunologically incompatible) in relation to a certain patient / individuum, and which is derived from an individuum of a different species. The starting material, i.e., the bone material used in step (i), must be decellularized. Decellularized bone material is commercially available. Moreover, the skilled person has standard means and methods at hand that allow to easily decellularize bone material.

[0087] The term "decellularized" basically means that the starting material, i.e., the bone material, is in a most preferred embodiment without any cells which are alive. It is not necessary that the bone material is completely free of living cells as long as the bone material is substantially free of living cells, i.e., the predominant part of the cells in the bone material are not alive.

[0088] Without being bound by theory, bone material can be decellularized as it is described in US patent 5,977,034.

[0089] In a nutshell, bone material can, e.g., be decellularized by using supercritical carbon dioxide (SC-CO2). SC-CO2 is commonly used for tissue decellularization due to its compatibility with biological materials and because it also does not leave toxins in the scaffolds. SC-CO2 has been shown to remove cells and genetic materials from bone tissue. Preferably, the incubation with SC-CO2 is performed at a pressure of 30 MPa and a temperature of 50°C.

[0090] Alternatively, "high hydrostatic pressure" (HHP) can be used as a physical decellularization procedure. HHP destroys cell membranes by increasing hydrostatic pressure. Using HHP, the structure of the tissue is significantly protected, while the genetic material and the nucleus are completely eliminated.

[0091] As a further alternative, chemical decellularization can be employed. Chemical decellularization commonly uses either acidic or basic detergents and chelating agents. As a non-limiting example, a non-ionic detergent like, e.g., Triton X-100 can be used as it not only destroys cells but also extracellular matrix glycosaminoglycans. Triton X-100 breaks down lipid-, protein- and protein-DNA bonds.

[0092] Chelating agents can also be used. As non-limiting examples, chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol-bis(|3-aminoethyl ether)- A / ,A / ,A / ',A / '-tetraacetic acid) can be used for decell ularizing bone tissue. Chelating agents bind metal ions such as Ca2+and Mg2+, leading to the separation of cells. EDTA is commonly used in combination with trypsin and other enzymes that disrupt cell-to-cell or cell-to-substrate interactions.

[0093] In accordance with step (i) of the method of the present invention, the bone material, once decellularized, is subjected to a decalcification or demineralization step.

[0094] The terms "decalcification" and "demineralization" are known to the skilled person. Moreover, the skilled person knows means and methods for how to decalcify or demineralize bone material.

[0095] Decalcification or demineralization of bones generally refers to the softening of bones due to the removal of calcium ions. Decalcification is often performed in science as a histological technique to, e.g., for studying bones or for extracting DNA. Indeed, since calcium-rich bones are exceedingly difficult to study, bone decalcification is commonly used to prepare specimen for research. Generally, there are two categories of decalcifying agents for removing calcium ions: chelating agents and acids.

[0096] Correspondingly known means and methods can also be used to decalcify bone material in the method of the present invention.

[0097] Acids that can be used for decalcification are further divided into weak (picric, acetic and formic acid) and strong acids (nitric and hydrochloric acid). The acids help to produce a solution of calcium ions while the chelating agents take up the calcium ions.

[0098] Chelation is a type of binding ions and molecules to metal ions. A variety of chelating agents are commonly known in the art with different affinities for different metals, different physical characteristics and different biological mechanism of action. Some common chelating agents are ethylenediaminetetraacetic acid (EDTA), 2,3-dimercaptopropanesulfonic acid (DMPS), thiamine tetra hydrofurfuryl disulfide (TTFD) and EGTA (ethylene glycol-bis(|3-aminoethyl ether)- / V, / V, / V', / V'-tetraacetic acid).

[0099] The chelating agents which are most frequently used in the decalcification / demineralization of bone material are ethylenediaminetetraacetic acid (EDTA) and EGTA (ethylene glycol-bis(P- aminoethyl ether)- / V, / V, / V', / V'-tetraacetic acid). Accordingly, in preferred embodiments, the decalcification / demineralization of decellularized bone material in accordance with step (i) of the method of the present invention is effected with either EDTA or EGTA.

[0100] The decalcification / demineralization with either an acid or a chelating agent is preferably effected in an aqueous solution.

[0101] Optionally, the aqueous solution comprises a buffering agent, preferably at a concentration of about 0.1 mM to about 100 mM.

[0102] Moreover, the aqueous solution has a certain pH, i.e., a "nearly physiological pH" as defined in more detail further below.

[0103] Examples of buffering agents that will control the pH in this range include glycine, acetate, phosphate, succinate, gluconate, glutamate, histidine, citrate, glycylglycine, aspartate, MES (2-( / V-morpholino)ethanesulfonic acid) and TRIS (Tr / s(hydroxymethyl)aminomethane). Preferred buffering agents include glycine, histidine, glutamate, succinate, phosphate, acetate, gluconate and aspartate.

[0104] The term "buffering agent" as used herein also includes the salts of the above buffering agents, such as the hydrochloride salt, the acetate salt, the phosphate salt and the sulfate salt. As stated above the aqueous solution for use as defined herein, may optionally comprise the buffering agent at a concentration of about ImM to about 50mM. Thus, the concentration of the buffering agent in the aqueous solution may be about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15mM, about 16mM, about 17mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM or about 50 mM. The concentration of the buffering agent in the aqueous solution may preferably be about 5mM to about 40 mM, about 8 mM to about 20 mM, particularly about lOmM.

[0105] In preferred embodiments, the aqueous solution for the decalcification / demineralization in the method of the present invention may comprise the chelating agent EDTA or EGTA at a concentration of about 0.001 M to about 0.7 M. Thus, the concentration of the chelating agent EDTA or EGTA in the aqueous solution may be about 0.001 M, about 0.005 M, about 0.008 M, about 0.01 M, about 0.02 M, about 0.03 M, about 0.04 M, about 0.05 M, about 0.06 M, about 0.07 M, about 0.08 M, about 0.09 M, about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.6 M or about 0.7 M. The concentration of the chelating agent EDTA or EGTA in the aqueous solution may preferably be about 0.3 M to about 0.4 M, 0.5 M, 0.6 M or 0.7 M, particularly preferred about 0.5 M.

[0106] The term "about" (and, similarly, the term "nearly") as used herein in the context of the entire invention and its disclosure preferably refers to ±10% of the indicated numerical value, more preferably to ±5% of the indicated numerical value, and in particular to the exact numerical value indicated. If the term "about" is used in connection with the endpoints of a range, it preferably refers to the range from the lower endpoint -10% of its indicated numerical value to the upper endpoint +10% of its indicated numerical value, more preferably to the range from the lower endpoint -5% to the upper endpoint +5%, and even more preferably to the range defined by the exact numerical values of the lower endpoint and the upper endpoint. If the term "about" is used in connection with the endpoint of an open-ended range, it preferably refers to the corresponding range starting from the lower endpoint -10% or from the upper endpoint +10%, more preferably to the range starting from the lower endpoint -5% or from the upper endpoint +5%, and even more preferably to the open-ended range defined by the exact numerical value of the corresponding endpoint.

[0107] As used in the present invention, the terms "comprising", "which comprises" or equivalent expressions encompass three alternatives, namely (i) "comprising", (ii) "consisting essentially of" and (iii) "consisting of". It is understood that in the context of the present invention, specifications such as "mM" or "% (w / w)" refer to the total volume or total weight of the resulting liquid aqueous composition.

[0108] The term "water" as used herein preferably refers to distilled, demineralized water, (highly) purified water or aqua ad iniectabilia (water for injections). The use of aqua ad iniectabilia is particularly preferred.

[0109] It is understood that the term "liquid" relates to a composition that is liquid at about 20°C and about 1,01325 bar = 1 atm.

[0110] It is understood that the term "aqueous composition" or "aqueous solution" refers to a composition essentially comprising water as the solvent (i.e., more than 80% (w / w) of the total solvent). Preferably the composition is essentially free of any organic solvents, wherein essentially free means less than <l%(w / w). More preferably the composition is free of any organic solvents. Most preferably the composition is an aqueous solution.

[0111] Without being bound by theory, the decalcification process can preferably be carried out with ethylenediaminetetraacetate (EDTA 0.05 mol / liter), can preferably also be carried out with EDTA 0.01 mol / liter and it can also preferably be carried out with EGTA. In a preferred embodiment, the pH is in the range of 6.7-7.6.

[0112] Many chemical bonds between sugars, lipids and proteins, as well as organic and inorganic compounds of ECM are ester compounds, which are hydrolyzed both base-catalyzed and acid- catalyzed.

[0113] As stated above, the decalcification / demineralization in accordance with step(i) of the method of the present invention is effected at a "nearly physiological pH value". As mentioned, this step is preferably effected in an aqueous solution, more preferably an aqueous solution comprising a buffering agent that will control the pH at a "nearly physiological pH value". Examples of buffering agents that will control the pH at a "nearly physiological pH value" have already been described above.

[0114] A "physiological pH value" in general terms is a pH value or a range of pH values wherein a normal cell or component of a cell functions. As an example, in the absence of pathological states, the pH of the human body ranges between 7.35 to 7.45, with the average at 7.40.

[0115] In the present invention, the term "physiological pH value" is, however, not particularly limited to this narrow range. Other pH values or pH value ranges may be used in the context of the present invention.

[0116] As mentioned above, the physiological range of pH required for the constant bone extracellular matrix / blood serum equilibrium is 6.8-7.4. The buffer capacity of H2PO4 ranges from 6.2 to 8.2. Any shift of the pH below 6.2 or beyond 8.2 exceeds the buffer capacity of phosphate. Thus, in a preferred embodiment, the near physiological pH is in the range of from about pH f 6.2 to 8.2.

[0117] In preferred embodiments, the "physiological pH value" in accordance with the present invention, preferably in an aqueous solution as defined herein, has a pH in the range of about pH 4.5 to about pH 8.5. Thus, the pH may be about 4.5, about 4.6. about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.3, about 8.4, or about 8.5.

[0118] Preferably the pH is in the range of from about pH 4.5 to pH 8.5, preferably in the range of pH 5.0 to pH 7.6, more preferably in the range of pH 6.7 to 7.6 or pH 6.7 to pH 7.4. In an even more preferred embodiment, the pH is in the range from 6.5 to 7.5. In a particularly preferred embodiment, the pH is a pH of 7.3.

[0119] The decalcification / demineralization in accordance with step(i) of the method of the present invention can be effected at a temperature from above 0°C to about 42°C. Preferably, temperatures above 42°C should be avoided if it is desired to avoid denaturation processes to occur. Thus, in more preferred embodiments, the temperature is in the range of about 1°C to about 42°C. Thus, the temperature may be about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, bout 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, or about 42°C.

[0120] Preferably the temperature is in the range of from about 22°C to about 39°C, about 22°C to about 38°C, about 25°C to about 38°C, about 30°C to about 38°C about 32°C to about 38°C, and more preferably in the range of about 33°C to about 38°C. In a preferred embodiment, the temperature is about 33°C. In a particularly preferred embodiment, the temperature is about room temperature, i.e., about 22°C.

[0121] Decalcification / demineralization is usually a lengthy procedure, as bone material has to be left in the decalcifying agent for at least some hours up to days to weeks, depending on the size of the bone.

[0122] Thus, the incubation of the decalcification / demineralization of step (i) of the method of the present invention is carried out for a time sufficient to allow the decalcification / demineralization, depending on various factors, like, the size of the bone, the temperature, etc..

[0123] There are numerous methods to monitor or to test how bone decalcification progresses and when bone decalcification is complete, such as X-ray examination, chemical analysis, and measurement of specimen flexibility. Corresponding methods are known to the person skilled in the art.

[0124] Preferably, the incubation of the decalcification / demineralization of step (i) of the method of the present invention is carried out until at least 90%, more preferably at least 95%, even more preferably at least 98% of the bone is decalcified / demineralized.

[0125] The incubation of step (i) of the method according to the present invention can be carried out in any kind of container that is suitable to hold the bone material as well as the aqueous solution and, accordingly, is not limited. Without being bound by theory, the incubation of step (i) of the method according to the present invention can, e.g., be carried out in a vessel, a test tube, a (tissue) culture dish, or in a Petri dish.

[0126] The second step (ii) is the fragmentation of the decalcified / demineralized bone material of step (i).

[0127] The term "fragmentation" generally refers to the action or process of breaking something, in the present case the decalcified / demineralized bone material of step (i), into smaller parts. The fragmented bone material is also often referred to herein below as "debris".

[0128] Bone material can be fragmented by various means known in the art. As examples, the bone material can be crushed, shredded and / or pulverized. Means and methods for crushing, shredding and / or pulverizing bone material are known in the art. Without being bound by theory, a means for crushing, shredding and / or pulverizing bone material is a device called "bone mill" or "bone crusher". Bone mills or bone crusher are known in the art. Any bone mill or bone crusher can be used in accordance with the fragmentation step (ii) for the fragmentation of the bone material. Accordingly, in a preferred embodiment, in the fragmentation step (ii), the fragmentation can be accomplished by using a bone mill or a bone crusher.

[0129] Preferably, in the fragmentation step (ii) of the method of the present invention, the bone material is fragmented so as to achieve fragmented bone material having a size in the range between 0.1 mm3to about 5 mm3.

[0130] Thus, the fragmented bone material may have a size of about 0.1 mm3, about 0.2 mm3, about 0.3 mm3, about 0.4 mm3, about 0.5 mm3, about 0.6 mm3, about 0.7 mm3, about 0.8 mm3, about 0.9 mm3, about 1.0 mm3, about 1.1 mm3, about 1.2 mm3, about 1.3 mm3, about 1.4 mm3, about 1.5 mm3, about 1.6 mm3, about 1.7 mm3, about 1.8 mm3, about 1.9 mm3, about 2.0 mm3, about 2.1 mm3, about 2.5 mm3, about 2.8 mm3, about 3.0 mm3, about 3.3 mm3, about 3.5 mm3, about 3.8 mm3, about 4.0 mm3, about 4.3 mm3, about 4.5 mm3, about 4.5 mm3, about 4.8 mm3, or about 5.0 mm3.

[0131] Preferably the fragmented bone material may have a size in the range of from 0.5 mm3to about 5 mm3, about 0.5 mm3to about 3 mm3, about 0.7 mm3to about 2 mm3, and more preferably in the range of about 0.7 mm3to about 1.5 mm3. In a particularly preferred embodiment, the size is about 1 mm3.

[0132] The third step (iii) is the incubation of the fragmented bone material of step (ii) in an aqueous solution at a nearly physiological pH value.

[0133] The terms "aqueous solution" and "nearly physiological pH value" have already been defined above in the context of step (i) of the method of the present invention. As regards these features as well as preferred embodiments of these features, the same applies, mutatis mutandis, as has been set forth above in the context of step (i) of the method of the present invention.

[0134] Moreover, as regards suitable temperatures for this incubation step (iii), as preferred embodiments of these features, the same applies, mutatis mutandis, as has been set forth above in the context of step (i) of the method of the present invention.

[0135] The incubation step (iii), i.e., the incubation of the fragmented bone material of step (ii) in an aqueous solution at a nearly physiological pH value is carried out for a sufficient time to allow the liquid composition for supporting reconstitution or regeneration of bone substance to form.

[0136] The formation of the liquid composition for supporting reconstitution or regeneration of bone substance of the present invention can be monitored or tested by using a refractometer or a UV / VIS meter.

[0137] In a preferred embodiment, the progression of the incubation step can be monitored by using a refractometer by measuring the liquid composition's refraction index by using methods known in the art and as shown in the Examples.

[0138] As shown in the appended Examples (see, in particular, Table 1), the supernatant obtained by the method, i.e., the composition of the present invention, has a refraction index of around 1.335. This refraction index is higher than that of pure water (1.333) and lower than that of protein-containing solutions. Accordingly, in preferred embodiments, the supernatant obtained by the method, i.e., the composition of the present invention, preferably has a refraction index between (but not including) the values of 1.333 and 1.3336, thereby excluding potential undesirable contaminations by proteins, lipids, and / or sugars. Preferably, the refraction index of the supernatant obtained by the disclosed method, i.e., the composition of the present invention, is about 1.335.

[0139] Preferably, the incubation of the fragmented bone material of step (ii) in an aqueous solution at a nearly physiological pH value in accordance with step (iii) of the method of the present invention is carried out for about 1 to 5 days, 2 to 5 days, 3 to 5 days or 4 to 5 days. In other preferred embodiments, the incubation is carried out for about 1 to 4 days, 1 to 3 days or 1 to 2 days. In more preferred embodiments, the incubation is carried out for 2 to 4 days. In other preferred embodiments, the incubation is carried out for about 1 day, 2 days, 3 days, 4 days or 5 days. In a most preferred embodiment, the incubation is carried out for 3 days.

[0140] In preferred embodiments, for this incubation step, a Ringer's solution or a (physiological) 0.9% NaCI solution can be used.

[0141] Ringer's solution is a solution of several salts dissolved in water for the purpose of creating an isotonic solution relative to the body fluids of an animal. Ringer's solution typically contains sodium chloride, potassium chloride, calcium chloride and sodium bicarbonate, with the last one being used to balance the pH. Different variants of Ringer's solution are known in the art and are commonly used. The precise proportions of these compounds vary from species to species.

[0142] The incubation of step (iii) of the method according to the present invention can be carried out in any kind of container that is suitable to hold the fragmented bone material of step (ii) and the aqueous solution at a nearly physiological pH value used in step (iii) and, accordingly, is not limited. Without being bound by theory, the incubation of step (iii) of the method according to the present invention can, e.g., be carried out in a vessel, a test tube, a (tissue) culture dish, or in a Petri dish.

[0143] The fourth step (iv) is the separation of the liquid supernatant from the fragmented bone material / debris.

[0144] The above incubation step (iii) (wherein the fragmented bone material of step (ii) is incubated in an aqueous solution at a nearly physiological pH value) takes place in a suspension wherein liquid parts and solid parts are present. The liquid part is separated from the solid part in step (iv) of the present invention.

[0145] Means and methods for the separation of a liquid component from the solid component / particles of a mixture are known in the art. For example, centrifugation is commonly used to separate a liquid component from the solid component / particles.

[0146] Without being bound by theory, the separation of the liquid supernatant from the fragmented bone material / debris may be carried out by centrifugation or by simple sedimentation, just to mention two possibilities as non-limiting examples. In the context of the present invention, in relation to step (iv) of the method of the present invention, sedimentation is preferred.

[0147] Centrifugation is a mechanical process which involves the use of the centrifugal force to separate particles from a solution according to their size, shape, density, medium viscosity and rotor speed. The denser components of the mixture migrate away from the axis of the centrifuge, while the less dense components of the mixture migrate towards the axis.

[0148] Sedimentation is generally the deposition of sediments. It takes place when particles (i.e., the fragmented bone material / debris in the method of the present invention) in suspension settle out of the fluid in which they are entrained and come to rest against a barrier. This is due to their motion through the fluid in response to the forces acting on them: these forces can, in general, be due to gravity (in a sedimentation in more specific terms) or due to centrifugal acceleration (as described above). The process of sedimentation is often referred to as settling which is the falling of suspended particles through the liquid, whereas sedimentation is the final result of the settling process.

[0149] In a preferred embodiment of the present invention, the separation of the liquid supernatant from the fragmented bone material / debris is carried out by sedimentation wherein, as explained, the solid part travels quickly and, preferably, fully, to the bottom of the container or vessel in which the separation is carried out. The precipitate that is formed is commonly known as a pellet. The remaining liquid that lies above the precipitate is called a supernatant.

[0150] The fifth step (v) is the recovery of the liquid supernatant of step (iv), thereby obtaining a liquid composition for supporting reconstitution or regeneration of bone substance.

[0151] Once the separation of the liquid supernatant from the fragmented bone material / debris is effected in step (iv), the liquid supernatant of step (iv) can be recovered, thereby obtaining a liquid composition for supporting reconstitution or regeneration of bone substance.

[0152] The liquid supernatant thus obtained in step (v), i.e., the thus obtained liquid solution / composition for supporting reconstitution or regeneration of bone substance in terms of the present invention is also referred to herein as an "exudate" (while the even more precise term is "transudate"). Yet, for the purpose of the present invention, the terms "exudate" and "transudate" are used interchangeably herein (as it also often interchangeably referred to in the art).

[0153] Means and methods for the recovery of the liquid supernatant are known in the art.

[0154] Preferably, upon recovery of the liquid supernatant, it is avoided that the recovered liquid contains parts of the precipitate.

[0155] For example, the liquid supernatant of step (iv) can be recovered in step (v) by using a pipette, e.g., a glass pipette or a micropipette known in the art. A pipette is a laboratory tool commonly used in chemistry, biology and medicine to transport a certain volume of liquid. Many pipette types work by creating a partial vacuum above the liquid-holding chamber and selectively releasing this vacuum to draw up and dispense liquid. As outlined above, in preferred embodiments, the liquid supernatant, i.e., the liquid composition recovered in step (v) preferably comprises electrolytes containing HPO3 / H2PO3 / Glucose and / or Ribose, more preferably, containing HPCh / I^PCh / Glucose and / or Ribose of about 1%.

[0156] In other preferred embodiments, the liquid supernatant, i.e., the liquid composition recovered in step (v) preferably comprises electrolytes containing HPO3 / H2PO3 and / or Glucose, more preferably, containing HPO3 / H2PO3 and / or Glucose of about 1%.

[0157] More preferably, the liquid supernatant, i.e., the liquid composition recovered in step (v) preferably comprises an electrolyte exudate containing HPO3 / H2PO3 and / or Glucose, more preferably, HPO3 / H2PO3 and / or Glucose 1%.

[0158] In more preferred embodiments, the liquid composition comprises the electrolytes HPO3 / H2PO3 / Glucose and / or Ribose in a concentration of about 0.1% to about 10.0 %. Thus, in even more preferred embodiments, the electrolytes HPCh / I^PCh / Glucose and / or Ribose may be in a concentration of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.0%, 2.0%, 2.5%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0% or about 10%. In a particularly preferred embodiment, the electrolytes HPO3 / H2PO3 / Glucose and / or Ribose are in a concentration of about 1.0%.

[0159] The concentrations of the above electrolytes HPCh / I^PCh / Glucose and / or Ribose may independently vary in the liquid composition comprising said electrolytes HPO3 / H2PO3 / Glucose and / or Ribose. Accordingly, the concentrations of the above electrolytes HPO3 / H2PO3 / Glucose and / or Ribose in the liquid composition comprising said electrolytes may independently be as follows:

[0160] In more preferred embodiments, the liquid composition comprises the electrolyte HPO3 in a concentration of about 0.1% to about 10.0%. In an even more preferred embodiments, the liquid composition comprises the electrolyte HPO3 in a concentration of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.0%, 2.0%, 2.5%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0% or about 10%. In a particularly preferred embodiment, the electrolyte HPO3 is in a concentration of about 1.0%. In more preferred embodiments, the liquid composition comprises the electrolyte H2PO3 in a concentration of about 0.1% to about 10.0%. In an even more preferred embodiments, the liquid composition comprises the electrolyte H2PO3 in a concentration of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.0%, 2.0%, 2.5%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0% or about 10%. In a particularly preferred embodiment, the electrolyte H2PO3 is in a concentration of about 1.0%. In more preferred embodiments, the liquid composition comprises the electrolyte Glucose in a concentration of about 0.1% to about 10.0%. In an even more preferred embodiments, the liquid composition comprises the electrolyte Glucose in a concentration of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.0%, 2.0%, 2.5%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0% or about 10%. In a particularly preferred embodiment, the electrolyte Glucose is in a concentration of about 1.0%.

[0161] In more preferred embodiments, the liquid composition comprises the electrolyte Ribose in a concentration of about 0.1% to about 10.0%. In an even more preferred embodiments, the liquid composition comprises the electrolyte Ribose in a concentration of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.0%, 2.0%, 2.5%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0% or about 10%. In a a particularly preferred embodiment, the electrolyte Ribose may be in a concentration of about 1.0%.

[0162] Once obtained the liquid supernatant of step (iv), i.e., the liquid composition for supporting reconstitution or regeneration of bone substance, can then either be subjected to a drying step (vi) explained in the following or can be used for supporting the reconstitution or regeneration of bone substance in a patient in need thereof as described in more detail further below, preferably, in the form of a pharmaceutical composition as described further below.

[0163] In a preferred embodiment, the present invention relates to a method for preparing a composition for supporting the reconstitution or regeneration of bone substance in a patient in need thereof as defined herein, wherein the method comprises the following additional step:

[0164] (vi) drying the liquid supernatant recovered in step (v) of the method of the present invention, thereby obtaining a dried form of a composition for supporting the reconstitution or regeneration of bone substance.

[0165] Means and methods for drying the liquid supernatant are known in the art.

[0166] Without being bound by theory, the liquid supernatant can simply be incubated at a certain temperature for a time sufficient to allow the drying of the liquid supernatant until the liquid is evaporated while the remainder is the dried form of the composition of the present invention.

[0167] In fact, drying is commonly known as a mass transfer process consisting of the removal of water or another solvent by evaporation from a semi-solid or liquid. This step can be used as a final production step before selling, packaging or storing the composition of the present invention. To be considered "dried", the final product is solid, preferably, in the form of a powder.

[0168] In a preferred embodiment, a simple way to dry the liquid supernatant in accordance with step (vi) of the method of the present invention is to let the liquid supernatant just incubate at room temperature without any further contribution. Alternatively, a source of heat and / or an agent to remove the vapor produced by the process are often involved.

[0169] In the most common case, a gas stream, e.g., air, applies the heat by convection and carries away the vapor as humidity. Without being limited, other possibilities are vacuum drying, where heat is supplied by conduction or radiation (or microwaves), while the vapor thus produced is removed by the vacuum system. Another indirect technique is drum drying, where a heated surface is used to provide the energy, and aspirators draw the vapor outside the room.

[0170] If heat is applied, preferred temperatures for the drying step (vi) range from room temperature to about 42°C, i.e., at a temperature in the range from from above about 20°C to about 42°C. Preferably, temperatures above 42°C should be avoided if it is desired to avoid denaturation processes to occur. Thus, in more preferred embodiments, the temperature is in the range of about 20°C to about 42°C. Other temperatures are also possible. Thus, the temperature may be about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, bout 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, or about 42°C.

[0171] Preferably the temperature is in the range of from about 22°C to about 39°C, about 22°C to about 38°C, about 25°C to about 38°C, about 30°C to about 38°C about 32°C to about 38°C, and more preferably in the range of about 33°C to about 38°C. In a more preferred embodiment, the temperature is about 40°C. In a particularly preferred embodiment, the temperature is about room temperature, i.e., about 22°C.

[0172] In a further embodiment, the liquid supernatant can be dried by lyophilization, also known as freeze drying. This is a low temperature dehydration process that involves freezing the product and lowering pressure, removing the ice by sublimation. This is in contrast to dehydration by most conventional methods that evaporate water using heat.

[0173] It is easily possible to monitor or to test how the drying of the liquid supernatant recovered in step (v) of the presently claimed method progresses and when the drying is complete. For instance, a simple supervision by eye is possible.

[0174] Alternatively, residual liquid parts can be measured by methods known to the person skilled in the art.

[0175] Preferably, the drying is carried out until at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably 100% of supernatant is dried, i.e., until a complete drying has been achieved. Without being bound by theory, in preferred embodiments, depending on the volume, the liquid supernatant can, e.g., be dried at about 40 °C for about 6 hours. Alternatively, the liquid supernatant can also be dried at about room temperature for about 24 hours. The times are indicated for an amount of approximately 8milliliters of liquid to be dried of. Yet, the times may vary depending on the volume of the liquid.

[0176] In alternative embodiments, depending on the volume, the liquid supernatant can, e.g., also be dried at about 70 °C for about 2 hours under supervision. The times are indicated for an amount of approximately 8m i II i liters liquid to be dried. Yet, the times may vary depending on the volume of the liquid.

[0177] The dried form of the composition is particularly suitable for storing the composition obtained by the method of the present invention.

[0178] For storage, the dried composition should, in preferred embodiments, be stored at a temperature range from about 3°C to about 29°C.

[0179] Once obtained, the dried form of a composition for supporting reconstitution or regeneration of bone substance, can be used for supporting the reconstitution or regeneration of bone substance in a patient in need thereof as described in more detail further below.

[0180] In a preferred embodiment, the present invention relates to a method for preparing a composition for supporting the reconstitution or regeneration of bone substance in a patient in need thereof as defined herein, wherein the decalcification / demineralization in step (i) is effected by incubating the decellularized bone material with an aqueous solution comprising a chelating agent, preferably, wherein said chelating agent is ethylenediaminetetraacetic acid (EDTA) or EGTA (ethylene glycol-bis(|3-aminoethyl ether) V, / V, / V', / V'-tetraacetic acid).

[0181] The terms "aqueous solution", "chelating agent", "EDTA" and "EGTA" have already been defined above in the context of step (i) of the method of the present invention. As regards these features as well as preferred embodiments of these features, the same applies, mutatis mutandis, as has been set forth above in the context of step (i) of the method of the present invention.

[0182] Similarly, suitable "nearly physiological pH values" as well as other conditions like, e.g., suitable temperatures, have also already been defined above. As regards these features as well as preferred embodiments of these features, the same applies, mutatis mutandis, as has been set forth above in the context of step (i) of the method of the present invention.

[0183] In a preferred embodiment, the present invention relates to a method for preparing a composition for supporting the reconstitution or regeneration of bone substance in a patient in need thereof as defined herein, wherein the fragmentation of the decalcified / demineralized bone material in step (ii) is effected by crushing, shredding and / or pulverizing the bone material.

[0184] The terms "fragmentation" as well as possible ways to fragment bone material by, e.g., crushing, shredding and / or pulverizing as well as preferred sizes of the thus obtained fragmented bone material have already been defined above in the context of step (ii) of the method of the present invention. As regards these features as well as preferred embodiments of these features, the same applies, mutatis mutandis, as has been set forth above in the context of step (ii) of the method of the present invention.

[0185] In a preferred embodiment, the present invention relates to a method for preparing a composition for supporting the reconstitution or regeneration of bone substance in a patient in need thereof as defined herein, wherein the incubation of the fragmented bone material in step (iii) is effected in an isotonic aqueous solution, preferably in Ringer's solution or in a 0.9% NaCI solution.

[0186] "Isotonic" generally refers to a solution that has similar concentrations of salt and / or sugars to the concentrations found in the human body.

[0187] The terms "aqueous solution", "Ringer's solution" and "0.9% NaCI solution" have already been defined above in the context of step (iii) of the method of the present invention. As regards these features as well as preferred embodiments of these features, the same applies, mutatis mutandis, as has been set forth above in the context of step (iii) of the method of the present invention.

[0188] Moreover, any other suitable isotonic aqueous solution can be used in step (iii) of the present invention, like, e.g., any commonly known infusion solution suitable for use for infusion into the human body. Such infusion solutions are commonly based on Ringer's solution or a physiological 0.9% NaCI solution as already described herein above.

[0189] In a preferred embodiment, the present invention relates to a method for preparing a composition for supporting the reconstitution or regeneration of bone substance in a patient in need thereof as defined herein, wherein the nearly physiological pH value in step (i) and in step (iii) is a pH in the range of pH 4,5 to pH 8.5, preferably in the range of pH 5.0 to pH 7.6, or more preferably in the range of pH 6.7 to pH 7.4. In a particularly preferred embodiment, the pH is a pH of 7.3.

[0190] The term "nearly physiological pH value" has already been defined above in the context of step (i) and step (iii) of the method of the present invention. As regards this feature and the indicated preferred ranges, as well as preferred embodiments thereof, the same applies, mutatis mutandis, as has been set forth above in the context of step (i) and step (iii) of the method of the present invention. In a preferred embodiment, the present invention relates to a method for preparing a composition for supporting the reconstitution or regeneration of bone substance in a patient in need thereof as defined herein, wherein the separation in step (iv) is effected by sedimentation or centrifugation.

[0191] The terms "separation", "sedimentation" and "centrifugation" have already been defined above in the context of step (iv) of the method of the present invention. As regards these features as well as preferred embodiments thereof, the same applies, mutatis mutandis, as has been set forth above in the context of step (iv) of the method of the present invention.

[0192] In a preferred embodiment, the present invention relates to a method for preparing a composition for supporting the reconstitution or regeneration of bone substance in a patient in need thereof as defined herein, wherein the decellularized bone material in step (i) is allogenic or xenogeneic in relation to the patient.

[0193] The terms "allogenic" and "xenogeneic" have already been defined above in the context of step (i) of the method of the present invention. As regards these features as well as preferred embodiments thereof, the same applies, mutatis mutandis, as has been set forth above in the context of step (i) of the method of the present invention.

[0194] In a preferred embodiment, the present invention relates to a method for preparing a composition for supporting the reconstitution or regeneration of bone substance in a patient in need thereof as defined herein, wherein the liquid composition obtained in step (v) or the dried composition obtained in step (vi) is supplemented with metal or metal alloy particles, compounds that support or enhance osteosynthesis, additional bone material, preferably additional allogenic bone material, or pharmaceutically active compounds associated with metabolic diseases.

[0195] The liquid composition obtained in step (v) of the method of the present invention, or the dried composition obtained in step (vi) of the method of the present invention can be supplemented with one or more further compounds. The nature of the one or more compounds which can be used to supplement the liquid composition obtained in step (v) or the dried composition obtained in step (vi) is not particularly limited and can be any compound that has an additional effect in supporting the reconstitution or regeneration of bone substance in a patient in need thereof. The skilled person can easily choose or select one or more additional compounds that can be supplemented, depending on the desired (additional) effect.

[0196] Without being bound by theory, in preferred embodiments, the liquid composition obtained in step (v) of the method of the present invention or the dried composition obtained in step (vi) of the method of the present invention is supplemented with metal or metal alloy particles, compounds that support or enhance osteosynthesis, additional bone material, preferably additional allogenic bone material, or pharmaceutically active compounds associated with metabolic diseases.

[0197] In a preferred embodiment, the present invention relates to a composition for supporting reconstitution or regeneration of bone substance, obtainable by the method as defined herein, preferably, wherein said liquid composition comprises electrolytes containing HPO3 / H2PO3 / Glucose and / or Ribose.

[0198] In a preferred embodiment, the composition of the present invention for supporting reconstitution or regeneration of bone substance can, e.g., be a liquid composition that is directly obtained in step (v) of the method of the present invention or the dried composition obtained in step (vi) of the method of the present invention. Optionally, in other preferred embodiments, the composition of the present invention for supporting reconstitution or regeneration of bone substance can, e.g., be the liquid composition that is obtained in step (v) of the method of the present invention or the dried composition obtained in step (vi) of the method of the present invention which is, furthermore, supplemented with one or more compounds as defined above.

[0199] As outlined above, in preferred embodiments, the liquid supernatant, i.e., the liquid composition recovered in step (v) preferably comprises electrolytes containing HPO3 / H2PO3 / Glucose and / or Ribose, more preferably, containing HPCh / I^PCh / Glucose and / or Ribose 1%.

[0200] Thus, the present invention relates to a composition for supporting reconstitution or regeneration of bone substance, obtainable by the method as defined herein, preferably, wherein said liquid composition comprises electrolytes containing HPCh / I^PCh / Glucose and / or Ribose.

[0201] As regards preferred embodiments of said liquid composition comprising electrolytes containing HPCh / I^PCh / Glucose and / or Ribose, the same applies, mutatis mutandis, to the composition for supporting reconstitution or regeneration of bone substance that is obtainable by the method as defined herein, as has been set forth above in the context of the methods of the present invention.

[0202] Thus, once obtained, the liquid supernatant of step (v), i.e., the liquid composition of the present invention, can be used for supporting reconstitution or regeneration of bone substance (either directly or supplemented with one or more compounds as defined above) in a patient in need thereof.

[0203] Moreover, once obtained the dried form of a composition of step (vi), it can be used for supporting reconstitution or regeneration of bone substance (either directly or supplemented with one or more compounds as defined above) in a patient in need thereof. The dried form of the composition can be used for supporting reconstitution or regeneration of bone substance as defined herein can be used upon rehydratation with a suitable buffer prior to use. Suitable buffers are preferably buffers which are commonly used in the preparation of pharmaceutical compositions and that are known to the skilled person. Examples of suitable buffers are described herein.

[0204] Accordingly, the composition for supporting reconstitution or regeneration of bone substance, obtainable by the method as defined herein, is particularly useful in medical settings.

[0205] If used in medical settings as described in more detail in the following, the composition for supporting reconstitution or regeneration of bone substance is preferably termed "pharmaceutical composition". Yet, the term "composition" for supporting reconstitution or regeneration of bone substance and the term "pharmaceutical composition" for supporting reconstitution or regeneration of bone substance are used interchangeably in the following.

[0206] Accordingly, in preferred embodiments, the present invention relates to the (pharmaceutical) composition of the present invention as defined herein for use in a method of supporting reconstitution or regeneration of bone substance in a patient.

[0207] Moreover, in another preferred embodiment, the present invention relates to a pharmaceutical composition of the present invention comprising an effective amount of the composition as defined herein, and at least one pharmaceutically acceptable excipient.

[0208] Further, in another preferred embodiment, the present invention relates to a pharmaceutical composition of the present invention comprising an effective amount of the composition as defined herein, and at least one pharmaceutically acceptable excipient, for use in a method of supporting reconstitution or regeneration of bone substance in a patient.

[0209] Hence, in a preferred embodiment, the present invention relates to a pharmaceutical composition of the present invention comprising an effective amount of the composition as defined herein, for use as a drug.

[0210] Further, in another preferred embodiment, the present invention relates to a pharmaceutical composition of the present invention comprising an effective amount of the composition as defined herein, and at least one pharmaceutically acceptable excipient, for use as a drug in a method of supporting reconstitution or regeneration of bone substance in a patient.

[0211] The terms "reconstitution or regeneration of bone substance in a patient" have already been described above. As regards the definitions as well as the preferred embodiments, the same applies, mutatis mutandis, to the medical settings / pharmaceutical compositions as has been set forth above.

[0212] Terms like "reconstitution or regeneration of bone substance in a patient", "treatment" and / or "prevention" and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. Accordingly, the reconstitution or regeneration of bone substance in a patient may relate to (acute) states of a certain disease / disorder or condition of the bone as described above but may also relate to the prophylactic treatment in terms of completely or partially preventing a certain disease / disorder or condition or symptom thereof of the bone. Preferably, the term "treatment" is to be understood as being therapeutic in terms of partially or completely curing a disease and / or adverse effect and / or symptoms attributed to the disease.

[0213] However, as already explained above, in the context of the present invention, "a patient in need of supporting the reconstitution or regeneration of bone substance" is, in its broadest sense, a patient that suffers from some kind of condition and / or disorder and / or disease that led to the destruction and / or degradation of the bone and / or the structure of the bone. The above are only given as examples in order to illustrate that there are many conditions / disorders / diseases that lead to destruction and / or degradation of the bone and / or the structure of the bone.

[0214] As also already explained above, in the context of the present invention, the "destruction" and / or "degradation" may be gradual and may include all stages or grades a given bone tissue or bone tissue may have in terms of an alteration and / or destruction and / or weakening and / or distortion.

[0215] In certain embodiments, as also already explained above, in the context of the present invention, the destruction and / or degradation is a destruction and / or degradation of a part of the structure of a certain bone. In certain other embodiments, the destruction and / or degradation is a destruction and / or degradation and / or distortion of the entire or full structure of a certain bone.

[0216] Thus, as also already explained above, in the context of the present invention, the bone tissue / bone structure that is destroyed, degraded, altered, distorted and / or weakened in terms of the present invention may be prone to the supporting of the reconstitution or regeneration of said bone substance in a patient in need thereof.

[0217] As also already explained above, in the context of the present invention, the terms "reconstitution" and "regeneration" in the present invention have the same meaning as the terms have in the art. "Regeneration" in biology and in medicine generally refers the process of renewal, restoration and tissue growth that makes cells or tissues resilient to natural fluctuations or events that cause disturbance or damage. Every species is capable of regeneration. Regeneration can either be complete where the new tissue is the same as the lost tissue, or incomplete after which the necrotic tissue becomes fibrosis. As also already explained above, the present invention is not particularly limited to a certain degree of reconstitution or regeneration of bone substance in a patient in need thereof. The composition prepared in accordance with the method of the present invention has the capability of "supporting" reconstitution or regeneration of bone substance in a patient in need thereof.

[0218] The term "supporting" not only includes a full reconstitution or regeneration of bone substance (in terms of a full recovery) but all stages or degrees of reconstitution or regeneration, even if there is only a gradual, but still significant, improvement into the direction of a regeneration or reconstitution in terms of the present invention. Thus, in its broadest sense, the term "supporting" means that the degree of reconstitution or regeneration is slightly, but still significantly, advanced compared to a patient in need thereof who has not been in contact with the composition prepared in accordance with the present invention. As will be outlined in more detail further below, therapeutic progress in terms of the degree of reconstitution or regeneration of bone substance in a patient in need thereof can be monitored by periodic assessment.

[0219] The pharmaceutical composition or drug of the present invention may be administered via a large range of classes of forms of administration known to the skilled person. Administration may be systemically, locally, orally, through aerosols, including but not limited to tablets, needle injection, the use of inhalators, creams, foams, gels, lotions and ointments.

[0220] Preferably, the pharmaceutical composition of the present invention for use in a method of supporting reconstitution or regeneration of bone substance in a patient as described herein is to be administered intravenously, topically, intradermally, subcutaneously, intra- cutaneously, intramuscularly and / or intrathecally.

[0221] These routes of administration, i.e., via an intravenously, topically, intradermally, subcutaneously, intra-cutaneously, intramuscularly and / or intrathecally are known to the skilled person.

[0222] In a preferred embodiment, the composition for supporting reconstitution or regeneration of bone substance of the present invention is administered to the bone substance / bone defect in need of reconstitution or regeneration as such, i.e., the liquid composition for supporting reconstitution or regeneration of bone substance of the present invention is administered directly to the bone substance.

[0223] In another preferred embodiment, the composition for supporting reconstitution or regeneration of bone substance of the present invention is administered to the bone substance / bone defect in need of reconstitution or regeneration by a sponge-like or cotton swab-like device that has absorbed the liquid composition for supporting reconstitution or regeneration of bone substance of the present invention. In another preferred embodiment, the composition for supporting reconstitution or regeneration of bone substance of the present invention is administered to the bone substance / bone defect in need of reconstitution or regeneration by a membrane or a dressing that has absorbed the liquid composition for supporting reconstitution or regeneration of bone substance of the present invention.

[0224] An excipient or carrier is an inactive substance formulated alongside the active ingredient, i.e., the pharmaceutical composition of the present invention for use in a method of supporting reconstitution or regeneration of bone substance in a patient for the purpose of bulking-up formulations that contain potent active ingredients. Excipients are often referred to as "bulking agents," "fillers," or "diluents". Bulking up allows convenient and accurate dispensation of a drug substance when producing a dosage form. They can also serve various therapy-enhancing purposes, such as facilitating drug absorption or solubility, or other pharmacokinetic considerations. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-sticking properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. The selection of appropriate excipients also depends on the route of administration and the dosage form, as well as the active ingredient and other factors.

[0225] Thus, the pharmaceutical composition of the present invention for use in a method of supporting reconstitution or regeneration of bone substance in a patient as described herein may be in solid, liquid or gaseous form and may be, inter alia, in a form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s). It is preferred that said pharmaceutical composition optionally comprises a pharmaceutically acceptable carrier and / or diluent.

[0226] These compositions / pharmaceutical compositions of the present invention for use in a method of supporting reconstitution or regeneration of bone substance in a patient as described herein can be administered to the subject at a suitable dose. Administration of the suitable compositions may be affected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration.

[0227] The compositions of the invention may also be administered directly to the target site, e.g., by biolistic delivery to an external or internal target site.

[0228] The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depend on many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The pharmaceutically active matter may be present in amounts between 1 ng and 10 mg / kg body weight per dose (preferably, in the range of 1%, 0,1% to 5%, more preferably, at 1% in case the dried composition of the present invention is used); however, doses below or above this exemplary range are envisioned, especially when considering the aforementioned factors. If the regimen is a continuous infusion, it should also be in the range of 1 pg to 10 mg units per kilogram of body weight per minute.

[0229] Examples of suitable pharmaceutical carriers, excipients and / or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose, i.e., in "an effective amount" which can easily be determined by the skilled person by methods known in the art. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depend on many factors, including the patient's or subject's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.

[0230] Thus, preferably, the pharmaceutical composition of the present invention for use in a method of supporting reconstitution or regeneration of bone substance in a patient as described, is / are included in an effective amount. The term "effective amount" refers to an amount sufficient to induce a detectable therapeutic response in the subject to which the pharmaceutical composition is to be administered. In accordance with the above, the content of the composition of the present invention in the pharmaceutical composition is not limited as far as it is useful for treatment as described above, but preferably contains 0.0000001-10% (preferably, between 0,1% to 1% of the dried composition of the present invention) by weight per total composition. Further, the composition of the present invention described herein is preferably employed in a carrier. Generally, an appropriate amount of a pharmaceutically acceptable salt is used in the carrier to render the composition isotonic. Examples of the carrier include but are not limited to saline, Ringer's solution and dextrose solution. Preferably, acceptable excipients, carriers, or stabilisers are non-toxic at the dosages and concentrations employed, including buffers such as citrate, phosphate, and other organic acids; salt-forming counter-ions, e.g., sodium and potassium; low molecular weight (> 10 amino acid residues) polypeptides; proteins, e.g., serum albumin, or gelatine; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids such as histidine, glutamine, lysine, asparagine, arginine or glycine; carbohydrates including glucose, mannose or dextrins; monosaccharides; disaccharides; other sugars, e.g., sucrose, mannitol, trehalose or sorbitol; chelating agents, e.g., EDTA; non-ionic surfactants, e.g., Tween, Pluronics or polyethylene glycol; antioxidants including methionine, ascorbic acid and tocopherol; and / or preservatives, e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, e.g., methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol). Suitable carriers and their formulations are described in greater detail in Remington's Pharmaceutical Sciences, 23rded., 2020.

[0231] Therapeutic progress can be monitored by periodic assessment.

[0232] The pharmaceutical composition / drug for use in a method of supporting reconstitution or regeneration of bone substance in a patient as described above may be in sterile aqueous or non-aqueous solutions, suspensions, and emulsions as well as creams and suppositories. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Preservatives and other additives may also be present such as antimicrobials, anti-oxidants, chelating agents and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may comprise further agents depending on its intended use. Said agents may, e.g., be polyoxyethylene sorbitan monolaurate, available on the market with the commercial name Tween, propylene glycol, EDTA, citrate, sucrose as well as other agents being suitable for the intended use of the pharmaceutical composition that are well-known to the person skilled in the art.

[0233] In accordance with this invention, the term "pharmaceutical composition" relates to a composition for administration to a patient, preferably a human patient.

[0234] As outlined above, the pharmaceutical composition / drug for use in a method of supporting reconstitution or regeneration of bone substance in a patient may be supplemented with one or more additional compounds. Thus, in a preferred embodiment, a combination of the pharmaceutical composition / drug for use in a method of supporting reconstitution or regeneration of bone substance in a patient with one or more additional compounds is envisaged.

[0235] Thus, the term "combination" in this context relates to a combination of two components, i.e., the pharmaceutical composition / drug for use in a method of supporting reconstitution or regeneration of bone substance in a patient as defined herein as well as the one or more additional compounds (as well as the preferred embodiments as described herein in the context of the (method for preparing the) composition of the present invention and the one or more additional compounds, respectively). In a preferred embodiment, a simultaneous application is envisaged. This applies when the liquid compound obtained in step (v) or the dried composition obtained in step (vi) is (directly) supplemented with the one or more additional compounds as described herein above.

[0236] Yet, in particular, in the context of medical settings described herein, the combination also encompasses a subsequent application of the two components. Thus, one of these components may be administered before, simultaneously with or after the other one of the combination, or vice versa.

[0237] Accordingly, "in combination" as used herein does not restrict the timing between the administration of the two components. Thus, when the two components are not administered simultaneously with / concurrently, the administrations may be separated by 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours or 72 hours or by any suitable time differential readily determined by one of skill in art and / or described herein. In a preferred embodiment, when the two components are not administered simultaneously with / concurrently, the administrations may be separated by 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours or 72 hours or by any suitable time differential readily determined by one of skill in art and / or described herein.

[0238] The invention also relates to methods of supporting the reconstitution or regeneration of bone substance in a patient in need thereof, wherein the (pharmaceutical) composition of the present invention as defined herein is administered to a subject, preferably, in a therapeutically effective amount as defined herein.

[0239] As regards the preferred embodiments of the methods for treatment the same applies, mutatis mutandis, as has been set forth above in the context of the composition or the pharmaceutical composition for uses as defined above.

[0240] In the present invention, the subject is, in a preferred embodiment, a mammal such as a dog, cat, pig, cow, sheep, horse, rodent, e.g., rat, mouse and guinea pig, or a primate, e.g., gorilla, chimpanzee and a human. In a most preferable embodiment, the subject is a human.

[0241] Other aspects and advantages of the invention will be described in the following examples, which are given for purposes of illustration and not by way of limitation. Each publication, patent, patent application or other document cited in this application is hereby incorporated by reference in its entirety.

[0242] Figure 1: 80x magnification. On the left lower side is a rib transplant (1), on the right-side cancellous bone matrix (2), both decalcified with EDTA prior to the incubation as described. The picture was taken on day three of the incubation. Between both bone matrix particles, a spheric structure (3) developed attached to both pieces. On the left lower side adjacent to the rib transplant and in the cancellous region such filaments developed as well.

[0243] Figure 2: 135x magnification. Image shows the filaments solved in the medium in more detail.

[0244] Figure 3: 60x magnification. Image taken on day six of the incubation. On the right side is a part of the rib transplant (1), the spheric structure (2) from picture one is now flaky, with structured inside.

[0245] Figure 4: 30x magnification. Image shows incubated bone matrix dried at air. Right down is a solid structured piece leaked out of the collagen bone matrix particle (2) parts and structures during incubation. Geometrically placed filaments with smaller side chains. An intermediate zone (3) separates the dense part of the air-dried bone matrix from a structured exudate (4) visible on the upper side of the picture.

[0246] Figure 5: shows in more detail main (1) and side chain (2) filaments linked to each other in centric structures. The main chains are tied together in centers (3).

[0247] Figure 6: shows the more solid part of the compartment. Densely condensed polymerized backbones (1), branches of polymerized main side chains (2), small side chains (3).

[0248] Figure 7: 80-fold magnification of a bone matrix rib transplant (1), decalcified with EDTA.

[0249] The transplant was air dried after being incubated for 11 days in an aqueous solution. The filaments (2) sorting out of the transplant are geometrically sorted. At the leaving site of the rib transplant the filaments are conical, the interstitial space translucent (3). The structures arose from the transplant in cell-free aqueous medium. The underlying process is an autocatalytic hydrolysis.

[0250] Figure 8: The main filaments (2) are organized in a cross or star shaped manner. A center is always present. As well as parallel side chains. EDTA-decalcified rib transplant (1), The interstitial space (3) is translucent, in this picture black, due to the black background used for photography. Figure 9: Patterns of cross-like and star-like, organelle-like structures consisting of a rectangular center (1) with main arms (2) defined by being linked to the rectangular center and side arms (3) defined by being linked to the main arms, the interstitial black space consisting of HPO3- salts and Glucose.

[0251] Figure 10: Reaching the side walls (1) of the plates the filaments form thickened conical apices (2).

[0252] Figure 11: 2-D figure image of a centre, leaf-like extensions with a central string, a dark cytoplasm with a brighter, yellow border, where phosphonates continuously react with Fe, building a membrane-like structure.

[0253] Figure 12: Air dried bone matrix was incubated in aqueous solution with Fe. Attached to Fe-metal objects > 3mm3, structures (1) are visible in large quantities. In 3-D view the structures are spherical with different sizes, are covered with red brown leaf with yellow edges.

[0254] Figure 13: shows a large example of such a structure (2) on Fe-metal objects (1).

[0255] Figure 14: an Fe-metal object (1) was made with 3 mm3and incubated with bone matrix particles. The substrates covering the bottom of the plate leaked out of a bone matrix particle outside of that picture. The arrow indicates the direction of expansion. The expanding matrix attached to the metal object from all sides and fixed it to the bottom of the plate.

[0256] Figure 15: The Fe-metal object in the middle of the picture (1) and the flat 2-D extensions (2), corresponding to the main and side extensions in pictures (7-9). The extensions are little structured, covered by a yellow border. One of the constituents of the yellow borders, are water soluble meta-phosphate compounds, with a pH of HPO3- about 7.8. The yellow color comes from the Fe attached to the HPO3- / H2PO3 system. The HPO3- / Glucose containing cover facilitates the chemo attractive behavior of the appendices.

[0257] Figure 16: 80-fold magnification. Fe-metal object, the arrowhead like apex (2) of the filamentous extensions is lined up, attached to each other through the membrane forming cover (3).

[0258] Figure 17: Visible is the circle in the middle of the picture (1), where the Fe metal object is placed. The white opaque layer under the Fe metal object consists of phosphorous / glucose compositions, the ring structured gap (2) surrounds the object, radially permeated by brown strings, corresponding to the Fe colored phosphorous compounds.

[0259] Figure 18: shows the same object as Figure 19, yet the background in picture twenty is white, in picture nineteen black. As the background now is white, the brown colored gap (1) permeating phosphorous filaments (2) are good to see.

[0260] Figure 19: The filaments on the right side of the picture consisting of a center (1), main arms (2), side chains (3) leaked from the bone matrix into the aqueous solution and polymerized to long filaments cross shaped, white short and long side chains on all main filaments. On the left side is a metal object of Titanium Oxide (TiCh) (5). The main filament directed against the foreign object reacts with substantial swelling of its content (6). The tubular structures around the swollen filament are typical meta-phosphorous (Na-meta-phosphate) condensations in aqueous solutions. Also, the radially against the metal directed tubules show these characteristic features.

[0261] Figure 20: TiO2 metal object, the Gap (2), shows the drop-like, swollen appendices (3) leaked out of the bone matrix during incubation in a cell-free medium under physiological conditions. The surfaces of the drop-like apices reach towards the foreign body surfaces of the metals, chemically attracted.

[0262] Figure 21: shows a filamentous extension (1) swelling at the apex and attaching to the metal object (2) by chemoattraction.

[0263] Figure 22: the picture of the bone matrix (l) / TiO2-metal object (2) was taken from the bottom of the plate after the probe dried on room air. Dark shadowed in the center is the metal object. The object is surrounded and undermined as well by a translucent matrix (3) which is poorly structured and homogenous, looking like a gap. The borders of the gap are arrow shaped extensions (4). At the frontier towards the gap the apices of the arrows are increasingly swollen. These arrows headed apices appear drop-like as long as they are in a humid environment. The appearance of the filamentous structure is mainly influenced by the composition of the salts contained in the physiological solution. Center of the starlike filaments. The thin filaments within the bone matrix are either glucose or meta-phosphate polymers (5). Figure 23: Display of a rib transplant, demineralized cortical bone and a TiCh object attached to each otherafter being incubated together in an isotonic, pH-neutral aqueous solution after 7 days. This picture exemplifies autonomous, cell independent abilities of the bone matrix in regard to transplant- or implant osseointegration.

[0264] Figure 24: On the upper side of the picture a rib-transplant is seen which was incubated together with a bone matrix particle. The extensions directed against the ribtransplant derive from a second bone matrix particle. The extensions display arrowhead like structures, which are seen if the solution for the incubation contains isotonic concentrations of NaCI, KCI, and MgCl. The interstitial space contains isotonic meta-phosphate and Glucose concentrations, the micro tubular structures are polymerisations of meta-phosphates, when the solubility product is exceeded, the filaments are polymerisation products of Glucose.

[0265] Figure 25: 120x magnification of the gap, showing the translucent space with filaments of polymerized Glucose.

[0266] Figure 26: Diagram showing the pH vs. days. Bone matrix was incubated in water for 12 days. The rib transplant was decalcified with EDTA prior to the incubation in water, the other transplants were demineralized with HCI. The curves displaying the pH over time for the EDTA decalcified Bone was significantly different from those bones demineralized with HCI as HCI demineralized bones led to a further acidification of the solution, which recovered slowly and became neutral only on day seven to nine. In contrast, EDTA-decalcified bone never dropped into acidic conditions and remained under neutral conditions over time.

[0267] Figure 27: Protein concentrations rose above 0,2 mg / mL in the solution only on day 4 of the incubation of decalcified bone matrix. The protein concentrations increased steadily to 0,5 mg / mL within 12 days.

[0268] Figure 28: The highly acidic solution contained non-essential amino acids (25 mg of non- essential amino acids in 100 milliliter). Incubating decalcified bone matrix (0,1 g) in 50 mL of the solution resulted in an almost complete absorption of the amino acids within 24 hours.

[0269] Figure 29: An allograft rib transplant was incubated in either H2O, EDTA, C2H5OH 30% over a period of 12 days. Every day, a 200-microliter probe was taken, performing a quantitative protein measurement. The used method is sensitive for protein concentrations > 0,2 milligram / millilitres. Values below must be neglected as random artefacts. Incubating rib transplants in H2O dilute protein into the supernatant, while EDTA and C2H5OH inhibit enzymes intrinsic to the bone matrix responsible for degradation.

[0270] Figure 30: SDS-PAGE using bis-Tris as running buffer maintaining a neutral pH. A 20- microliter probe was taken every day from 0,1-gram allograft incubated in fifty mL H2O. The numbers at the top of each lane represents the day of incubation, indicating that probes were taken on days 1, 2, 3, 4, 5, 6, 8, 9, 11. In the gel, a single band is visible from day one until day nine. Only on day eleven a second lane between 460 and 368 kDa appears. Silver staining was used, which is a very sensitive method, staining mainly charged amino acids of proteins, and is sensitive down to 250 nano grams (0,25 micrograms) of protein. Keeping environmental conditions in a range of pH 6.8 - 7.4 ester bonds remain intact; protein does not appear in the supernatant.

[0271] Figure 31: SDS-PAGE using Tris-Acetate as running buffer maintaining a pH >8.4. A 20- microliter probe was taken every day from the same sample of 0,1-gram allograft incubated in fifty milliliter H2O like shown in Figure 30. The numbers at the top of each lane represents the day of incubation, indicating that probes were taken on days 1, 2, 3, 4, 5, 6, 8, 9, 11. In the gel, one band is seen continuously from day one to day 10 getting weak at day 12. Yet, additional bands appear on day 5 between 71 and 117 kDa, and in the range of 238-268 kDa. The next bands appear on day 6, 8, 10 and day 12, showing that ester bond breaking of macromolecules (>460 kilo Dalton) happens within a very short period (40 minutes) if these molecules are exposed to an environment above or below a neutral pH. Silver staining was used, as explained in Figure 30. Keeping environmental conditions in at a pH of >8.4ester bonds hydrolyse; protein do appear in the supernatant.

[0272] Figure 32: lOx magnification against a black background. The yellow-rimmed structures correspond to sedimented polymerisates sticking to the bottom of the plate after extracellular matrix of the bone has been incubated in aqueous solution for 3 days. After that, eight milliliters of the supernatant were pipetted into a plate and dried at room air. Yellow-rimmed structures. The yellow staining of the (borders) = "surfaces of the polymerisates" is caused by the addition of Fe to the incubation solution. The content (2) of the small structures (polymerisates) is transparent, the content of the larger structures (polymerisates) is white (2). The large structures (polymerisates) form centromeres, with peripheral and sub-peripheral appendices (3), which are recognizable as black continuations.

[0273] Figure 33: The image is dominated by a polymerisate. The center (1) is determinable, which sticks as sediment to the bottom of the dried Petri dish. The center is surrounded by fanned, leafy structures surrounded by a yellow line. The contents are milky whitish in color (3). The leaf-like milky white structures are hinted at by elongated, axial, hypotense formations (2). In the elongation of the hypotense, elongated structures, slimmer extensions (4) emerge, without milky, whitish coloration, which, however, are also yellow-brown bordered. The hypotense, transparent extensions, hereinafter referred to as appendices, are abundantly branched. These hypotense, yellow-brown bordered appendices are associated with brown-colored vesicles.

[0274] Figure 34: 120x magnification against a black background. 0.1g ECM was incubated in

[0275] Ringer's solution Fresenius 7mL together with Fe for 3 days at 35°C. Fe stained the membrane surface of the polymerisates (M) golden-yellow, vacuoles (V) inside the centromere analogue, which in this illustration have a surface. Parts of the cytoskeleton (Z) were also stained golden-yellow. The image shows the peripheral part of a centromere analogue at the junction with two peripheral appendix analogues (U). Outside the membrane in the interstitium there are red-brown colored vesicles. The membrane defines an inside and outside, i.e., a cytoplasm and an interstitium (I).

[0276] Figure 35: 80x magnification of two parallel extensions of the organelle-like structure (1).

[0277] Sub-peripheral extensions (2) of different sizes and shapes. Antenna-like subperipheral extensions (3), which are different from others and from which reticulo-tubular networks emerge (4) and connect the organelle-like structures to each other. Along the tubules, which are also likely to have a consistent surface against the interstitial fluid, Fe-OH-stained vesicles are visible (4). The surface of the organelle-like structure is clearly recognizable against a white background brown or gray. Vacuolar-reticular cytoplasm-like structures.

[0278] Figure 36: Native image of a dried supernatant at 40x magnification, edge of the liquid film (1), centre of an organelle-like structure (2), extensions of the organelle-like structure (3), sub-peripheral extensions (4), peripheral extensions (5); centriole analogues can be identified in this image since a long extension is opposed by a short one from the same centre of the organelle-like structure, with a central indicated square, and since sub-peripheral extensions cannot be identified in the diagonal of the sub-peripheral. Strong Fe2Os and FeOH stains can be detected in the centromere analogues, sub-peripheral appendix analogues towards the edge, not on this side. Peripheral appendix analogues form a 3-D marginal wall with Fe inclusions that are significantly lower than in the upstream structures.

[0279] Figure 37: 50x magnification against a black background. Centre (2) of an organelle-like polymerisate from which four extensions (2a, 2b, 2c, 2d) emerge. These centres have very different shapes. Centre (2a) points from the centre (1) into the interior of the shell, is by far the shortest, with paired and further appendices. The centromeres 2b & 2c run parallel to the edge of the Petri dish, both centromeres have appendices protruding on both sides. The first subperipheral appendix of centromere 2d appears longer than its centromere and forms another sub-peripheral appendix that appears almost as voluminous as its parallel neighbour 2d. Between the appendix and the centromere 2d there is another appendix. The centromeres, directed against the edge of the Petri dish, and their appendices are especially voluminous, swollen. Towards the edge, the membranes become more prominent. The peripheral ends of the organelle-like polymerisates end with rectilinear arrowhead-like ends (4). The interstitial space between parallel centromeres has almost elapsed, in many places the membranes are glued together or connected by interstitial tubules. Along the central hypotense axis there are white-gray membrane-surface- bound, reticular cisterns. Interstitial space (5).

[0280] Figure 38: 50x magnification against a black background. The prominent hypotense axis

[0281] (1) proceeds towards the edge of the Petri dish. At right angles to these subperipheral appendices (2), there are extended interstitial space (3) in the periphery. Arrowheads on the border with the edge of the Petri dish. The surface to the periphery is becoming increasingly prominent. The surface is distinctly yellow due to the Fe, yellow-staining Fe compounds in the transudate are not visible below this magnification. Vesicles in the interstitium, interconnecting filaments in the interstitium.

[0282] Figure 39: 20x magnification against a white background. A centrosome (1), centromere

[0283] (2), peripheral appendices (3). Crucial to note is that the iron-intoxicated centromeres have formed regular peripheral appendices. The aqueous medium in which the polymerized transudate expanded was absorbed by it. In this way, the Fe dissolved in the medium reacted with the polymerizing transudate. Via its tubular system, the polymerisate transported the medium and the Fe dissolved in it from its center to its periphery. In this process, da Fe Fe-OH formed bonds along the tubular structures, which stained them yellow and brown. At the moment of this image, only small Fe residues were left in the center of the centromere (2), which were deposited in the distal centromeres near the periphery at the time of recording. It is particularly noteworthy that the polymerisate "piled up" water at its peripheral edge, which enclosed it with its peripheral appendices. In this way, the peripheral appendices form a peripheral marginal wall.

[0284] Figure 40: Coronal slide from a DVT (digital volume tomography) made one month after the operation of a patient who underwent tooth extraction cystectomy and socket preservation in the molar region of the left side of the mandible using cortical fibers as bone replacement material demineralized with HCI. 1: the hypotense bone replacement material filling out the extraction hole. 2: local spongious bone of the patient's mandible adjacent to the site of reconstruction. 3: cortical bone of the patient's mandible. The brighter grey colour indicates the higher density of the cortical structures compared to the cancellous bone sites. 4: The channel of the inferior alveolar nerve. 5: dark grey coloured soft tissue of the tongue. 6: dark grey coloured soft tissue of the patient's cheek.

[0285] Figure 41: Axial view of the mandible of the same patient as in Figure 40. The patient underwent tooth extraction on both sides of the mandible in the molar region. 1: the hypotense bone replacement material filling out the extraction hole.

[0286] Figure 42: Coronal slide from a DVT (digital volume tomography) 14 days after the operation of a patient who underwent tooth extraction, cystectomy and socket preservation using a cortico-cancellous block, decalcified with EDTA for 3 days, fragmented into small pieces, incubated in the "solution", i.e., the supernatant as obtained by the method of the present invention as exemplified in the appended Examples, i.e., the composition of the present invention. Bone density of the used bone replacement material (3) equals to the patient's proper cancellous bone (1). 2 is the cortical structure of the upper alveolar ridge. 4: dark grey coloured structure of the palate. 5: dark grey coloured soft tissue of the nose. 6: black colour of the air in the maxillary sinus. 7: black colour of the air in the oral cavity.

[0287] Figure 43: Axial slide from a DVT (digital volume tomography) of the patient's DVT shown in Figure 42. 1: black coloured air in the maxillary sinus. 2: part of the patient's proper cancellous bone of the upper alveolar ridge. 3: hyperintense cortical rim of the alveolar rim. 4: hyperintense metal of dental implants used for reconstruction of teeth. 5: bone replacement material as described in Figure 42, showing normal to slightly hyperintense bone replacement material. 6: hyper intense structure of a rib cortical structure decalcified with EDTA for 3 days used as a membrane to cover the reconstruction site according to the art of reconstruction of bone defects in the jaws.

[0288] Figure 44: Coronal slide from a DVT (digital volume tomography) performed immediately after the operation of a patient who underwent tooth extraction, cystectomy and socket preservation using a cortico-cancellous block, decalcified with EDTA for 3 days, fragmented into small pieces, incubated in the "solution" ", i.e., the supernatant as obtained by the method of the present invention as exemplified in the appended Examples, i.e., the composition of the present invention. Bone density of the used bone replacement material (3) equals to the patient's proper cancellous bone (1). 2 is the cortical structure of the upper alveolar ridge. 4: dark grey coloured structure of the palatinale gingiva. 5: dark grey coloured soft tissue of the nose. 6: black colour of the air in the maxillary sinus. 7: black colour of the air in the oral cavity.

[0289] Figure 45: This image shows the corresponding sagittal plane of the coronal slide shown in Figure 44. 1: air in the maxillary sinus appearing black. 2: nose. 3: roots of the teeth in the upper jaw, which are hyperintense compared to the cancellous bone of the alveolar ridge. 5: air in the oral cavity. 6: bone replacement material, an allograft bone block decalcified for 3 days with EDTA, then fragmented, mixed with the "solution" and used to fill out the cavity in the maxilla which remains after tooth extraction.

[0290] EXAMPLES

[0291] MATERIAL AND METHODS

[0292] Demineralization, Incubation, Documentation, Staining

[0293] Preparations: The allogeneic bone substitute material further mentioned as bone matrix is delivered decellularized and sterile packaged. The package of cortico- cancellous bone block measuring 10x18x25 mm is opened under sterile conditions and disassembled into three parts of approximately equal size. These 3 bone blocks are decalcified together in a tube with a volume of 50mL in neutral pH (>6.5<7.4) over three days. After that, the bone blocks are ground. 0.1 grams of the fibrous-mushy bone mixture are incubated in a plate with ten milliliters of Ringer's solution or 0.9% NaCI or deionized water (H2O) together with different metal objects for three days, then dried in room air.

[0294] Incubation: The incubator is made for cell cultures and is set to 35 °C Celsius and 5% CO2. Unlike cell cultures, the medium is not changed during the incubation period.

[0295] For quantitative protein measurements and for investigations with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two hundred microliters of sample were taken daily, which was not refilled.

[0296] The media used is 0.9% NaCI or Ringer's solution Fresenius.

[0297] Documentation: The photo and film documentation were carried out in the medium and sample filled Petri dish, as well as the sediment during and after drying.

[0298] Staining: After drying, samples were stained with eosin, hematoxylin, Sirius pikrin acid, Alcian blue, or Schiff's reagent, or the samples were documented natively. A special coloration was created by incubating the samples in aqueous solution together with colloidal iron, as described above.

[0299] Documentation: The film and photo documentation were carried out by the research microscope Nikon SMZ18 stereo microscope, with a fixed Nikon D800E photo camera via the Camera Control Pro2 software of Nikon in jpg and RAW format. With the help of microscopic photo documentation, both photos and films were made. During the incubation of the matrix in Ringer's solution Fresenius, titanium oxide (TiC>2) objects with a diameter of > / = 3 millimeters and a height of 5 millimeters and as TiO2 particles with a size <=90 micrometers, with zirconium oxide (ZirCh) particles of 12 micrometers in size, with iron (Fe) < 90 micrometers in size were placed in the liquid film. The objects with which the interactions were studied were also incubated at the beginning of the incubation period, unless otherwise highlighted in the results. The bone was either decalcified with EDTA or with HCI according to the protocol. If the bone matrix was decalcified with HCI, the procedure is further called "demineralization" because it could be shown that the exposition of bone matrix to HCI does not specifically remove Ca2+but also breaks ester-bonds, thus removes un- specifica lly different compounds and minerals and Ca2+as well. In contrast, if EDTA is used to remove Ca2+from the bone matrix, Ca2+and some other minerals are removed rather specifical, thus this procedure merits the term "decalcification" much more. EDTA is produced to chelate specifically Ca2+, yet some other minerals as well. But EDTA is used in a physiological range of pH from 7-7,4 avoiding ester bond break off.

[0300] During incubation, two or more pieces of bone were placed in the liquid film. After three days, the sample was dried in room air and photographed. This examination protocol was modified so that the drying phase was filmed under a microscope. The interaction of the decalcified matrix was carried out with allogeneic bone parts of another batch, either decalcified or non-decalcified.

[0301] EXAMPLE 1: SEDIMENTS

[0302] 1. Sediments

[0303] Allografts were incubated in either H2O, NaCI 0.9%, Ringer's solution, different versions of minimal essential media, made for cell cultures, for 12 days.

[0304] Over 12 days the pH-values were measured, the protein concentration of the SN (supernatant) and an SDS-PAGE was made with the daily probes using either Tris- Acetate or a BIS-TRIS buffer. Photos were taken.

[0305] Change is a prerequisite for healing. Either the local bone or the used bone replacement material has a potential for changing its proper constitution, best would be both. Figures 1, 2 and 3 are part of a series of pictures taken at distinct stages of changing bone replacement material incubated in salt solution for several days. 3 days afterthe cell free bone was put into the aqueous solution, spheric, yet undefined structures extended from the solid piece of bone into the liquid space (Figure 1).

[0306] Figure 2 shows in detail at 100-fold magnification thin filaments tide together at the center of the sphere. In picture three after a time of 9 days a flocking within the spheric organized filaments occurred, yet the flocks rarely left those spheric structures. The probes were air-dried before further investigating such bone-borne structures. The solid, collagenous bone structures remained microscopically unaffected by the incubation in the aqueous solution. It was surprising to observe expanding structures from the bone matrix which were organized with a constantly repeating pattern (Figure 4).

[0307] The observations of constantly repeating spheric patterns were unexpected. So far, the extracellular matrix of bone consists of a collagen structure kept together by several proteoglycans and glycoproteins. So far, cells are supposed to deliver the enzymatic equipment for reorganizing the bone matrix. It is, thus, unclear in how far degrading enzymes of the bone extracellular matrix are an intrinsic constituent of the extracellular matrix of bone itself.

[0308] Current literature counts multiple structural proteins of the bone extracellular matrix, proteoglycans, glycoproteins, and mitogens. What cannot be found in current literature is that compartments of the bone matrix form a coherent structure woven into the collagen matrix which dissolves from the collagen network of the bone matrix strongest by incubation in distilled water or aqueous, isotonic and pH neutral solutions at temperatures between 35 and 42 °C, as shown below.

[0309] Figures 4, 5 and 6 show components of the bone matrix at low magnification. In Figure 4 in the left upper part a structure is visible separated from the corresponding collagen matrix by a semicircle like transition zone convex to the left upper angle of the picture. Figures 5 and 6 show matrix filaments and their side chains. Downright shows such a side chain structure of the matrix is visible in high magnification. In Figure 6, parts of the corresponding collagen matrix.

[0310] For the next investigations, decalcified bone matrix was used. In the Figures referred to in the following, patterns extracted from a decellularized and an entirely demineralized rib-transplant are shown.

[0311] In the decalcified rib, structures reach out of the rib transplant (Figure 7), 12 days afterthe start of the incubation in salt solution and air-dry procedure afterwards. The cone-like structures stand in rectangular position to the rib collagen piece (Figure 8). In this special rib the structures extended over a multifold plane (Figure 9) and expanded to the borders of the plate (Figure 10).

[0312] The bone matrix expands significantly under physiological, yet cell-free conditions in vitro. The constantly appearing patterns of expansion indicate underlaying, regulatory, enzymatic mechanisms intrinsic to the bone matrix. Although the underlaying mechanisms are unclear, there are strong indications that enzymatic hydrolysis is the leading mechanisms for this. The expanding substrates are part of the invention and described further below. A spherical organelle-like structure in the sediments of bone matrix incubated together with Fe

[0313] In the following, the observation of a spherical organelle-like structure in the sediments of bone matrix incubated together with Fe (Eisenpulver, particles having a size of <90 micrometer; Werth-Metall; CAS-Nummer: 7439-89-6) is described.

[0314] In contrast to the procedure described above, a second procedure was used to investigate the spontaneous interactions in cell-free medium between EDTA- decalcified bone matrix and metal objects. Therefore, the incubation procedure was done as described above, yet Fe particles were added to the medium during incubation. After 7 days, the supernatant was removed carefully with a pipette and the remaining sediments air dried.

[0315] Figure 11 of the dried sediment revealed both lobed shaped objects, attached to the bottom of the plate, surrounded by yellow borders, like cell membranes. All these objects have a clearly defined center from which the lobes extended peripheral. Many objects were not flat fixed to bottom of the plate but attached to Fe particles and reached as spherical organelle-like objects (Figures 12 and 13) three dimensionally into the air. The center of the spherical object is always fixed to a metal object added to the solution. The spherical organelle-like structures appears brown, red if the metal object is Fe. The leaves of the spherical organelle-like structures are always directed away from the metal objects. The leaves are red brown, the edges of the leaves yellow.

[0316] In the presence of Fe particles having a size of <90 micrometer compounds of the bone matrix react with Fe2+ / 3+to 2-D and 3-D organelle like spherical objects. The color is specific for compounds of Fe2+ / 3+reacting with bone matrix constituents and does not appear if the bone matrix is incubated in aqueous solutions with any other proved metal particles like TiCh, ZirCh, CuCh, KH2PO4, Glucose-6-PO4.

[0317] To find out which constituents of the bone matrix could be involved in the reactions with Fe and cause these brown colored objects the following procedures were performed.

[0318] 500 milligram Na-meta-phosphate (HPOs(-2)) (Carl Roth; CAS: 68915-31-1) were solved in fifty milliliter distilled water, corresponding to a 1% Na-meta-phosphate solution. No organic compounds like bone matrix were added to this solution additionally. Then, ten milliliters of this solution were given into two different plates each. Five hundred milligram KH2PO4 were mixed with fifty milliliters of deionized water obtaining a 1% solution KH2PO4, ten milliliters of that solution were given in two separate plates each. The same was done with a 1% solution of Glucose. Fe was then added to one plate with Na-meta-phosphate, one plate with KH2PO4 and in one of the two plates with Glucose 1% and incubated at 35°C over 24 hours. The plates were the dried at room temperature.

[0319] Na-meta-phosphate 1% + Fe forms specific spheric, red-brown leaf-like structures as well as brown membranes and yellow fluorescent and green vesicles. In contrast, the KFhPC -Fe solution as well as the Glucose 1% solution with Fe remain colorless (Picture), (Picture). Na-meta-phosphate (HPO3) specifically binds with Fe, exerting a red brown to yellow-brown colored leaf-like structure. Na-meta-phosphates react with Fe to a red brown, yellow brown compound. This fact (finding) is used to identify the presence of meta-phosphates in the bone matrix successfully.

[0320] The result of these investigations showed that neither sugar compounds nor KH2- phosphate is the reason for brown colored 2-D and 3-D lobed and spheric objects. Na-meta-phosphates specifically form brown colored 2-D and 3-D objects, which resemble to those seen microscopically in the bone matrix sediments. Glucose incubated with Fe in Ringer's solution dried at air room temperature left colorless white branched crystals, KH2PO4 reacted with Fe to complex but white crystal structures, Glucose-6-phosphate attached to Fe intensively, yet the reaction of the phosphate with Fe caused black spheric structures. A spherical organelle-like structure in the sediments of bone matrix incubated together with Fe metal objects >3 millimeter3

[0321] Fe metal objects >3 millimeter3(Eisenpulver, particles having a size of <90 micrometer; Werth-Metall; CAS-Nummer: 7439-89-6) were used. The above-described procedure was done with Fe-metal objects > 3mm3. After the probes dried at room temperature the bone matrix particles and the metal objects added to the solution are fixed to the plate and to each other by the substrates leaking out of the bone matrix particles during incubation (Figure 14). The metal objects were surrounded by a translucent zone which established between arrowlike extensions of the organelle-like structure and the bone matrix particles (Figures 15 and 16). The content of the translucent zone is strongly connected with the metal object (Figures 17 and 18).

[0322] The organelle-like extensions are organized, arrow-like and directed against the metal objects in a structured way. These patterns of the arrow-like extensions towards the metal object occurred repeatedly and are not random.

[0323] In contrast to the interface of bone matrix derived organelle-like structure incubated with TiC>2 objects, the organelle-like structures were covered with a yellow to brown like membrane. Previously, it was shown that the only substrate leaking the bone matrix, which we were able to stain brown to yellow by incubating the bone matrix with Fe, were meta-phosphates. In the presence of Fe meta-phosphates and related compounds appear specifically brown to yellow. The surface and different structures within the appendices expanding out of the organelle-like structures appeared in this specific brown, yellow color, while the content, meaning the cytoplasm of the organelle-like structure is always translucent. The cytoplasm of the organelle-like structure contains different substrates, one of which is glucose, yet not significant amounts of protein (less than 0,2 micrograms / milliliter) within the first three days (results shown in later parts), while one constituent of the membrane and the cytoskeleton is meta-phosphate, or any meta-phosphate containing compound, which enables meta-phosphates react with Fe. Meta-phosphates easily solve in water or aqueous solutions, as does Fe2+, while Fe3+does not. Yet, Fe3+linked to metaphosphates does solve in aqueous solutions, according to investigations in this matter.

[0324] Bone matrix incubated under physiological conditions provides solvents into the aqueous solution which form organelle-like structures. These structures direct their expansion towards foreign bodies like metal of plastics. The structures are covered by a membrane which contains significant concentrations of meta-phosphorous compounds and sugar. Both substrates are significantly involved in the antigen recognition and fixation process of the extracellular matrix. Phosphorous compounds were stained with Fe yellow to brown. Interactions between the bone matrix and titanium oxide objects > 3 mm3

[0325] Several implants in orthopedic, trauma- and dental medicine are made of TiCh. Consistent data on how metal objects osseo-integrate are not available. General confidence in the use of titanium for osteosynthesis is evidence-based success. Bone matrix and TiCh Objects incubated in any cell-free aqueous solutions were air dried after seven days. The bone matrix leaked solvents into the solution which covered the bottom of the plate. The solvent was well organized in rectangular filamentous structures with long main and short side chains (Figure 19), independent of whether TiO2 objects were added to the solution during incubation.

[0326] The DROPS (Figures 20 and 21): the peripheral ends of the main extensions of the organelle-like structures were always directed either against the wall of the plate, and, if present, against the TiO2 objects. Close to the metal object the thin filaments changed into droplet like structures which bonded with the surface of the TiO2 object (Figure 20). The drop-like structures are main filaments linked to the center of the cross-shaped organelle-like systems either or were side chains from the main filaments. Figure 21 shows a special filament attached to a TiO2 particle.

[0327] The GAP (Figures 17, 18 and 21): Gaps are called translucent zones without the typical geometrical patterns, always surrounding metal objects as soon as the bone matrix solvents reached these metal objects. These gaps are specifically mentioned, because they appear constantly in the bone matrix / metal object interactions.

[0328] Incubating bone matrix under physiological conditions develops organelle-like structures which do always display a center from which main filaments expand to the periphery. From the main filaments side chains develop into the interstitial space. The organelle-like structures appear cross-shaped or starshaped. Towards foreign objects, like metal objects, the main filaments and the side chains develop drop-like shape, covered with a membrane. The membrane stains brown due to HPO3 as a main constituent of the membrane. Swollen filaments have enlarged surfaces which attach to the foreign body metal objects. Chemically, the presence of HPO3 and glucose are significant constituents for the foreign body reactions of attachment. 5. Interactions between different bone matrix particles incubated together in Ringer's solution

[0329] Different bone matrix particles (Figures 23, 24 and 25) incubated together displayed similar processes as seen in the interaction of bone matrix with either Fe-objects or TiO2-objects. The bone matrix particles directed the expanding characteristics against each other, like arrow headed extensions or gaps and never developed other specific characteristics. These processes ended in stable attachments of bone.

[0330] No specific differences between the mechanism of attaching of the bone matrix to metal objects and attaching of particles of the bone matrix to each other can be detected. Molecular constituents of the bone matrix solve into an aqueous environment and expand. The molecular constituents polymerize to organelle-like structures forming a membrane which contains significant concentrations of sugar and HPO3. Both constituents contribute significantly to the interaction of any given bone matrix particle with its environment.

[0331] EXAMPLE 2: THE SUPERNATANT

[0332] 1. pH values during incubation of ECM demineralized with EDTA and HCI

[0333] 0,1-gram bone matrix was incubated in 50 mL different aqueous solutions or distilled water. The initial pH values of all probes were + / - 6 but decreased to values below 5 in transplants demineralized in HCI within the first 24 hours, while increased to values of + / - 7.4 within 24 hours without decreasing into acid conditions when the transplants were decalcified with EDTA (Figure 26).

[0334] From day 1 to day 12, the pH values of supernatants from transplants demineralized with HCI increased slowly from values + / -5 on day 1 to values + / - 7.5on day 12. The supernatants of these transplants reached values above + / - 6.8 only from day 9 onwards, thus remain in an acidic environment for these 9 days. On the other hand, solutions of transplants decalcified with EDTA remained stable with pH values from day 1 to day 12 at values + / - 7.6. No buffers were added to the solutions, yet the pH remained quite stable increasing in all probes to values between 7, 3-7,8, indicating, that buffer agents are intrinsic to the bone matrix. One conjugate acid-base pair in bone matrix is, according to our invention, meta-phosphate (HPO3-) the phosphorous oxo-anion H3PO3 as the conjugate base of phosphonic acid and phosphonate (2-) as the conjugate acid. Decalcifying transplants with EDTA avoids exposure to acids, preventing the danger of acid-catalyzed ester-bond hydrolysis, meta-phosphate (HPO3-), the phosphorous oxo-anion H3PO3, phosphonate (2-) are constituents of an acid-base buffer system contributing to keep the environment within a physiological range. These phosphor compounds are highly soluble in water and quickly responsive the changes in pH. Development of protein concentrations in the supernatant of incubated bone matrix

[0335] Measurements of protein concentrations in the supernatant of transplants incubated in either H2O or aqueous solution displayed continuously increasing protein values from day 1 to day 12 reaching highest values on day 11 with 0.49 mg / mL + / - 0.013 independent of whether the transplants were incubated in distilled water or isotonic solutions, and independent of whether transplants were decalcified with HCI or EDTA (Figure 27).

[0336] In ethanol solution 30% protein measurements remained all the time below 0.15 mg / milliliter, incubating transplants in 0.5M EDTA for 12 days was associated with no increase in protein levels in the supernatant as well (Figure 28) compared to protein levels in H2O. Transplants exposed to strong acids, as NEAA (Non-Essential-Amino- Acids) (Carl Roth) (Figure 28) containing twenty-five milligrams / milliliter of AAs, pH 1.8 over 12 days, absorbed almost all Amino acids from the solution. The protein concentration dropped from 25 mg / mL to 2.03 + / - 0.038 within 24 hours. Then the protein concentration remained stably low until day eight, then values increased sharply again.

[0337] Incubating bone matrix in ethanol 30% keeps pH levels above 8 and proteins at low level, impacting inhibitory on the autonomous metabolism of the bone matrix.

[0338] Due to hydrolysis of bone matrix in physiological conditions protein appears in the supernatant increasingly over 12 days, yet media remain hypo-proteinaceous within the first 3 days of incubation. The bone matrix absorbs up to 25 mg / mL of Amino acids or protein under acid conditions (pH<2). EDTA and Ethanol inhibit metabolic turn-over of the bone matrix. SDS-PAGE of the supernatant

[0339] 0,1-gram bone matrix was incubated in 50 mL water over 12 days. From day zero onwards a 200-microliter probe was taken for SDS-PAGE. Probes were analyzed using two different systems.

[0340] First ThermoFisher NuPAGE Bis-Tris Mini Gels were used. For the samples preparations LDS Sample Buffer (4x) was used and 50 mL 20x NuPAGE MES SDS Running Buffer keeping the pH in a neutral range in 950 mL deionized water. Second, NuPAGE Tris-Acetate Mini Gels were used, NuPAGE LDS Sample Buffer (4x) and Tris- Acetate (20x) was used as running buffer, with a pH at 8,4. Standard silver stain kit was used for detection of the proteins. Bis-Tris buffer performs the SDS-PAGE under neutral conditions, Tris-Acetate buffers maintains a pH environment of 8.4.

[0341] While Bis-Tris SDS-PAGE displayed one protein band over 12 days (Figure 30), SDS- PAGE run under basic conditions displayed increasing bands from day four onwards until a broad range of bands appeared constantly between day 9 and 11 (Figure 31).

[0342] These findings demonstrate that:

[0343] First, any change of the environment around any transplant incubated in any aqueous solution is independent from protein leaking from the transplant into the aqueous environment. Silver staining is a very sensitive staining, displaying protein at levels as low as 0,25 microgram / milliliter. Yet only one such band appeared in all probes from the beginning. Otherwise, enzymes, hormones or cytokines or other protein attached to the bone matrix through ester bonds appear in the supernatant not earlier the on day four of the incubation.

[0344] Second, from day four onwards, increasingly divers protein bands are displayed in the Tris-Acetate SDS-PAGE reaching the exposition of a large range of proteins and protein fragments around day ten. Thus, deviations from a neutral pH during the time of incubation causes ester bond breaks within the bone-matrix associated with the risk of inactivating the transplants with exposing them to any environment outside of neutral conditions. Measurements of the refraction Index of the supernatant

[0345] The refraction index shows a physical property of any given substrate according to the extent the given substrate slows down the speed of light in reference to the speed of light in vacuum. Different substrates were measured with a refractometer listing these substrates according to the Index measured (see the following Table 1).

[0346]

[0347]

[0348] Table 1: This Table displays a series of determined refraction indices of different substrates which either contain salts (KCI, KH2PO4, NaCI, or supernatants of incubated allografts in Ringer's solution, sugar compounds in varying concentrations (glucose 1%, glucose 33%, fructose 1%, galactose 1%), tensides, acid solutions, protein rich solutions, fatty liquids. Salt solutions display specific refraction indices depending on their concentrations. Bone supernatant also displays a very specific number as refraction index, which differs only slightly but significantly from the Ringer's solution used for incubation. This refraction index is a reliable indicator for the ongoing bone diluting process. Investigating transplants and the supernatant according to Rl values in a refractometer is displayed in the following. The cortico-cancellous matrix decalcified with EDTA displays constantly an Rl value of 1,3343. This value stands close to the Rl values of KH2PO4 (1.3340), NaCI 0.9% (1.3343), Ringer's solution Fresenius (1.3344), Na-meta-phosphate (1.3344), F2 in Water 1% (1.3345). Lactose 2% (1.336), Glucose 1% (1.347), EDTA 0.5M / L (1.362), the disinfectant Octenisept (1.365), CaCI (1.371) are slightly but clearly higher, proteinaceous solutions like milk (1.374), soja (1.396) are significantly higher and oil like olive oil distinguishes clearly with Rl values around 1.469.

[0349] The supernatants of all transplants incubated in aqueous solutions display constantly a value of 1.335, which is lower than that of Glucose -6-P 9% (1.3463) and that of Glucose 1% (1.347) but slightly higher than that of HPO3 (1.3344), KH2PO4 (1.3340) solutions.

[0350] Refraction Indices of the bone matrix and the supernatant of incubated bone matrix provide constantly a value of 1,3345. That proves, that no substrates appear in the supernatant within 7 days which impact on the refraction index negatively. That excludes significant concentrations of substrates of protein, lipids, and different groups of sugar compounds. The provided refraction index proves that the content of the supernatant is mainly composed of salts and glucose. Formation of main arms and extensions correlates with the size of the polymerizing transudate

[0351] Supernatant from EDTA-decalcified bone matrix, incubated in an aqueous solution at 35°C, was given in a separate plate and dried in room air. Samples were incubated with Fe addition to the medium (Figure 32) yellow-rimmed leaf-like objects with similar shapes but varied sizes were displayed at lOx magnification. Objects of all sizes displayed a yellow membrane (I) (lower right of the picture). The small objects are round to rectangular with translucent cytoplasm (II) (lower right half of the image). Expansion of the content develop into a lobed configuration, yet the cytoplasmic membrane remains always uninterrupted. The yellow color of the membrane like structure is due to a reaction of HPOsH polymer content reducing Fe3+to Fe2+(OH). Metaphosphoric acids are highly soluble and reduce Fe in the solution. This surface reaction forms a stable yellow band around the HPO3- polymerisates forming translucent leave-like structures. In strongly acid solutions the HPO3- / H2PO3- buffer system exceeds the buffer capacity of the phosphorous system, making these parts of the ECM unreactive towards environmental objects. Polymerized condensates expand geometrically forming lobed extensions with rectangular side branches expanding from a center. The transudate condensate to polymerisates when exceeding the solubility product, forming translucent flat, red brown structures with yellow borders. In the cytoplasm of each transudate, a center is determinable. During the increase in size, extensions expand from the corners of the center. These appendices expand as elongated structures with almost right- angled sprouting side arms. From the peripheral ends of the main and lateral arms, paired, elongated, leaf-like extensions, perpendicular to each other, expand. Figure 33 shows an example of a more developed transudate. It has a definable center from which four main arms expand. These four main arms have a hypotense longitudinal axis, the cytoplasm is milky white, and is surrounded by a yellow membrane. The hypotense longitudinal axes of these main arms expand as peripheral appendices, which are also bounded by membranes stained yellow by Fe but differ from the main arms in that their cytoplasm is transparent and not milky white.

[0352] Incubating bone ECM in an aqueous solution within a physiological range of pH (6.5- 7.5) releases an electrolyte exudate containing HPCh / PCh / Glucose 1%, polymerizing to leave-like organelles. The refraction index of the content is constantly 1.335, excluding significant concentrations of protein, lipoprotein, and glycoprotein. Formation of the organelle like structures does not take place if the pH of the solution exceeds into an acidic or basic range. Structures of the polymerising transudate that can be found by light microscopy

[0353] At magnifications of thirty times or more (Figure 34), the hypotense center can be seen in the cytoplasm, several extensions of different lengths in four directions, which have a hypotense longitudinal axis. In the cytoplasm, a tubulo-reticular network can be seen and membrane-covered vacuoles, such as structures of a cytoskeleton. Figure 35 shows an exemplary section of the interstitial space between main extensions, which are connected by a tubulo-reticular network, and along which, brown-colored vesicles are stored. The expansion

[0354] The transition from the original, smallest possible representable form of the polymerizing transudate to its maximum observable expansion takes place in repeatable patterns and leads to definable manifestations. At the stage of maximum expansion, opposing main arms are formed parallel to the target of the expanding polymerisation of the transudate. Be it because this is the border of the liquid film, or the border of the plate, or an arbitrarily inflicted object (see Figures 36, 37 and 38).

[0355] Two recurring, characteristic features of the polymerisation process of the transudate occur: tubulo-reticular networks sprouting at right angles, in the form of lateral arms, branch strongly, are clearly swollen. The second feature is the "leading edge", a characteristic shape of the surface of the of the polymerizing transudate (Figures 37, 38 and 39). Polymerizing transudates form a 3-dimensional boundary wall at the edge of the expansion

[0356] In a further study, five milliliters of the supernatant were filled into a plate so that the border of the liquid film does not reach the side wall of the plate, so that the liquid itself forms the edge.

[0357] The polymerisation of the transudate in this case occurred in the center of the aqueous medium and expanded centrifugally towards the outer border. About five millimeters before the end of the liquid film, a new center was formed with two thin, long main arms parallel to the border of the liquid. From the new nucleated center, a main arm expands towards the edge of the lateral arms and appendices that sprout from it. From the peripheral end of the main arm expanding appendices, which form a 3-D marginal wall (Figures 36 and 39) shows that the HPCh / Fe stays in high concentration in the main arm and its lateral arms staining it dark brown and passes only in low concentration into the marginal appendices forming the marginal wall. Figure 39 is an enlarged close-up image of the perimeter wall with yellow and green, fluorescent patterns created by Fe staining. The temporal sequence of the expansion of the polymerizing transudate and their occurring forms (is there diversity?)

[0358] The shown Figures are screenshots which were extracted from films documenting the essential stages of development of the organelle-like structures.

[0359] The plate was filmed with 5 mL of supernatant from each bone sample. The drying of the supernatant was filmed. Incubation was conducted in a) without Fe, in b) together with Fe particles having a size of <90 microns. Fe particles having a size of <90 microns stained the distinct structures of the polymerizing transudate and organic sediment yellow green. Pictures from two different investigations a) and b) are shown. a) shows the expansion of a single polymerisate from the center to the edge of a liquid film. The distinctive feature of the polymerizing transudate in a) is the excessive formation of an interstitial tubulo-reticular network. b) shows an example of the early developmental processes of polymerisation in the center of a liquid film.

[0360] What all polymerisates have in common is that they develop characteristic features during size growth and expansion, which can be seen directly or indirectly through the light microscope at 10-100x magnification.

[0361] With the increase in the size of the polymerisates, the curved sides of the rectangle pass into rectilinear ones. The rounded corners become pointed ones. On the sides, intussusceptions are formed, parallel secondary tubules, which merge into cisterns and whose membranes become visible through HPO3(-) / Fe metabolism. As a result of these activities, a tubular compartment is formed peripherally to the central cytoplasm. This tubular compartment is visible by hypotense (black) and yellowgreen colored lines parallel to the lateral edges. The membranes are yellow green in color due to Fe incubation.

[0362] Fluid flows are visible through light reflections. These can be seen not only within the angular boundaries, but also around the outer edges, within the cytoplasmic membrane. The space between the outer edges of the tubular compartment and the cytoplasmic membrane is what we call the peripheral cytoplasm.

[0363] The ECM transudate polymerizes in the elongation of its diagonals. The first signs of this expansion are the exogenous extension of the diagonal, central tubules into the interstitial, moist space. These exogenous expansions can be rectilinear or saccular. During expansion, they always form sub-peripheral and peripheral appendices several centimeters long.

[0364] EXAMPLE 3: THE SUPERNATANT IS CAPABLE OF RECONSTITUTING, HEALING OR REGENERATING BONE SUBSTANCE

[0365] In order to test the capability of the above-described supernatant (i.e., the composition of the present invention obtained by the method of the present invention) with respect to its impact on reconstituting, regenerating and / or healing bone substance, the following study has been performed.

[0366] 60 reconstructive bone augmentation operations of the upper or the lower jaw were carried out, using either non-demineralized bone replacement material, HCI demineralized bone replacement material or EDTA-decalcified bone replacement material.

[0367] Eleven times cortico-cancellous granules, which were not demineralized, were used as bone replacement material, twenty-seven times cortical fibers demineralized with HCI, and 22 times cortical fibers demineralized with EDTA as described above. 11 times two different bone replacement materials were used.

[0368] The obtained supernatant, i.e., the composition of the present invention, was added 11 times to the bone replacement material. Rehydration of the bone replacement material was carried out according to established practice in the field, usually with aqueous solutions. 11 times the supernatant, i.e., the composition of the present invention was used for rehydration.

[0369] A score was established defining radiological visibility according to the bone density of the bone replacement material after the operation. The score "0" was attributed to results when the bone replacement material displayed the same density as soft tissue which is dark grey, "1" was attributed to results when the bone replacement material distinguished positively from soft tissue, yet was clearly less dense than either cortical or cancellous local bone (i.e., the patient's resident, intact bone). "2" was attributed to results as soon as the bone replacement material displayed the same grey as local bone (i.e., the patient's resident, intact bone).

[0370] Non-decalcified bone displayed a score of "2" (high quality bone density) in 100% (8 patients), EDTA-demineralized cortical and cancellous bone displayed a score "2" (high quality bone density) in 95%, corresponding to 18 out of 19 patients. Due to decalcification of the bone replacement material radiological density did not decrease. Cortical fibers demineralized with HCI could be distinguished post- operatively from local bone but this was less pronounced (compared the treatment with the composition of the present invention derived from EDTA-demineralized bone material), yet, detectable from local soft tissue, score "0" very insufficient bone density quality in 19% (3 patients), and only weakly visible score "1" in 56% (9 patients), only 4 out of 16 patients (25%) in this group displayed appropriate results.

[0371] Figures 40 and 41 show a radiograph of a mandible after tooth extraction one month after the operation. The extraction site was filled with HCI demineralized cortical fibers. One month after the operation the bone replacement material could only weakly be recognized, if at all. Figures 42 and 43 are radiograph images one month after tooth extraction, cystectomy, and immediate reconstruction of the lesion with EDTA-demineralized bone replacement material in the upper premolar region. The additive (i.e., the obtained supernatant as described above, i.e., the composition of the present invention) was added as rehydration solution. Arrows indicate the region filled with the one replacement material, which was clearly visible and not different from the local bone of the recipient.

[0372] Figures 44 and 45 are slices of a 3-D radiograph taken on the same day immediately after tooth extraction, cystectomy and filling of the defect side with EDTA- demineralized allogens bone replacement material.

[0373] Bone density in radiographs is a reliable means to estimate bone quality. Cortical structures appear light grey, cancellous bone in darker grey shades compared to cortical structures. Metal and otherforeign bodies appear white. Soft tissue appears dark grey and air appears black.

[0374] Bone replacement material demineralized with HCI appears most likely in dark grey equal to soft tissue, while every bone replacement material decalcified with EDTA appears either in bright white equal to cortical or cancellous bone.

[0375] In summary, the obtained supernatant as described above, i.e., the composition of the present invention, is capable of reconstituting, regenerating and / or healing bone substance, while the composition of the present invention obtained from bone material (also termed bone replacement material above) demineralized with EDTA turned out to have an even more pronounced effect in reconstituting, regenerating and / or healing bone substance compared to the composition of the present invention obtained from bone material (also termed bone replacement material above) demineralized with HCI.

[0376] SUMMARY

[0377] The bone matrix expands significantly under physiological, yet cell-free conditions in vitro. The constantly appearing patterns of expansion indicate underlaying, regulatory, enzymatic mechanisms intrinsic to the bone matrix. Although the underlaying mechanisms are unclear, there are strong indications that enzymatic hydrolysis is the leading mechanisms for this. The expanding substrates are part of the invention and described further below. In the presence of Fe particles having a size of <90 micrometer compounds of the bone matrix react with Fe2+ / 3+to 2-D and 3-D organelle-like spherical objects. The color is specific for compounds of Fe2+ / 3+reacting with bone matrix constituents and does not appear if the bone matrix is incubated in aqueous solutions with any other proved metal particles like TiC>2, ZirC>2, CuC>2, KH2PO4, Glucose-6-PC>4.

[0378] Bone matrix incubated under physiological conditions provides solvents into the aqueous solution which form organelle-like structures. These structures direct their expansion towards foreign bodies like metal of plastics. The structures are covered by a membrane which contains significant concentrations of meta-phosphorous compounds and sugar. Both substrates are significantly involved in the antigen recognition and fixation process of the extracellular matrix. Phosphorous compounds were stained with Fe yellow to brown.

[0379] Incubating bone matrix under physiological conditions develops organelle-like structures which do always display a center from which main filaments expand to the periphery. From the main filaments side chains develop into the interstitial space. The organelle-like structures appear cross-shaped or starshaped. Towards foreign objects, like metal objects, the main filaments and the side chains develop drop-like shape, covered with a membrane. The membrane stains brown due to HPO3 as a main constituent of the membrane. Swollen filaments have enlarged surfaces which attach to the foreign body metal objects. Chemically, the presence of HPO3 and glucose are significant constituents for the foreign body reactions of attachment.

[0380] No specific differences between the mechanism of attaching of the bone matrix to metal objects and attaching of particles of the bone matrix to each other can be detected. Molecular constituents of the bone matrix solve into an aqueous environment and expand. The molecular constituents polymerize to organelle-like structures forming a membrane which contains significant concentrations of sugar and HPO3. Both constituents contribute significantly to the interaction of any given bone matrix particle with its environment.

[0381] Decalcifying transplants with EDTA avoids exposure to acids, preventing the danger of acid-catalyzed ester-bond hydrolysis. Meta-phosphate (HPO3-), the phosphorous oxo-anion H3PO3, phosphonate (2-) are constituents of an acid-base buffer system contributing to keep the environment within a physiological range. These phosphor compounds are highly soluble in water and quickly responsive the changes in pH. Due to hydrolysis of bone matrix in physiological conditions protein appears in the supernatant increasingly over 12 days, yet media remain hypo-proteinaceous within the first 3 days of incubation. The bone matrix absorbs up to 25 mg / mL of Amino acids or protein under acid conditions (pH<2). EDTA and Ethanol inhibit metabolic turnover of the bone matrix.

[0382] These findings demonstrate, that

[0383] 1): any change of the environment around any transplant incubated in any aqueous solution is independent from protein leaking from the transplant into the aqueous environment. Silver staining is a very sensitive staining, displaying protein at levels as low as 0,25 microgram / milliliter. Yet only one such band appeared in all probes from the beginning. Otherwise, enzymes, hormones or cytokines or other protein attached to the bone matrix through ester bonds appear in the supernatant not earlier the on day four of the incubation.

[0384] 2): from day four onwards, increasingly divers protein bands are displayed in the Tris- Acetate SDS-PAGE reaching the exposition of a large range of proteins and protein fragments around day ten. Thus, deviations from a neutral pH during the time of incubation causes ester bond breaks within the bone-matrix associated with the risk of inactivating the transplants with exposing them to any environment outside of neutral conditions.

[0385] Refraction Indices of the bone matrix and the supernatant of incubated bone matrix provides constantly a value of 1,3345. That proves, that no substrates appear in the supernatant within 7 days which impact on the refraction index negatively. That excludes significant concentrations of substrates of protein, lipids, and different groups of sugar compounds. The provided refraction index proves that the content of the supernatant is mainly composed of salts and glucose.

[0386] Incubating bone ECM in an aqueous solution within a physiological range of pH (6.5- 7.5) releases an electrolyte exudate containing HPCh / PCh / Glucose 1%, polymerizing to leave-like organelle-like structures. The refraction index of the content is constantly 1.335, excluding significant concentrations of protein, lipoprotein, and glycoprotein. Formation of the organelle-like structures does not take place if the pH of the solution exceeds into an acidic or basic range.

[0387] The organelle-like structure expands in the elongation of its diagonals. The first signs of this expansion are the exogenous extension of the diagonal, central tubules into the interstitial, moist space. These exogenous expansions can be rectilinear or saccular. During expansion, they always form sub-peripheral and peripheral appendices several centimeters long.

[0388] The expanding parts of the organelle-like structure form rectilinear structures, with leading edges as soon as the expansion is directed against an object larger than 1 millimeter, or implicitly against any object that cannot be phagocytosed by the organelle-like structure because of its size or holds up a surface against it that it perceives as an antigen for chemical, physical reasons.

Claims

CLAIMS1. A method for preparing a composition for supporting the reconstitution or regeneration of bone substance in a patient in need thereof, wherein the method comprises the following steps:(i) decalcification / demineralization of decellularized bone material at a nearly physiological pH value;(ii) fragmentation of the decalcified / demineralized bone material of step (i);(iii) incubation of the fragmented bone material of step (ii) in an aqueous solution at a nearly physiological pH value;(iv) separation of the liquid supernatant from the fragmented bone material / debris; and(v) recovery of the liquid supernatant of step (iv), thereby obtaining a liquid composition for supporting reconstitution or regeneration of bone substance, preferably, wherein said liquid composition comprises electrolytes containing HPO3 / H2PO3 / Glucose and / or Ribose.

2. The method of claim 1, wherein the method comprises the following additional step:(vi) drying the liquid supernatant recovered in step (v) of claim 1, thereby obtaining a dried form of a composition for supporting the reconstitution or regeneration of bone substance.

3. The method of claim 1 or claim 2, wherein the decalcification / demineralization in step (i) is effected by incubating the decellularized bone material with an aqueous solution comprising a chelating agent, preferably, wherein said chelating agent is ethylenediaminetetraacetic acid (EDTA) or EGTA (ethylene glycol-bis(|3-aminoethyl ether) V, / V, / V', / V'-tetraacetic acid).

4. The method of any one of claims 1 to 3, wherein the fragmentation of the decalcified / demineralized bone material in step (ii) is effected by crushing, shredding and / or pulverizing the bone material.

5. The method of any one of claims 1 to 4, wherein the incubation of the fragmented bone material in step (iii) is effected in an isotonic aqueous solution, preferably in Ringer's solution or in a 0.9% NaCI solution.

6. The method of any one of claims 1 to 5, wherein the nearly physiological pH value in step (i) and in step (iii) is a pH in the range of pH 4,5 to pH 8.5, preferably in the range of pH 5.0 to pH 7.6, more preferably in the range of pH 6.7 to pH 7.4, even more preferably in the range of pH 6.5 to pH 7.5, and most preferably a pH of 7.3.

7. The method of any one of claims 1 to 6, wherein the separation in step (iv) is effected by sedimentation or centrifugation.

8. The method of any one of claims 1 to 7, wherein the decellularized bone material in step (i) is allogenic or xenogeneic in relation to the patient.

9. The method of any one of claims 1 to 8, wherein the liquid composition obtained in step (v) or the dried composition obtained in step (vi) is supplemented with metal or metal alloy particles, compounds that support or enhance osteosynthesis, additional bone material, preferably additional allogenic bone material, or pharmaceutically active compounds associated with metabolic diseases.

10. A composition for supporting reconstitution or regeneration of bone substance, obtainable by the method according to any one of claims 1 to 9, preferably, wherein said liquid composition comprises electrolytes containing HPCh / PCh / Glucose and / or Ribose.

11. The composition of claim 10 for use in a method of supporting reconstitution or regeneration of bone substance in a patient.

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