Composite material for application to a bone defect site

Abstract

The present disclosure relates to a composite material for application to a bone defect site to promote new bone growth at the site comprising: a combination of freeze-dried mineralized bone allograft particles; and a monophasic gel comprising cross-linked hyaluronic acid; wherein the hyaluronic acid is cross-linked by a cross-linking agent, the cross-linking agent being a diglycidyl ether. The present disclosure also relates to a method to obtain a composite material for application to a bone defect site to promote new bone growth at the site comprising mixing freeze-dried mineralized bone allograft particles with a monophasic gel comprising hyaluronic acid; and to a bone regenerative material comprising the composite material as defined herein for use in a method of treating a patient suffering from a degenerative bone condition, bone loss, bone deficiency or bone atrophy.

Description

[0001] COMPOSITE MATERIAL FOR APPLICATION TO A BONE DEFECT SITE

[0002] TECHNICAL FIELD

[0003] The present disclosure is related to a composite material for application to a bone defect site to promote the growth of new bone on the site.

[0004] BACKGROUND ART

[0005] Currently, bone regeneration of the maxillary or mandible bone is usually performed with biomaterials or bone substitutes, used alone or in combination with the patient's own autogenous bone; whose tendency is to reduce harvesting it, reducing morbidity.

[0006] The most commonly used biomaterials or bone substitutes for bone regeneration are xenografts (bone of animal origin), allografts (human bone from a living donor or cadaver) and alloplastics (of synthetic origin). In general, xenografts are osteoconductive and their particles hardly undergo resorption, but the formation of new bone around them requires 6 to 9 months. Alloplastic materials are usually hydroxyapatites and calcium phosphates, which have limited osteoconductive capacity and undergo very marked resorptions or encapsulation into the surrounding tissue. Human donor bone offers good osteoconductive qualities and, depending on processing, can also be inductive. Healing times with the allograft are around 4 to 6 months.

[0007] The three types of bones described, alone or combined with autogenous bone, are commonly used covered with collagen membranes.

[0008] In recent years, these bone substitutes have also been combined with different platelet concentrates to stimulate and accelerate the healing of the new bone or bone to be regenerated, with little success when evaluated histologically and clinically.

[0009] For instance, patent application EP1127581 A1 discloses a sterile malleable bone composition for application to a bone defect site comprising a mixture of demineralized osteogenic bone powder, in a hydrogel carrier; the bone powder having a particle size of from about 100 to about 850 microns; the bone powder comprising from about 25 to about 35% of the weight of the composition; the carrier being selected from the group consisting of sodium hyaluronate, chitosan and N,O-carboxymethyl-chitosan in water solution, the sodium hyaluronate, chitosan or N,O-carboxymethyl-chitosan having a high molecular weight ranging from five hundred thousand to three million Daltons and ranging from 1 % to 4.5% by weight of the carrier solution. Patent application W02005051446 A1 discloses a composite material comprising hyaluronic acid and / or hyaluronic acid derivatives, a matrix of demineralized bone and / or biocompatible and biodegradable ceramics and / or bone of autologous or allogenic or animal origin.

[0010] Patent application US6437018 B1 discloses a sterile formable bone composition for application to a bone defect site to promote new bone growth at the site comprising a mixture of demineralized osteoinductive and osteoconductive bone powder with a particle size ranging from about 100 to about 850 microns in an aqueous carrier solution, the bone powder ranging from about 25 to about 35% of the weight of the composition, the carrier comprising a hydrogel component of sodium hyaluronate in a phosphate buffered aqueous solution, said hydrogel component having a high molecular weight ranging from over six hundred thousand to three million Daltons with a stable viscosity at a temperature range from about 22 °C to about 37 °C and ranging from about 0.75% to about 5.0% by weight of the aqueous carrier solution, said composition having a pH ranging from about 6.8 to about 7.

[0011] Moreover, Cerabone® plus (Botiss Biomaterials) is a trademark name for a material combining bovine bone grafting with non-cross-linked hyaluronic acid used in implantology, periodontology and oral and CMF surgery.

[0012] However, there are many aspects in the composite materials available up to date that need to be addressed and have not been completely solved in the prior art. For instance, there is a need to improve the mechanical properties of those materials by obtaining a compact and easy-to-manipulate (handling) material and to a material that presents optimal hardening. There is also a need for achieving high quality bone in terms of density and consistency after the healing time. Moreover, current solutions involve a long time of new bone formation (from 6 to 9 months). Furthermore, today’s biomaterials might not prevent the heterologous graft being susceptible to early resorption, losing the dimensional / volumetric stability.

[0013] The present disclosure attempts to solve at least the problems mentioned above.

[0014] SUMMARY OF THE INVENTION

[0015] I n order to address one or more of the foregoing problems, one aspect of the present disclosure relates to a composite material for application to a bone defect site to promote new bone growth at the site comprising a combination of freeze-dried mineralized bone allograft particles and a monophasic gel comprising cross-linked hyaluronic acid, wherein the hyaluronic acid is cross-linked by a cross-linking agent, the cross-linking agent being a diglycidyl ether.

[0016] In a second aspect, the present disclosure is related to a method to obtain a composite material for application to a bone defect site to promote new bone growth at the site comprising mixing freeze-dried bone mineralized allograft particles with a monophasic gel comprising hyaluronic acid, the hyaluronic acid being cross-linked by a cross-linking agent, the cross-linking agent being a diglycidyl ether.

[0017] In a third aspect, the present disclosure is related to a bone regenerative material comprising the composite material as disclosed herein for use in a method of treating a patient suffering from bone loss or bone deficiency or bone atrophy, the method comprising cleaning a bone defect site and at least filling the bone defect site with a bone regenerative material that facilitates formation of new, regenerated bone material in the site.

[0018] BRIEF DESCRIPTION OF THE FIGURES

[0019] FIG. 1 depicts images of the composite material according to the present disclosure.

[0020] FIG. 2 depicts images in relation to a patient under orthodontic treatment with tooth #36 for extraction due to a periapical infection treated with the composite material disclosed herein.

[0021] FIG. 3 depicts images in relation to a patient with recommended extraction of tooth #14 and subsequent treatment with the composite material disclosed herein.

[0022] FIG. 4 depicts images in relation to a patient with a failed root canal treatment and subsequent treatment with the composite material disclosed herein.

[0023] FIG. 5 depicts images in relation to a patient with stage 3 periodontitits and subsequent treatment with the composite material disclosed herein.

[0024] FIG. 6 depicts images in relation to a patient with a large apical cyst and subsequent treatment with the composite material disclosed herein.

[0025] FIG. 7 depicts images of bone regeneration after 6 months of healing using a xenograft.

[0026] FIG. 8 depicts images of bone regeneration after 4 months of healing using a mineralized cortico-cancellous allograft.

[0027] FIG. 9 depicts images of bone regeneration after 3 months of healing using the composite material disclosed herein.

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029] The present disclosure is described more fully hereinafter through reference to different preferred realizations, embodiments and examples.

[0030] In the present disclosure, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise. The present disclosure relates to a composite material for application to a bone defect site to promote new bone growth at the site comprising a combination of mineralized bone allograft particles; and a monophasic gel comprising cross-linked hyaluronic acid; wherein the hyaluronic acid is cross-linked by a cross-linking agent, the cross-linking agent being a diglycidyl ether.

[0031] In a particular embodiment, the composite material for application to a bone defect site to promote new bone growth at the site may consist of a combination of mineralized bone allograft particles; and a monophasic gel comprising cross-linked hyaluronic acid; wherein the hyaluronic acid is cross-linked by a cross-linking agent, the cross-linking agent being a diglycidyl ether.

[0032] In the present disclosure, the term “gel” is to be understood as a semi-solid material that exhibits no flow in the steady state. A gel is a dispersion of molecules of a liquid within a solid medium. In the present invention, the gel comprising cross-linked hyaluronic acid has to be understood as a dispersion of water within cross-linked hyaluronic acid.

[0033] In the present disclosure, a “monophasic cross-linked hyaluronic acid gel” is a homogeneous, single-phase gel where the hyaluronic acid molecules are uniformly cross-linked throughout the gel matrix. There is no distinct separation between different phases within the gel and does note separate into different components (as in the case of i.e. biphasic gel). A monophasic cross-linked hyaluronic acid gel has its hyaluronic acid chains interwoven in a continuous, uniform network. Monophasic cross-linked hyaluronic acid gels therefore are smooth, easily injectable and flowable, have a high stability and maintain their integrity for long periods, providing consistent volume and shape, making them applicable in the i.e. dermatological field in fine line treatment, facial volume restoration, and wrinkle filling.

[0034] In the present disclosure, the term “degree of cross-linking” refers to the extent to which individual hyaluronic acid molecules are chemically bonded together through cross-links. In particular, the degree of cross-linking of cross-linked hyaluronic acid is defined as the ratio between the number of cross-linked hyaluronic acid disaccharides and the total number of hyaluronic acid disaccharides (see e.g. Kenne et al. (2013) Carbohydrate Polymers).

[0035] Preferably, the mineralized bone allograft particles might be cortico-cancellous, cortical and / or cancellous and / or a combination thereof. Moreover, the freeze-dried mineralized bone allograft particles may have a mean average particle size (D50) of between 250 to 1.250 pm, preferably 500 pm.

[0036] The freeze-dried mineralized bone allograft particles in the composite material are preferably in an amount of between 25 to 95% by weight with respect to the amount of the composite material. Preferably, the bone allograft particles might be in an amount of between 60 and 90% by weight with respect to the amount of the composite material. Even more preferably, the bone allograft particles might be in an amount of between 50 to 70% by weight with respect to the amount of the composite material.

[0037] Alternatively, the proportion between freeze-dried mineralized bone allograft particles and cross-linked hyaluronic acid are between 1 :1 and 3:1 by weight, respectively. More preferably, the proportion might be between 1 :1 and 2:1.

[0038] The cross-linking agent cross-linking the hyaluronic acid as disclosed herein may preferably be compounds that have two epoxide groups, that allow the compound to form covalent crosslinks with the hyaluronic acid residues. Preferably, the cross-linking agent might be a diglycidyl ether. Preferably, the diglycidiyl ether might be 1 ,4-butanediol diglycidyl ether (BDDE), Ethylene Glycol Diglycidyl Ether (EGDE), or combinations thereof. Other cross-linking agents such as diatomaceous earth, glutaraldehyde, genipin, carbodiimides, sodium periodate, photocrosslinking agents and combinations thereof might also be envisaged.

[0039] Moreover, the monophasic gel comprising cross-linked hyaluronic acid may comprise residual cross-linking agent. By residual it is understood that the cross-linking agent has not completely reacted with the hyaluronic acid, and, therefore, it might be present in the gel. In the present disclosure, the cross-linking agent present in the monophasic gel is less than 0.5 ppm, preferably less than 0.4 ppm, more preferably less than 0.3 ppm.

[0040] The hyaluronic acid being cross-linked having the degree of cross-linking disclosed herein and in the amount disclosed herein renders what is known in the art as a highly cross-linked hyaluronic acid, as opposed to low or non-cross-linking hyaluronic acids, wherein the crosslinking agent is present at a lower ratio or is completely absent.

[0041] Preferably, the monophasic gel comprising cross-linked hyaluronic acid may have a concentration of between 20 to 30 mg / ml of hyaluronic acid in water. Preferably, the concentration of cross-linked hyaluronic acid may be 23 mg / ml. Preferably, the monophasic gel comprising cross-linked hyaluronic acid may be buffered to a pH of between 6.8 and 7.4 with any available buffer, for instance sodium phosphate buffer.

[0042] Preferably, the cross-linked hyaluronic acid may have a molecular weight of at least 2500000 Dalton (2500 kDa). Preferably, the cross-linked hyaluronic acid may have a molecular weight of between 2500000 Da (2500 kDa) and 3000000 (3000 kDa).

[0043] In the present disclosure, the term “hyaluronic acid” is interchangeable to “sodium hyaluronate”. The skilled person understands that those terms are equivalent.

[0044] The composite material as disclosed herein may further comprise antibiotics or antimicrobial agents.

[0045] Moreover, the composite material may further include bone morphogenic proteins.

[0046] The present disclosure also relates to a method to obtain a composite material for application to a bone defect site to promote new bone growth at the site comprising: mixing freeze-dried mineralized bone allograft particles with a monophasic gel comprising hyaluronic acid; wherein the hyaluronic acid is cross-linked by a cross-linking agent, the cross-linking agent being a diglycidyl ether.

[0047] The proportion between the freeze-dried mineralized bone allograft particles and the gel comprising cross-linked hyaluronic acid is between 1 :1 and 3:1 by weight, respectively. More preferably, the proportion might be between 1 :1 and 2:1.

[0048] Preferably, the monophasic gel comprising cross-linked hyaluronic acid may have a concentration of between 20 to 30 mg / ml of hyaluronic acid, preferably 23 mg / ml.

[0049] The present disclosure also relates to a bone regenerative material comprising the composite material as disclosed herein, for use in a method of treating a patient suffering from a degenerative diseased bone condition, bone loss, bone deficiency or bone atrophy, the method comprising cleaning the bone defect site, optionally removing a portion of the degenerated bone material; and at least partially filling the site the bone regenerative material that facilitates formation of new, regenerated bone material in the bone defect site. Cleaning the bone defect site often involves clearing the granulation tissue and / or connective tissue from the bone compartment in the bone defect site. The degenerative condition might be characterized by a loss of bone mineral density (BMD) and lack of cortical bone. Prior to filling the bone defect site with the composite material, the site might be covered with a material that fixes the composite material onto the bone defect site. A resorbable membrane such as a collagen or a pericardium membrane are envisaged. Alternatively, a non-resorbable material such a Polytetrafluoroethylene (PTFE) or a titanium mesh could be used.

[0050] The present disclosure arises from the finding that the composite material as disclosed has several advantages over biomaterials used in bone regeneration, specifically in the dental technical field.

[0051] In particular, the composite material as disclosed herein covered when applied to a bone defect site with collagen or pericardium membrane has been used by the inventors for the last three and a half years in oral bone regeneration in more than two hundred patients in procedures such as: ridge preservation / reconstruction, guided bone regeneration prior or simultaneous to implant placement, sinus lifts, periodontal infrabony defects, horizontal and vertical GBR. Most of the mentioned procedures had the aim of restoring missing teeth with dental implant supported restorations, as well as restoring the lost periodontium when treating infrabony defects. Thus, the bone regeneration procedures aimed to place or allocate a dental implant in the maxillary bone, and / or recuperate the periodontal bone loss.

[0052] The outcomes of these procedures are clinically, radiographically, and histologically compared to the ones previously obtained with the use of a freeze-dried bone allograft (FDBA) cortico- cancellous covered by a collagen membrane (#1 combination used in the USA) and to the use of a xenograft covered by a collagen membrane (#1 combination used in Europe).

[0053] The inventors have unexpectedly found that using the composite material as disclosed herein significantly improves over other alternatives. For instance, the mechanical properties of the composite material lead to an improvement of the graft by generating a compact, easy-to- manipulate material and hardening of the same since its establishment. The composite material as disclosed herein promotes and improves the healing of hard and soft tissues, together with reducing the time of new bone formation, radically diminishing it to 2-3 months (as compared to typical 6-9 months of currently used materials). The new bone formed achieves high density and moreover, the nature of the composite material protects the heterologous graft and reduces early resorption, stabilizes the clot, has anti-inflammatory and angiogenic properties, is antibacterial, osteogenic and osteoinductive.

[0054] Usage / Handling The composite material according to the disclosure is obtained by mixing freeze-dried mineralized bone allograft particles and a monophasic gel comprising cross-linked hyaluronic acid by a cross-linking agent, the cross-linking agent being a diglycidyl ether. When mixing both components, after a few seconds, less than one minute, a malleable putty of sticky consistency is achieved. The composite material being a malleable putty is very convenient for its handling.

[0055] FIG. 1 shows the composite material being a mixture of mineralized bone allograft particles and the monophasic gel comprising cross-linked hyaluronic acid being malleable and of a sticky consistency. The composite material conveniently adheres to the i.e. metallic spatula used for mixing both components providing optimal mechanical properties for handling and subsequent application into the bone defect site.

[0056] Such a consistency provides several advantages in oral bone regeneration such as a very convenient handling and application of a “one piece” material from the preparation package to the recipient site, instead of having hundreds of bone particles scattered. The chair time procedure becomes faster for the patient, and more efficient and safer for the professional, without losing part of the material and avoiding the saliva contamination of it.

[0057] When working on the upper maxilla the sticky consistency of the material is even of more help as it prevents from falling. Likewise, when filling the frequently narrow cavities of sockets and / or sinuses, the nature of this material helps very much in these applications.

[0058] As indicated hereinabove, the composite material is typically applied onto a bone defect site. In general, bone regenerative materials are applied covered by resorbable membranes. Commonly used membranes are collagen, but pericardium is also used. Other alternatives are also envisaged, such as the use of non-resorbable materials such as PTFE membranes or titanium meshes. Other clinical scenarios do not require covering the composite material as such, when the bone defect site is self-contained preventing the composite material from falling apart from the bone defect. In these circumstances, the composite material can be solely covered by a collagen sponge type of material, or even a temporary restoration, until the coagulum is formed.

[0059] Clinical findings

[0060] Several advantages have been found when re-entering the regenerated sites after using the composite material as disclosed herein: Healing times (Re-entry timings): bone regeneration procedures at the oral cavity are expected to heal according to the existing scientific evidence in between a 6- to 9-months period (i.e. using xenograft). Procedures performed with freeze dried bone allograft as bone substitute may reduce the healing time to 4 to 6 months in some circumstances (i.e. in ridge preservation). Moreover, bone regeneration re-entries after waiting the healing time (6 to 9 months) often show the presence of an unattached mass comprised of bone particles surrounded by connective tissue or granulation tissue (encapsulation of the bone graft material). New bone formation does not occur in the entire grafted area, more commonly in the coronal third of the grafted area (most superficial / outer area).

[0061] However, the hundreds of clinical cases performed by the inventors have shown a tremendous acceleration in the bone maturation or healing, finding clinically and radiographically a mature bone 2,5 to 3 months after the grafting surgical procedure, compared to the standard 6 to 9 months of healing. In other words, the composite material as disclosed herein shows a faster maturation (2,5 to 3 months of healing) and incorporation / amalgamation with the neighboring bone.

[0062] Bone quality and consistency / density: often, when re-opening a bone regeneration treated site clinicians find a soft bone (type III to IV, being types I and II hard bone). This means that the regenerated bone is unmature and the healing time has been insufficient, as the cortical bone (outer layer, the last component of the bone to develop) has not formed yet. Therefore, the bone is unable to withstand mechanical forces or the dental implant insertion torque (25 N cm), thus increasing the risk for implant failure. On other occasions, the consistency of the regenerated bone is uneven, being softer in the upper part of the whole regenerated area with many visible bone graft particles not incorporated into the bone but encapsulated into the gum (connective tissue) above it.

[0063] By using the composite material according to the disclosure, the quality of the regenerated bone has been greatly improved by obtaining a more uniform and homogeneous regenerated bone such that there is no distinction between the native and the newly formed bone and the formation of cortical bone is obtained in a very short healing period and having a high density (type II bone)

[0064] Predictability: complications after bone regeneration procedures have been described up to 25% of the cases treated by experts. The disclosed composite material has shown very predictable results, without postoperative infections reported nor adverse healing events. No complications have been reported in any of the clinical scenarios and all planned dental implants have been placed according to the original treatment plan.

[0065] Radiographic findings

[0066] Before performing regenerative procedures, intraoral periapical radiographs, panoramic radiographs and / or, more importantly, Cone Beam Computerized Tomographs (CBCTs) are taken to diagnose and plan them. After the healing time, CBCTs are taken again to assess the bone gain achieved and simulate the dental implant placement.

[0067] In all treated cases using the disclosed composite material, CBCTs were taken before the surgical procedure and after the healing time before the implant surgery. Horizontal and vertical bone gains have been documented. Moreover, these post-operative CBCTs show a 3D radiographic image of the newly regenerated bone extremely alike the neighboring native bone, being for most of the cases undistinguishable. The cortical bone is also formed in early stages of the healing showing early maturation (2.5 to 3 months post-operation). In the medium to long-term (1 to 4 years of follow-up), CBCTs have shown bone dimensional stability of what was gained at 3 months of healing.

[0068] Histologic findings

[0069] For some of the patients treated with the described combination, biopsies of the grafted and healed areas were taken when bone drilling (for the implant placement) at 2.5 - 3 months after the regenerative surgery. These samples were analyzed and studied for evaluating the residual bone graft particles, the percentage of vital bone and the amount of connective tissue. Through the histomorphometry analysis it can be consistently seen that the amount of vital bone is superior at 2.5 - 3 months compared to other biopsies reported in the literature with FDBA alone or xenograft at 6 months. None of the bone allograft particles are encapsulated into connective tissue but rather assembled within newly formed vital bone. Osteocytes can be observed in the newly formed bone, being vital bone, and in the grafted particles as well. A reverse layer with osteoblasts can also be seen. An ideal amalgamation of the grafted particles and the newly formed bone is observed, too.

[0070] EXAMPLES

[0071] The following examples are provided with the intent of further illustrating the present disclosure but should in no case be interpreted as to be limiting of the present disclosure. Example 1 : Preparation of the composite material

[0072] 0,5 g of freeze-dried mineralized bone allograft (BIOBank, Lieusaint, France) was mixed with 0,5 mL of a gel comprising cross-linked hyaluronic acid (Apriline® FORTE, Suisselle). The mixture was performed on a sterile metallic tray. After a short time (typically less than 1 minute) a consistent putty-like mass is obtained.

[0073] The gel comprising cross-linked hyaluronic acid is monophasic and comprises highly crosslinked hyaluronic acid, the cross-linking agent being BDDE, as specified by the company providing the gel (Suisselle). The degree of crosslinking of the product Apriline® FORTE is 1- 3%

[0074] The composite material as disclosed herein, being a mixture of freeze-dried mineralized bone allograft and monophasic gel comprising cross-linked hyaluronic acid is then applied to a bone defect site, typically onto a defect site on maxillary bone.

[0075] The defect site might be covered or not, depending on the bone defect configuration, by a collagen membrane prior to application of the composite material to the defect site.

[0076] Example 2: Patient under orthodontic treatment with tooth #36 for extraction

[0077] A patient under orthodontic treatment had tooth #36 extracted, and a treatment of the composite material as disclosed herein was performed for ridge preservation. FIG. 2A is a panoramic radiograph showing tooth #36 with a periapical infection to be extracted, while FIG. 2B shows a follow-up CBCT after 4 months with already formed cortical bone and a completely regenerated bone defect.

[0078] Example 3: Patient with recommended extraction of tooth #14

[0079] A patient was recommended extraction of tooth #14 due to periapical infection with proximity to the sinus and possible communication. FIG. 3A are images (photography and radiography) of tooth #14 before operation. FIG. 3B are radiographic images (3D CBCT and radiography) immediately after the extraction and treatment with the composite material as disclosed herein. FIG. 3C are post-operative images (radiographies and 3D CBCTs) after 4 months of the operation, where it can be observed a complete bone healing with cortical bone already being present at the coronal and apical regions. Therefore, the composite material is not only a bone filler, but also a promoter of the natural ossifying process. At the same time, it is important to note that the ridge width and height are fully maintained, and the buccal bone defect healed.

[0080] Example 4: Patient with a failed root canal treatment

[0081] In this case, a patient had a failed root canal treatment with a periapical lesion in proximity with the mandibular nerve / canal causing a lingual fenestration. FIG. 4A is an image (radiography) of the tooth with its infection before operation. FIG. 4B is an image after tooth extraction and immediate placement of the composite material herein disclosed. FIG. 40 is an image showing complete bone regeneration with corticalization of the lingual bone and maturation of the alveolar newly formed bone after 3 months of treatment. FIG. 4D is a radiography of an implant placement at the treated site performed after 3 months of treatment with the composite material as disclosed herein.

[0082] Example 5: Patient with stage 3 periodontitis

[0083] In this case, a patient suffers from generalized stage 3 periodontitis with a hopeless tooth. Tooth #15 with type 2+ mobility is planned for extraction. Immediate implant placement is performed with simultaneous vertical guided bone regeneration (GBR) using the “tent pole abutment” technique to allow for vertical bone growth. FIG. 5A are images (radiography and CBCT) of the tooth before operation. FIG. 5B shows an image (radiography) of the implant placement together with the composite material as disclosed herein. FIG. 40 are 3 months post-operative images (radiography and CBCT) showing a clear radiographic vertical bone gain at the implant and neighboring tooth sites.

[0084] Example 6: Patient with large apical cyst

[0085] In this case, the patient underwent excision of a large apical cyst in tooth #16 that also affected tooth #15. A biopsy of #16 was obtained, as shown in FIG. 6A and 6B. Histological analysis shows neoformed bone, with osteoid cells and osteoblasts. In some of the neoformed bone fragments, while examination under polarized light, lines of bony opposition are insinuated. No revascularization, bone marrow or connective tissue is observed. FIG. 6C shows an image (radiography) of the teeth (#15 and #16) and the cyst before operation. FIG. 6D shows an image (radiography) of the site one month after excision of both teeth and the cyst and treatment with the material composite as disclosed herein. FIG. 6E shows an image (radiography) of the site two months after operation and treatment with the material composite as disclosed herein. FIG. 6F shows an image of the site obtained after placing two implants at the two bone sites at 16 and 15 N cm load torque after three months of excision and composite material treatment.

[0086] The inventors also performed a clinical comparison after oral bone regeneration using different types of particulate bone substitutes or bone graft biomaterials. The following examples (Example 7 to 9) show the results obtained.

[0087] Example 7

[0088] FIG. 7 relates to photographs of bone defects of three different patients (corresponding to FIGs. 7 A, 7B and 70) after 6 months of healing and bone regeneration using a xenograft (FIGs. 7A and 70) and an alloplastic biomaterial (FIG. 7B). FIG. 7A shows encapsulation of the xenograft particles into the soft tissue (gum) not achieving complete bone regeneration. FIG. 7B shows the 6 months healing after using an alloplastic biomaterial which is hardly incorporated into the bone and can be differentiated as a piece at the outer area. FIG. 70 shows several residual particles at the outer surface of the regeneration, not being part of the defect.

[0089] Example 8

[0090] FIG. 8 relates to photographs of bone defects of two different patients (corresponding to FIGs. 8A and 8B) after 4 months of healing and bone regeneration using a mineralized cortico- cancellous allograft. No residual graft particles can be observed, though the bone is immature, without the cortical component, and slightly soft in quality.

[0091] Example 9

[0092] FIG. 9 relates to photographs of bone defects of two different patients (corresponding to FIGs. 9A and 9B) after 3 months of healing and bone regeneration using the freeze-dried mineralized cortico-cancellous allograft combined with the cross-linked hyaluronic acid as disclosed herein. The healing time is completed earlier than in Examples 7 and 8, at 3 months, and the bone quality is greatly improved by the presence of a defined cortical plate, with a harder density (type 3 bone), coinciding with the histomorphometry findings (as shown above).

Claims

CLAIMS1. A composite material for application to a bone defect site to promote new bone growth at the site comprising: a combination of freeze-dried mineralized bone allograft particles; and a monophasic gel comprising cross-linked hyaluronic acid; wherein the hyaluronic acid is crosslinked by a cross-linking agent, the cross-linking agent being a diglycidyl ether.

2. The composite material according to claim 1 , wherein the freeze-dried mineralized bone allograft particles are cortico-cancellous, cortical and / or cancellous.

3. The composite material according to claim 1 or 2, wherein the freeze-dried mineralized bone allograft particles have a mean average particle size (D50) of between 250 to 1.250 pm.

4. The composite material according to any one of the previous claims, wherein the freeze- dried mineralized bone allograft particles are present in an amount of between 25 to 90% by weight with respect to the amount of the composite material.

5. The composite material according to any one of the previous claims, wherein the crosslinking agent is 1 ,4-butanediol diglycidyl ether.

6. The composite material according to any one of the previous claims, wherein the monophasic gel comprising cross-linked hyaluronic acid has a concentration of cross-linking agent of less than 0.5 ppm.

7. The composite material according to any one of the previous claims, wherein the monophasic gel comprising cross-linked hyaluronic acid has a concentration of between 20 to 30 mg / ml of cross-linked hyaluronic acid.

8. The composite material according to any one of the previous claims, wherein the monophasic gel comprising cross-linked hyaluronic acid is buffered to a pH of between 6.8 and 7.4.

9. The composite material according to any one of the previous claims, wherein the hyaluronic acid has a molecular weight of at least 2500000 Dalton.

10. The composite material according to any one of the previous claims, further comprising antibiotics or antimicrobial agents.11 . Method to obtain a composite material for application to a bone defect site to promote new bone growth at the site comprising:Mixing freeze-dried mineralized bone allograft particles with a monophasic gel comprising hyaluronic acid; wherein the hyaluronic acid is cross-linked by a cross-linking agent, the cross-linking agent being a diglycidyl ether.

12. The method according to claim 11 , wherein the proportion between the freeze-dried mineralized bone allograft particles and the gel comprising cross-linked hyaluronic acid is between 1 :1 and 3:1 by weight, respectively.

13. The method according to any one of claims 11 or 12, wherein the monophasic gel comprising cross-linked hyaluronic acid has a concentration of between 20 to 30 mg / ml of hyaluronic acid.

14. The method according to any one of claims 11 to 13, wherein the diglycidyl ether is 1,4- butanediol diglycidiyl ether.

15. A bone regenerative material comprising the composite material as defined in any one of claims 1 to 10, for use in a method of treating a patient suffering from a bone loss or bone deficiency or bone atrophy, the method comprising: cleaning a bone defect site; and at least filling the bone defect site with the composite material that facilitates formation of new, regenerated bone material in the site.

Images