Osseoinductive bone graft preparation from human hard tissues and methods for customizable bone regeneration

Abstract

This invention relates to the preparation of bone grafts from human hard tissues, such as teeth, bone, and cartilage, that retain osseoinductive potential. The grafts are processed through optimized lab procedures to preserve their biological activity, achieve sterilization, and minimize water content for enhanced shelf life. These grafts can be formulated into particulate, block, or dough / pastel - like forms. The particulate matter can be combined with body fluids containing growth factors to create a moldable material for packing into bone defects, promoting superior osteogenesis. Additionally, the particulate matter can be used in 3D printing to produce customized grafts with controlled porosity based on CT scans and computer-aided design (CAD). Block grafts can be machined into precise shapes using CAD models and computer-aided machining (CAM).

Description

[0001] TITLE: OSSEOINDUCTIVE BONE GRAFT PREPARATION FROM HUMAN HARD TISSUES AND METHODS FOR CUSTOMIZABLE BONE REGENERATION

[0002] CROSS REFERENCE

[0003] The patent application claims the priority date benefit of Indian provisional patent application no. 202411042074 filed on May 30, 2024.

[0004] FIELD OF THE INVENTION

[0005] This present invention relates to a novel method for preparation of bone grafts from human hard tissues, such as teeth, bone, and cartilage, in particulate or block form. These grafts retain osseoinductive potential through optimized processing that ensures sterilization and low water content, enhancing shelflife. The disclosed method provides a superior alternative to synthetic and animal-derived grafts, offering improved safety and effectiveness for bone regeneration.

[0006] BACKGROUND OF THE INVENTION

[0007] Osteoconduction, osteoinduction, and osteogenesis are the fundamental biological mechanisms required for successful bone grafting. Osteoconduction involves the formation of a scaffold that allows the intrusion of osteoblasts, undifferentiated mesenchymal cells, and blood vessels. Osteoinduction is the active stimulation of osteoprogenitor cells to differentiate into osteoblasts, while osteogenesis involves the housing of osteoblasts within the graft material, promoting new bone formation.

[0008] Generally, bone grafts can be classified into different types depending upon the source from where they are derived, such as:

[0009] Autografts: Bone harvested from the patient's own body.

[0010] Allografts: Bone taken from a donor of the same species (typically cadaveric).

[0011] Xenografts: Bone taken from a different species.

[0012] Synthetic grafts: Manufactured materials that mimic the properties of natural bone. Each type has significant limitations. Alloplasts and xenografts, despite their widespread use, often lack the biological activity necessary for optimal bone regeneration and carry risks of disease transmission and immune reactions.

[0013] Historically, autogenous bone grafts have been considered the "gold standard" among bone grafting materials due to their possession of all three properties: osteoconduction, osteoinduction, and osteogenesis. However, they have limitations such as limited availability, the need for additional surgery, donor site morbidity, increased surgery duration, intraoperative blood loss, pain, and extended recovery time.

[0014] Demineralized freeze-dried bone allograft (DFDBA) is one of the top alternatives to autogenous bone. It offers advantages such as availability in adequate quantities, predictability, and the elimination of the need for additional surgery. DFDBA's edge over other alternatives lies in its osteoinductive potential, which is attributed to the exposure of growth factors like bone morphogenetic proteins (BMPs) during the demineralization process. Despite compromises in mechanical stability and resorption time, its space-maintaining ability can be enhanced by combining it with other materials.

[0015] Some of patent literature discussed herein are PCT patent publication no. WO 2012061024 Al discloses, Implants comprising a plurality of separate cortical bone units, which have been at least partially demineralized and are osteoinductive. US patent publication no. US 20080154379 Al discloses, sterile composite bone graft for use in implants comprising a central member constructed of biocompatible plastic with two end caps of cortical bone mated to opposite ends of the central member. US patent no. US 9999520 B2 discloses osteoimplant which comprises a coherent aggregate of elongate bone particles, the osteoimplant possessing predetermined dimensions and shape.

[0016] In 2019, approximately 16% of bone augmentation procedures in the United States used DFDBA as a grafting material, and this number is expected to rise with increased awareness and the proliferation of tissue banks. DFDBA finds extensive applications in orthopedics, dentistry, and traumatology, including periodontal tissue reconstruction, socket preservation, sinus elevation procedures, and other bone augmentation processes for implant placement.

[0017] DFDBA is derived from bone harvested from genetically dissimilar human donors, which is then cleaned, processed, and sterilized for use as grafting material. Despite its advantages, DFDBA faces challenges such as immune reactions, disease transmission, and graft contamination. These challenges are mitigated through stringent donor screening protocols, removal of cellular and fatty contents, and ensuring the graft is free of antigens responsible for immune reactions. Disease transmission is controlled by comprehensive donor medical, behavioral, and transmission history screening, as well as hematological screening for diseases like HIV, HBV, HCV, and syphilis.

[0018] Microbial contamination and enzymatic degradation of the graft may be prevented by removing or immobilizing free water content, which is crucial for their activity. Techniques such as ultrafreezing the bone below -40°C or lyophilization (removal of free water under vacuum conditions by sublimation) enable safe room temperature storage.

[0019] So, the processing methods can affect the osteoinductive potential of DFDB A, however there are limited disclosure on such processing methods which may have some actual positive impact. Therefore, due to lack of any such disclosure the understanding of such processing means is scarce.

[0020] The inventors in the present invention propose a solution to the above problem by providing an improved method for rapid processing of bone allografts without compromising their quality.

[0021] SUMMARY OF THE INVENTION

[0022] This present invention relates to the preparation of bone grafts from human hard tissues such as teeth, bone, and cartilage. These grafts can be in particulate or block form and possess osseoinductive potential, unlike synthetic materials or those of animal origin that merely act as scaffolds and carry a risk of transmitting animal diseases. The osseoinductive potential of these human tissues is preserved through optimized laboratory tissue processing procedures. This processing retains the osseoinductive properties, attenuates the tissue, and achieves sterilization to prevent disease transmission. Furthermore, the process ensures a low remnant water content, enhancing the shelf life of the graft.

[0023] The present solution offers allografts derived from human hard tissues, processed to retain osseoinductive potential. These grafts are formulated into a moldable dough using blood products with growth factors, or customized using CADCAM technology for precise defect repair. Embodiments of the present invention disclosure pertains to method for preparing a biologically active bone graft composition from human hard tissues, comprising the following steps:

[0024] 1. Collection of Human Hard Tissue: Obtaining human hard tissues such as extracted teeth, cortical / cancellous bone fragments, or cartilage from ethically sourced donors. The tissues may be autologous, allogeneic, or cadaveric in origin, screened and cleared for transmissible diseases as per medical standards.

[0025] 2. Cleaning and Initial Processing: Mechanically cleaning the obtained tissues to remove visible soft tissue residues, blood, and debris. Optionally soaking the tissues in an antiseptic or antibiotic solution to reduce initial bioburden.

[0026] 3. Demineralization and Decellularization: Subjecting the tissues to a controlled demineralization process using dilute acid solutions to expose bone morphogenetic proteins (BMPs) and enhance osteoinductive properties. Following demineralization, treating the tissues with enzymatic or other agents to remove cellular and fatty components while preserving the extracellular matrix (ECM).

[0027] 4. Drying and Size Reduction: Drying the processed tissues using lyophilization (freeze- drying) or vacuum drying to remove residual moisture. Milling or grinding the dried tissue into desired particle sizes (for powder, or into larger chunks for blocks), depending on intended clinical application.

[0028] 5. Sterilization: Sterilizing the processed graft material using gamma irradiation, ethylene oxide, or other suitable sterilization techniques to ensure asepsis without compromising biological activity.

[0029] 6. Formulation into Desired Product Form: o For powder or particulates: Packaging the sterilized material as ready-to-use graft powder for mixing with blood or other carriers. o For moldable dough: Mixing the sterile particulate with a biocompatible carrier such as platelet-rich plasma (PRP), fibrin glue, or autologous serum to form a pliable, moldable graft. o For blocks: Compacting the particulate into solid block form via compression molding or casting, followed by machining to desired dimensions. o For customized / 3D printed grafts: Combining the processed particulate with a printable biopolymer or adhesive and printing the graft based on patient-specific 3D imaging data.

[0030] 7. Packaging and Storage: Packing the final product in sterile containers, with appropriate labelling and storage instructions. The product may be stored in a lyophilized state, as a pre-mixed dough under refrigeration, or as a sterile, ready -to-print paste depending on form.

[0031] In one aspect the present disclosure provides a novel bone graft composition comprising of: a) Particulate matter derived from human hard tissues selected from teeth, bone, and cartilage; b) The particulate matter processed to preserve osseoinductive potential through demineralization, removal of cellular and fatty components, and sterilization; c) A carrier containing body fluids with growth factors, forming a moldable dough-like material suitable for packing into bone defects or augmentation sites.

[0032] In some aspects these approaches enhance clinical application, improve biological integration, and reduce patient morbidity, making the grafts versatile and effective for use in dental, orthopedic, plastic, neurosurgical, and ENT surgeries. The invention addresses limitations of existing graft materials, providing a superior, biologically active, and customizable solution for bone regeneration.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The invention will be better understood and objects other than those set forth above will become apparent when consideration is given to the following description thereof. Such description makes references to the annexed drawings wherein:

[0035] Figure.!: Shows pictorial images of particulate or block form of bone graft formed from human hard tissue.

[0036] Figure. 2: Shows grafting between sinus membrane and bony floor to augment a pneumatized maxillary antrum to place commercially pure titanium implants made in Banaras Hindu University. DETAILED DESCRIPTION OF THE INVENTION

[0037] The following description is merely exemplary in nature and is not intended to limit the enumerated embodiments or the application and uses of the embodiments. This description is not intended to be a detailed catalogue of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the scope of the instant invention.

[0038] The terms “for example” and “such as,” and grammatical equivalences thereof, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise.

[0039] As used herein, the term “about” is meant to account for variations due to any experimental errors which may be commonly accepted in the field. All measurements reported herein are understood to be modified by the term “about,” whether or not the term is explicitly used, unless explicitly stated otherwise. Further for the purposes of the present invention, ranges may be expressed as from “about” one particular value to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value to the other particular value. The recitation of numerical ranges by endpoints includes all the numeric values subsumed within that range.

[0040] As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials are described herein for use in the present disclosure; other suitable methods and materials known in the art can also be used. The materials, methods and examples are illustrative only and not intended to be limiting by any means. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict, the present specification, including definitions, will control.

[0042] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.

[0043] The term “including” is used to mean “including but not limited to” “Including” and “including but not limited to” are used interchangeably.

[0044] The present invention relates to a method for preparing a bone graft from human teeth, bone, and cartilage, involving demineralization to expose BMPs, removal of cellular components, sterilization, and lyophilization to reduce water content.

[0045] In one embodiment the present invention provides a bone graft composition comprising human-derived hard tissue in particulate, block, or moldable paste form, with preserved osseoinductive potential, optimized for enhanced shelf life and sterility.

[0046] In yet another embodiment the present invention provides a clinical application method using the bone graft for dental surgery, where the graft is applied to alveolar ridge defects, sinus lifts, or periodontal repairs, demonstrating superior osseoinductive and osteoconductive properties.

[0047] In certain embodiments according to the present invention the disclosure by use of sterilization techniques such as irradiation or ethylene oxide gas ensures the bone graft is free from pathogens, making it safe for transplantation in genetically non-identical individuals.

[0048] In some embodiments the present invention provides an improved bone graft product with low remnant water content of about 2-4%, achieved through freeze-drying, offering extended shelf life and stability for use in orthopedic, plastic, neurosurgery, and ENT procedures.

[0049] In one aspect of the above embodiment the bone grafts are derived from human hard tissues such as teeth, bone, and cartilage, which can be sourced from cadaveric donors or living donors through surgeries like tooth extractions.

[0050] In some embodiments according to the present invention grafts undergoes optimized lab processing procedures that preserve osseoinductive potential, such procedures include: exposure of growth factors such as bone morphogenetic proteins (BMPs);

[0051] - removal of cellular and fatty components to minimize immune reactions;

[0052] - use of techniques such as irradiation, ethylene oxide gas, or sterile processing to ensure the grafts are free from contaminants and pathogens; and formulation of the grafts into various forms to suit different clinical needs. In one aspect of the above embodiment formulation of the grafts into various forms to suit different clinical needs is selected from:

[0053] Particulate Form: For filling irregular bone defects.

[0054] Block Form: For structural support in larger defects.

[0055] Pastel or Dough Form: Moldable to fit specific defect shapes and sizes, enhancing ease of application.

[0056] In yet another aspect of the above embodiment the said processed grafts have a low remnant water content to improve shelf life and stability. Techniques such as freeze-drying (lyophilization) may be used to achieve this.

[0057] In some embodiments the present invention relates to a bone graft composition comprising particulate matter derived from human hard tissues such as teeth, bone, and cartilage. This particulate matter is processed to preserve its osseoinductive potential and is mixed with body fluids containing growth factors to form a dough / pastel like material.

[0058] In one aspect of the above embodiment the said osseoinductive dough / pastel can be packed into bone defects or augmentation sites to promote replacement osteogenesis, resulting in the formation of higher quality bone due to the combined effects of osseoinductive potential and growth factors.

[0059] In another embodiment according to the present invention the disclosure provides a method for creating customized bone grafts using particulate matter derived from human hard tissues. The particulate matter, prepared to retain osseoinductive potential, is used in 3D printing with a suitable adhesive material to create grafts with controlled porosity.

[0060] In one related aspect of the above embodiment the customized grafts are designed based on CT scans and computer-aided design (CAD), and are subsequently sterilized and fixed at the defect or augmentation site using screws. This method allows for precise repair and augmentation tailored to individual patient anatomy.

[0061] In a further embodiment according to the present invention the method for preparing bone graft blocks with osseoinductive potential from human hard tissues. The process involves obtaining CT scans of the defect site, creating a 3D model of the defect and the graft using CAD, and machining the prescribed grafts from the osseoinductive blocks using computer-aided machining (CAM). In one aspect of the above embodiment the said customized grafts is sterilized and fixed at the defect or augmentation site using screws. This approach ensures a precise fit and enhanced biological integration due to the osseoinductive properties of the graft.

[0062] In one aspect the aim of the present study is to histologically evaluate bone formation in extraction sockets following an 8-week socket preservation procedure, ensuring the processed graft maintains high osseoinductive potential and is biocompatible, safe, and effective for clinical use.

[0063] Embodiments of the present invention disclosure pertains to method for preparing a biologically active bone graft composition from human hard tissues, comprising the following steps:

[0064] 1. Collection of Human Hard Tissue:

[0065] Obtaining human hard tissues such as extracted teeth, cortical / cancellous bone fragments, or cartilage from ethically sourced donors.

[0066] The tissues may be autologous, allogeneic, or cadaveric in origin, screened and cleared for transmissible diseases as per medical standards.

[0067] 2. Cleaning and Initial Processing:

[0068] - Mechanically cleaning the obtained tissues to remove visible soft tissue residues, blood, and debris.

[0069] Optionally soaking the tissues in an antiseptic or antibiotic solution to reduce initial bioburden.

[0070] 3. Demineralization and Decellularization:

[0071] Subjecting the tissues to a controlled demineralization process using dilute acid solutions (e.g., hydrochloric or EDTA) to expose bone morphogenetic proteins (BMPs) and enhance osteoinductive properties.

[0072] - Following demineralization, treating the tissues with enzymatic or chemical agents (e.g., detergents, alcohols) to remove cellular and fatty components while preserving the extracellular matrix (ECM).

[0073] 4. Drying and Size Reduction: Drying the processed tissues using lyophilization (freeze-drying) or vacuum drying to remove residual moisture.

[0074] Milling or grinding the dried tissue into desired particle sizes (e.g., 100-500 microns for powder, or into larger chunks for blocks), depending on intended clinical application.

[0075] 5. Sterilization:

[0076] Sterilizing the processed graft material using gamma irradiation, ethylene oxide, or other suitable sterilization techniques to ensure asepsis without compromising biological activity.

[0077] 6. Formulation into Desired Product Form: i. For powder or particulates: Packaging the sterilized material as ready-to-use graft powder for mixing with blood or other carriers. ii. For moldable dough: Mixing the sterile particulate with a biocompatible carrier such as platelet-rich plasma (PRP), fibrin glue, or autologous serum to form a pliable, moldable graft. iii. For blocks: Compacting the particulate into solid block form via compression molding or casting, followed by machining to desired dimensions. iv. For customized / 3D printed grafts: Combining the processed particulate with a printable biopolymer or adhesive and printing the graft based on patient-specific 3D imaging data.

[0078] 7. Packaging and Storage:

[0079] - Packing the final product in sterile containers, with appropriate labelling and storage instructions.

[0080] The product may be stored in a lyophilized state, as a pre-mixed dough under refrigeration, or as a sterile, ready -to-print paste depending on form.

[0081] In one aspect of the above embodiment, some of the additional components which can be incorporated in the process may include:

[0082] Addition of growth factors (e.g., BMP-2, VEGF), antibiotics, or bioactive ceramics (e.g., hydroxyapatite, P-TCP) to improve regenerative outcomes. - Use of biodegradable binding agents or structural supports for load-bearing applications.

[0083] In certain embodiments according to the present invention the grafts as disclosed herein can be used for wide range of clinical applications including:

[0084] Dental Surgery: For alveolar ridge augmentation, sinus lifts, and periodontal defects.

[0085] Orthopedic Surgery: For spinal fusions, fracture repairs, and bone defect reconstructions.

[0086] Plastic and Reconstructive Surgery: For craniofacial reconstructions and cosmetic enhancements.

[0087] Neurosurgery: For spinal and cranial defect repairs.

[0088] ENT Surgery: For structural repairs within the ear, nose, and throat regions.

[0089] Some of the advantages of the presently disclosed processed grafts may be enumerated as:

[0090] - Eliminates the need for additional surgery to harvest autograft material.

[0091] - Provides an abundant source of graft material, suitable for large-scale bone defect repairs.

[0092] - Enhances the surgeon's ability to perform various reconstructive procedures without the limitations of autograft availability.

[0093] - Ensures the grafts are free from transmissible diseases through stringent donor screening and robust sterilization methods.

[0094] These grafts are more cost-effective compared to conventional autografts, reducing overall healthcare costs and making advanced surgical procedures more accessible.

[0095] Certain Exemplary embodiments of the present invention may be enumerated as:

[0096] A bone graft composition comprising particulate matter derived from human hard tissues selected from the group consisting of teeth, bone, and cartilage, wherein the particulate matter is processed to preserve its osseoinductive potential and mixed with body fluids containing growth factors to form a dough-like material suitable for packing into bone defects or augmentation sites. In one aspect of the above embodiment the wherein the processing of the particulate matter includes demineralization to expose bone morphogenetic proteins (BMPs), removal of cellular and fatty components, and sterilization.

[0097] In one embodiment the disclosure provides a method for creating a bone graft comprising: a) Obtaining particulate matter from human hard tissues; (as shown in figure -1) b) Processing the particulate matter to preserve osseoinductive potential; c) Mixing the processed particulate matter with body fluids containing growth factors to form a dough-like material; d) Applying the dough-like material to a bone defect or augmentation site.

[0098] In another embodiment the disclosure provides a method for preparing a customized bone graft comprising: a) Processing particulate matter derived from human hard tissues to preserve its osseoinductive potential; b) Using the processed particulate matter in a 3D printing process with a suitable adhesive material to create a graft with controlled porosity; c) Designing the graft based on CT scans and computer-aided design (CAD); d) Sterilizing the graft; e) Fixing the graft at the defect or augmentation site using screws, (see at fig. 2)

[0099] In one aspect of the said embodiment in the method the adhesive material used in the 3D printing process is biocompatible and facilitates integration of the graft with the host tissue.

[0100] In a further embodiment the method for preparing a bone graft block according to the present invention comprises: a) Processing human hard tissues to preserve osseoinductive potential; b) Forming blocks from the processed tissues; c) Creating a 3D model of the defect site using CT scans and computer-aided design (CAD); d) Machining the bone graft block to match the 3D model; e) Sterilizing the machined bone graft block; f) Fixing the bone graft block at the defect site using screws. In one aspect of the above embodiment the said bone graft blocks the processing of human hard tissues including demineralization to expose growth factors, removal of cellular components, and sterilization.

[0101] In one embodiment the present invention provides a bone graft product comprising: a) Particulate matter derived from human hard tissues processed to preserve osseoinductive potential; b) A carrier containing body fluids with growth factors; c) A form selected from the group consisting of dough-like material, 3D printed structures, and machined blocks, each tailored for specific bone defect or augmentation applications.

[0102] In one aspect of the above embodiment in the bone graft product the particulate matter is freeze- dried to reduce water content and enhance shelf life.

[0103] In some embodiments the present invention provides a method for enhancing bone regeneration comprising: a) Preparing a bone graft from human hard tissues through a process that includes demineralization, removal of cellular components, and sterilization; b) Forming the graft into a suitable form such as particulate, block, or dough-like material; c) Applying the graft to a bone defect or augmentation site; d) Ensuring the graft retains osseoinductive potential to promote osteogenesis and integration with host bone tissue.

[0104] In one embodiment the disclosure provides a method for preparing a moldable bone graft dough comprising: a) Processing particulate matter derived from human hard tissues to retain osseoinductive potential; b) Mixing the processed particulate matter with a blood product containing growth factors to form a dough-like consistency; c) Molding the dough-like material into a desired shape for application to a bone defect or augmentation site; d) Ensuring the dough-like graft promotes replacement osteogenesis and integrates with the host bone tissue. In one aspect of the above embodiment in the method the blood product is autologous blood, platelet-rich plasma (PRP), or any other suitable source of growth factors that enhance the osseoinductive properties of the graft.

[0105] In another embodiment the invention relates to a bone graft dough composition comprising:

[0106] 1. Particulate matter derived from human hard tissues processed to preserve osseoinductive potential;

[0107] 2. A blood product containing growth factors mixed with the particulate matter to form a moldable dough;

[0108] 3. The resulting dough being moldable into various shapes for application to bone defects, facilitating replacement osteogenesis.

[0109] In yet another embodiment the invention provides a method for creating customized bone grafts using computer-aided design and machining (CADCAM), comprising: a) Obtaining CT scans of the bone defect or augmentation site; b) Creating a 3D model of the defect and the graft using computer-aided design (CAD); c) Processing blocks of human hard tissue to preserve osseoinductive potential; d) Using computer-aided machining (CAM) to machine the processed blocks into customized grafts based on the 3D model; e) Sterilizing the customized grafts and fixing them at the defect or augmentation site using surgical screws; f) Promoting replacement osteogenesis to repair the defect.

[0110] In an aspect of the above embodiment in the said method the customized grafts are designed to have controlled porosity and specific structural features that enhance their integration with the host bone tissue.

[0111] In yet another related aspect of the above embodiments the bone graft product prepared using the CADCAM method, wherein the product comprises: a) Human hard tissue blocks processed to retain osseoinductive potential; b) Custom-machined by CADCAM for specific sites or to fit specific defect sites with 3D printed grafts by using particulate and adhesive fluids; c) Sterilized and designed for fixation with surgical screws; d) Promoting enhanced osseoinduction and osteoconduction at the application site.

[0112] In one embodiment the present invention provides a moldable bone graft dough composition, comprising:

[0113] Particulate matter derived from human hard tissues selected from the group consisting of teeth, bone, and cartilage;

[0114] The particulate matter being processed through a standardized protocol involving demineralization, removal of cellular and fatty components, lyophilization, and sterilization to retain osseoinductive potential;

[0115] A carrier comprising autologous or allogeneic blood products, such as platelet-rich plasma (PRP) or plasma fractions rich in growth factors;

[0116] The carrier and particulate matter together forming a dough-like, pliable material that is moldable to the shape of the defect site.

[0117] In another embodiment the present invention provides a method of preparing the bone graft dough, comprising the steps of:

[0118] Collecting human hard tissues such as extracted teeth, bone segments, or cartilage, from screened donors;

[0119] Processing the tissues through cleaning, demineralization, removal of cellular material, lyophilization, and sterilization (e.g., gamma irradiation or ethylene oxide treatment);

[0120] Grinding the processed material into fine particulate matter;

[0121] Mixing the particulate with autologous PRP or serum to form a homogeneous, moldable dough ready for immediate clinical use.

[0122] In yet another embodiment the present invention provides customized bone grafts via 3D printing, comprising:

[0123] Using processed human hard tissue particulates mixed with a biocompatible binder or adhesive suitable for 3D printing;

[0124] Designing the shape and structure of the graft based on CT or CBCT scans of the recipient site; Printing the graft with controlled porosity and dimensions, optimizing it for vascularization and cellular ingrowth;

[0125] Sterilizing the final printed graft using appropriate means and securing it at the defect site with surgical screws or plates.

[0126] In a further embodiment the present invention provides a machined bone blocks, comprising:

[0127] Fabricating solid blocks from processed human bone or teeth;

[0128] Designing the reconstruction site using computer-aided design (CAD) based on imaging data;

[0129] Machining the block using computer-aided manufacturing (CAM) tools (such as CNC milling);

[0130] Post-machining sterilization of the graft block and fixation at the target site using mechanical fixation tools like screws or mini plates.

[0131] In yet another embodiment the present invention provides a bone graft product comprising:

[0132] Human hard tissue processed into blocks, dough, or 3D-printed scaffolds;

[0133] Preserving osseoinductive and osteoconductive properties;

[0134] Designed for site-specific implantation via moldable application or pre-shaped CADCAM adaptation;

[0135] Facilitating replacement osteogenesis, where host bone gradually remodels and replaces the graft over time.

[0136] In another embodiment the present invention provides a method for enhancing bone regeneration in clinical procedures, comprising:

[0137] Selecting a graft form (dough, block, or printed scaffold) based on the clinical requirement;

[0138] Ensuring the graft material retains biological potential to induce bone regeneration;

[0139] Fixing the graft at the site of bone trauma, congenital defect, tumor resection, or reconstructive need; Supporting integration through osteoconduction and osseoinduction, with minimal donor site morbidity.

[0140] The processed grafts of the present invention are suitable for use in a wide range of surgical specializations, including:

[0141] Dental surgery (e.g., ridge augmentation, socket preservation);

[0142] Orthopedic surgery (e.g., long bone defects, j oint reconstruction);

[0143] Neurosurgery (e.g., cranial defects);

[0144] Plastic and reconstructive surgery (e.g., facial bone repair);

[0145] ENT surgery (e.g., mastoid reconstruction or sinus lift procedures).

[0146] In some embodiment according to the present invention the bone grafts may be formulated into different physical forms based on clinical needs, including:

[0147] Fine particles for sinus lift or socket grafting;

[0148] Putty or dough for irregular defects;

[0149] Blocks for load-bearing applications;

[0150] 3D-printed scaffolds for highly contoured anatomical reconstructions.

[0151] In some aspects of the above embodiment the dough formulation for preparation of the graft is further optimized with respect to carrier fluid and ratios of the ingredients. In one related aspect the carrier fluid may comprise platelet-rich plasma (PRP), fibrin glue, or serum, optionally enriched with bone morphogenetic proteins (BMPs) or other cytokines In yet another related aspect the ratio of particulate matter to carrier is optimized to provide maximum handling ease while maintaining structural integrity and biological activity.

[0152] Further according to various embodiments of the present invention sterilization and storage requirement for the processed grafts may be:

[0153] Sterilized via gamma irradiation, ethylene oxide, or supercritical CO2;

[0154] Packaged in sterile conditions and stored in lyophilized or frozen forms, depending on the form factor (e.g., frozen for dough, lyophilized for block or powder); Rehydrated with autologous fluids at the time of surgery, ensuring optimal performance.

[0155] In a related aspect the graft is also stable at room temperature.

[0156] In one embodiment, the bone graft composition as disclosed and prepared according to the present invention is used in the treatment of traumatic long bone defects, such as femoral or tibial fractures, by packing the dough-like bone graft material into the defect site after debridement. The composition promotes bone regeneration through the retained osseoinductive potential of the processed human hard tissues.

[0157] In another embodiment, the lyophilized particulate bone graft composition as disclosed and prepared in the present invention is rehydrated with platelet-rich plasma (PRP) and applied to alveolar bone defects resulting from tooth extraction or periodontal disease. The processed human tissue composition aids in bone regeneration and preparation of the jaw for future dental implant placement.

[0158] A further embodiment involves the use of a 3D-printed bone graft block according to the present invention, derived from demineralized and sterilized human teeth or bone, in reconstructive neurosurgery. The graft block is customized using CAD models from CT scans and fixed in place using biocompatible screws. This facilitates cranial vault reconstruction following trauma or tumor resection.

[0159] In yet another embodiment, the graft composition as disclosed in the present invention is used in paediatric patients with congenital bone deformities. The use of human-derived lyophilized graft ensures biocompatibility, while low immunogenicity and high osseoinductive activity support long-term integration and remodelling during growth.

[0160] In an embodiment, the bone graft composition according to the present invention is applied in treating benign cystic lesions (e.g., aneurysmal bone cysts or simple bone cysts) by curettage of the cyst cavity followed by packing with rehydrated, paste-form graft. This approach promotes ossification within the lesion site, minimizing recurrence.

[0161] The present invention provides means for the use of a lyophilized bone graft composition comprising demineralized human hard tissues in promoting bone regeneration in orthopaedic, dental, and craniofacial surgical applications. In some embodiments the present invention also relates to the use of processed and lyophilized human hard tissue in fabricating customized bone graft blocks for surgical implantation using additive manufacturing (3D printing) or subtractive machining methods guided by CT imaging data.

[0162] In one preferred embodiment the present invention further provides the use of freeze-dried bone graft material for preparation of a shelf-stable graft product that retains osseoinductive proteins, suitable for storage at ambient temperature for 3 to 5 years, eliminating the need for cold chain logistics.

[0163] In some embodiments according to the present invention, the composition as disclosed and prepared in the present invention is used for alveolar ridge augmentation prior to dental implant placement, where the lyophilized particulate is reconstituted with bone marrow aspirate or saline and applied at the site of planned prosthesis insertion.

[0164] The invention includes use of the bone graft composition in spinal fusion surgeries, where the dough-like graft is placed between vertebrae to promote osseous bridging and mechanical stability, especially in lumbar and cervical spine interventions.

[0165] The invention further provides for the use of an advanced fabrication process integrating computer-aided design (CAD), computer-aided manufacturing (CAM), and three-dimensional (3D) bioprinting techniques for the preparation of patient-specific bone grafts derived from human hard tissue sources such as teeth, bone, and cartilage. This integrated approach allows for the generation of grafts with precise geometrical conformity to the defect site, tailored internal porosity, and controlled surface topography. The process enables the inclusion of anatomical and mechanical parameters directly from medical imaging data (e.g., CT scans), facilitating superior fit, fixation, and osteointegration. Furthermore, the resulting grafts are characteristically processed to retain osseoinductive potential while maintaining minimal residual moisture content, thereby offering enhanced shelf stability and suitability for a wide range of orthopaedic, maxillofacial, and dental applications.

[0166] In some embodiments the invention provides for the use of a process combining 3D bioprinting and CAD-assisted modelling to fabricate bone grafts with controlled micro-architecture that mimics cancellous bone. This includes the deposition of demineralized and lyophilized hard tissue particulates within a printable biocompatible matrix to form interconnected porous structures optimized for cell infiltration, nutrient diffusion, and vascularization upon implantation. In one embodiment the present invention also includes the use of a CAM-guided subtractive machining process for producing dense cortical-like graft blocks from pre-processed human hard tissues. The CAM tooling system operates based on a CAD file derived from patient imaging data to achieve exact defect-matching geometry. The process ensures highly accurate dimensions and minimal micro-particulate shedding, making the graft ideal for load-bearing defect reconstruction.

[0167] In some embodiment the invention further provides for the use of a hybrid process wherein CAD-generated anatomical models are first 3D printed using a bio-paste composed of lyophilized tissue particles, and then post-processed through CAM finishing operations to enhance structural integrity and interface compatibility. This two-stage fabrication process enables both internal architectural control and external precision finishing in a unified workflow, delivering grafts with functional and aesthetic fidelity to native bone.

[0168] The aforesaid description is enabled to capture the nature of the invention. It is to be noted, however, that the aforesaid description illustrates only typical embodiments of this invention and are therefore not to be considered limiting of its scope.

[0169] EXAMPLES:

[0170] The following example includes only exemplary embodiments to illustrate the practice of this disclosure. It will be evident to those skilled in the art that the disclosure is not limited to the details of the following illustrative examples and that the present disclosure may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive.

[0171] Example 1: Preparation of DFDBA Bone Graft with Optimized Porosity

[0172] Materials and Methods: Cancellous bone segments were harvested from five human donors after obtaining written consent and following APASTB guidelines. All donors were screened twice for HIV, HBV, HCV, and syphilis, 180 days apart to mitigate the window period-related false negatives.

[0173] 1. Initial Processing and Cleaning: a) The harvested bone was washed with sterile phosphate-buffered saline (PBS) to remove visible blood and soft tissue. b) Defatting was performed using 70% ethanol, followed by sequential washes in sterile distilled water. c) Swabs from medullary canals were cultured to confirm absence of microbial growth.

[0174] 2. Demineralization: a) The bone chips were submerged in 0.6 N HC1 at 4°C for 2-4 hours to dissolve mineral content and expose BMPs. b) The acidic medium ensured microbial inactivation and enhanced porosity. c) Chips were then rinsed with sterile water until neutral pH was achieved.

[0175] 3. Freeze-Drying: a) Demineralized bone chips were rapidly frozen at -80°C. b) The samples were transferred to a freeze-dryer under vacuum (<0.1 mbar) for 24-36 hours to allow sublimation of water. c) Residual moisture content was determined gravimetrically and found to be within 2-4 wt%, suitable for long-term storage as compared to conventional grafts.

[0176] 4. Porosity Optimization: a) A proprietary sieving and sorting protocol was used to select particles with pore sizes predominantly in the 150-400 pm range, enabling both osteoconduction and vascular infiltration. b) Microporosity (<10 pm) was also characterized via mercury intrusion porosimetry to assess potential for protein retention.

[0177] 5. Packaging and Sterilization: a) Final products (Demineralized freeze-dried bone allograft (DFDBA)) were packed under sterile conditions and gamma-irradiated at 25 kGy to ensure sterility.

[0178] Example 2: Scanning Electron Microscopy (SEM) for Surface Morphology Analysis SEM analysis was conducted on processed DFDBA particles to evaluate trabecular integrity and pore characteristics.

[0179] The images revealed: preserved trabecular architecture post-demineralization.

[0180] - Enhanced surface roughness was evident, contributing to osteoblast attachment.

[0181] Interconnected macropores and micropores were observed, facilitating nutrient exchange and cellular migration.

[0182] - BMPs embedded in the exposed matrix were visualized, supporting the osteoinductive nature of the graft.

[0183] Example 3: Histomorphometric Evaluation of Bone Regeneration Post-Ridge Preservation

[0184] Procedure: Five human patients undergoing ridge preservation post-extraction were implanted with DFDBAfrom the prepared batches. Biopsy samples were harvested after 12-16 weeks for analysis.

[0185] Results: Table-1

[0186] Observation The histomorphometric data confirmed substantial new bone formation.

[0187] Connective tissue levels and graft resorption were within expected ranges, suggesting effective osteoconductive and osteoinductive behavior.

[0188] Example 7: Comparative study for preparation method for Bone Graft from Human Teeth:

[0189] 1. Collection: Extracted human teeth are collected from verified donors.

[0190] 2. Cleaning: Teeth are cleaned to remove debris and soft tissue. 3. Demineralization: Teeth are soaked in an acidic solution to remove minerals and expose bone morphogenetic proteins (BMPs).

[0191] 4. Decellularization: Cellular components are removed using enzymatic digestion and detergent washes.

[0192] 5. Sterilization: Material is sterilized using gamma irradiation.

[0193] Comparison step:

[0194] (a) Freeze drying: The material is rapidly frozen and subjected to vacuum drying to remove water via sublimation.

[0195] (b) Air Drying (instead of freeze drying): One sample is dried using conventional air drying.

[0196] Result: Table-2

[0197] Observation: The use of freeze drying in the preparation of bone grafts significantly enhances the shelf life, stability, and ease of storage, while preserving biologically active molecules. In contrast, non-freeze-dried grafts are more susceptible to degradation and require strict storage conditions.

[0198] Example 8: Comparative Analysis of Bone Graft Types:

[0199] A bone graft composition prepared from human-derived hard tissue (teeth, bone, or cartilage) in particulate, block, or moldable paste form. The graft is demineralized to retain osseoinductive BMPs, followed by complete decellularization, sterilization, and lyophilization (freeze drying) for stability as shown in Example 1.

[0200] Comparative assessment over conventional and experimental graft types is provided in below table: Table-3: Comparison of different graft types based on efficiency parameters:

[0201] Further comparative study on various other parameters as available form literature were assessed, below table depicts various properties / features of the bone grafts when compared with the present invention:

[0202] Table-4: Comparison of conventional grafts and autografts: The data in above tables 3 & 4 depicts that the present invention outperforms conventional grafts in terms of storage, usability, and biological potential. Some of the key advantages of Present Invention are

[0203] • Superior shelf life (up to 5 years) without need for cold chain logistics

[0204] • Preserved osseoinductivity via BMP exposure from demineralization

[0205] • Sterile and stable form, ideal for point-of-care use

[0206] • Available in versatile formats (particulate, block, or moldable paste)

[0207] The evaluation study depicted in examples 2-8, demonstrate the comprehensive preparation, sterility assurance, physicochemical characterization, and biological evaluation of the DFDB A bone graft developed according to the present invention, showing its suitability for clinical use in ridge preservation and other orthopedic applications.

[0208] REFERNECES:

[0209] [1] Z. Schwartz et al., “Ability of commercial demineralized freeze-dried bone allograft to induce new bone formation.,” J. PeriodontoL, vol. 67, no. 9, pp. 918-926, Sep. 1996, doi: 10.1902 / jop.1996.67.9.918.

[0210] [2] M. Zhang, R. M. J. Powers, and L. J. Wolfinbarger, “A quantitative assessment of osteoinductivity of human demineralized bone matrix.,” J. PeriodontoL, vol. 68, no.

[0211] 11, pp. 1076-1084, Nov. 1997, doi: 10.1902 / jop.1997.68.11.1076.

[0212] [3] A. Nather, Bone grafts and bone substitutes: Basic science and clinical applications. 2005

[0213] [4] J. N. Kearney, “Storage, Processing and Preservation BT - Essentials of Tissue Banking,” G. Galea, Ed. Dordrecht: Springer Netherlands, 2010, pp. 95-107.

[0214] [5] A. Nather, N. Yusof, and N. Hilmy, Radiation in Tissue Banking: Basic Science and Clinical Applications of Irradiated Tissue Allografts . World Scientific, 2007.

[0215] [6] M. R. Urist and B. S. Strates, “Bone morphogenetic protein.,” J. Dent. Res., vol. 50, no. 6, pp. 1392-1406, 1971, doi: 10.1177 / 00220345710500060601.

Claims

Claims:

1. A bone graft composition comprising particulate matter derived from human hard tissues selected from the group consisting of teeth, bone, and cartilage, wherein the particulate matter is demineralized to expose bone morphogenetic proteins (BMPs), decellularized, sterilized, and lyophilized to reduce water content and enhance shelf life.

2. The bone graft composition as claimed in claim 1, wherein the lyophilized particulate matter is rehydrated with autologous or allogenic body fluids comprising growth factors to form a dough-like material suitable for application to bone defects or augmentation sites.

3. A method of preparing a bone graft comprising: a. collecting human hard tissues selected from the group consisting of teeth, bone, and cartilage; b. processing said tissues by demineralization to expose bone morphogenetic proteins (BMPs), removal of cellular and fatty components, and sterilization; c. subjecting the processed tissues to freeze drying to reduce water content; and d. forming the processed tissues into particulate, block, or paste form.

4. The method of claim 3, further comprising rehydrating the freeze-dried particulate with autologous blood, platelet-rich plasma (PRP), bone marrow aspirate, or saline to form a moldable material for direct clinical application.

5. A method for producing a patient-specific bone graft comprising: a. processing human-derived hard tissue to preserve osseoinductive potential; b. designing a graft model using CT imaging and computer-aided design (CAD); c. fabricating the graft using 3D printing or CNC machining techniques; d. subjecting the graft to sterilization and lyophilization; e. implanting the graft using mechanical fixation.

6. The method of claim 5, wherein the 3D-printed graft includes a biocompatible adhesive or binder facilitating host tissue integration and controlled porosity.

7. A bone graft block comprising lyophilized, processed human hard tissue, customized based on patient imaging, wherein the block maintains structural integrity, osseoinductive potential, and is capable of being stored at ambient conditions for atleast three years.

8. A bone graft product comprising: a) processed particulate matter derived from human teeth, bone, or cartilage, b) preserved osseoinductive proteins, c) a lyophilized form having low residual moisture, d) a carrier fluid comprising biologically active growth factors, wherein the product is available in a form selected from dough-like paste, pre-formed block, or 3D-printed scaffold.

9. The bone graft product of claim 8, wherein the lyophilized form provides a shelf life ofat least three years and ranging to 5 years at room temperature without degradation of biological activity.

10. A method for enhancing bone regeneration at a defect site comprising: a. preparing a bone graft by demineralization, decellularization, sterilization, and freeze drying of human-derived hard tissues; b. rehydrating the graft with body fluid comprising growth factors; c. applying the graft to a bone defect or augmentation site, wherein the graft retains osseoinductive potential and promotes osteogenesis and host integration.

11. The method as claimed in claim 3, further comprising the step of customizing the bone graft into patient-specific geometries using computer-aided design (CAD) and computer- aided manufacturing (CAM) based on medical imaging data.

12. The method as claimed in claim 11, wherein the customized graft is prepared using three- dimensional (3D) bioprinting techniques with a printable medium containing the lyophilized particulate matter and a biocompatible adhesive.

13. The method as claimed in claim 3, wherein the processed particulate matter is incorporated into a moldable paste by mixing with autologous body fluids selected from the group consisting of blood, plasma, or platelet-rich fibrin to enhance biological integration upon application.

14. Use of the bone graft composition as claimed in claim 1 for the preparation of patientspecific bone implants through CAD-assisted modelling and CAM-based shaping for orthopaedic, craniofacial, or dental reconstruction.

15. Use of the bone graft composition as claimed in claim 1, in combination with 3D bioprinting and image-guided customization, for regenerating bone tissue in complex or irregular defects requiring high structural fidelity and osseoinductive potential.

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