CN110507854B

CN110507854B - Bone graft implant and method for preparing the same

Info

Publication number: CN110507854B
Application number: CN201910778873.8A
Authority: CN
Inventors: 李智峰; 盖增; 苟元彬; 李湘杰; 李次会
Original assignee: Beijing Kejian Biotechnology Co ltd
Current assignee: Beijing Kejian Biotechnology Co ltd
Priority date: 2019-08-22
Filing date: 2019-08-22
Publication date: 2021-11-09
Anticipated expiration: 2039-08-22
Also published as: CN110507854A

Abstract

The invention provides a bone graft implant and a preparation method thereof. The decalcified bone matrix is used as a basic material, the collagen in the decalcified bone matrix is subjected to a thermal crosslinking reaction, and the collagen in the decalcified bone matrix and glycosaminoglycan are subjected to a thermal crosslinking reaction to prepare the bone graft implant, so that the mechanical strength and the degradation period of the bone graft implant are improved, the cartilage cell transformation is promoted, the bone graft implant can be used as the bone graft implant for nasal plastic surgery and other surgeries, and the bone graft implant can be used for nasal comprehensive plastic surgery by replacing autologous cartilage tissue under the condition that a patient does not want to take the autologous cartilage tissue or the autologous cartilage tissue is not applicable.

Description

Bone graft implant and method for preparing the same

Technical Field

The present invention relates to a bone graft implant, and more particularly, to a bone graft implant prepared using a demineralized bone matrix as a basic material. The invention also relates to a preparation method of the bone graft implant. The bone graft implant can be used for comprehensive reshaping of the nose and other parts.

Background

The comprehensive reshaping of the nose is one of the common plastic and cosmetic surgery operations, namely, the operation for improving the appearance of the nose is achieved by filling various autologous or allogeneic tissues or tissue substitutes to cushion the outer nose. The quality of the nose augmentation material is a key factor in determining the effectiveness of the surgery. Silica gel and expanded polytetrafluoroethylene are the most commonly used prosthesis augmentation nasal materials in clinic, but in recent years, many scholars point out the defects and shortcomings of silica gel prostheses through clinical tests, for example, the movement of the prosthesis and the protrusion degree of the nose tip are not enough after the silica gel is placed in the operation, and the silica gel can generate light transmission phenomenon under strong light to influence the operation effect due to the material problem; the expanded polytetrafluoroethylene is a novel medical high polymer material, is prepared from polytetrafluoroethylene resin by special processing methods such as stretching and the like, and is an ideal biological tissue substitute. However, the expanded body is difficult to take out after transplantation, and bacteria are easily hidden in the pores, so that the incidence rate of postoperative chronic infection is high, thereby limiting the clinical application. Autologous cartilage is the safest orthopaedic filling and shaping material, is the first choice for nasal structure reconstruction, has wide application, and covers congenital malformation repair, defect repair after trauma, repair and reconstruction in tumor surgery, treatment in beauty surgery and the like. In the comprehensive reshaping of the nose, the costal cartilage and the nasal septum cartilage are taken as the nasal septum extension graft and the columella support, but the chest of the patient who takes the costal cartilage has incision scars and postoperative pain, and the calcification of the aged cartilage is serious, so that the usable amount is less, thereby bringing much pain to partial patients, influencing the operation effect and the operation comfort of beauty seeking people.

To circumvent these drawbacks, in recent years, emerging allogeneic decalcified bone matrix grafts have been developed. It can provide enough graft material, and compared with autologous costal cartilage, it can reduce operation time and supply area complication. The allogeneic decalcified bone matrix is a compound natural bone graft material consisting of collagen, non-collagen, growth factors with low concentration and the like, and mainly comes from human skull, femoral shaft and tibial stem. As a bone grafting material, the decalcified bone is applied for more than 20 years in a plurality of countries around the world, and a large amount of literature data prove the effectiveness and the safety of the decalcified bone in orthopedics and maxillofacial plastic application.

The Decalcified Bone Matrix (DBM) is prepared by using cortical bone of allogeneic limbs and backbones and carrying out processes of degreasing, deproteinization, decalcification, sterilization and the like. The material has moderate hardness, is convenient for shape trimming and random cutting, and simultaneously has certain toughness and certain mechanical stability. Can be biologically combined with autologous tissue after surgery, allowing vascularization of the tissue and ingrowth of autologous tissue.

The decalcified bone has the following advantages: has sufficient source, easy cutting and shaping, safe use, no toxicity, no harm, no hemolysis and no teratogenesis to human bodies, and preliminarily shows that the cartilage or bone forming capability is realized. The decalcified bone-nose comprehensive plastic surgery can be applied, local anesthesia can be realized, the surgery time is short, the risk is reduced, and beginners can easily understand and operate the surgery. The decalcification bone supporting force is stable without damaging the nasal septum cartilage, secondary damage and scar of the chest wall caused by taking the costal cartilage are avoided, the pain of a beauty-seeking person is greatly reduced, the operation comfort level of the beauty-seeking person is obviously improved, the satisfaction is high, and the nasal septum cartilage and the autologous costal cartilage can be completely replaced in the comprehensive shaping of the nose.

However, the decalcified bone matrix has high absorption rate and is easy to deform and collapse, and the clinical application of the material is severely limited. The allogeneic decalcified bone matrix promotes tissue growth at a much slower rate than the degradation rate of the material itself, which is the most important reason for high absorption and deformation. Furthermore, the local severe inflammatory reaction caused by the partially implanted decalcified bone matrix can also accelerate the degradation and absorption of materials.

Disclosure of Invention

The inventors of the present invention found that a thermal crosslinking (DHT) method is used to perform a crosslinking reaction of a collagen matrix in an allograft decalcified bone matrix material (DBM), and a collagen matrix with a glycosaminoglycan such as hyaluronic acid, chondroitin sulfate, heparin, etc.; through the cross-linking reaction among the collagen matrixes, the degradation time of the DBM material can be obviously prolonged, meanwhile, the mechanical property of the material is enhanced, and the cell-mediated shrinkage degree of the collagen matrixes is weakened. Glycosaminoglycan crosslinked on the DBM can promote the proliferation of Mesenchymal Stem Cells (MSCs) and the formation of chondrocytes in vivo, and finally promote the formation of cartilage tissues. The shape of the orthopedic implant can be effectively maintained by crosslinking within the matrix of the DBM, preventing collapse or over-resorption. And the conversion of the implant into elastic cartilage tissue is promoted by the crosslinked glycosaminoglycan.

The present invention has been made based on the above-mentioned studies.

The present invention firstly provides a bone graft implant, which is prepared by using a demineralized bone matrix as a basic material, allowing collagen in the demineralized bone matrix to undergo a thermal crosslinking reaction, and allowing collagen in the demineralized bone matrix to undergo a thermal crosslinking reaction with glycosaminoglycan.

Specifically, the bone graft implant of the present invention comprises:

decalcified bone matrix as a skeleton; and

a glycosaminoglycan;

wherein collagen in the decalcified bone matrix is polymerized by a thermal crosslinking reaction; and collagen in the demineralized bone matrix and the glycosaminoglycan are polymerized through a thermal crosslinking reaction.

The decalcified bone matrix may be selected from bone graft materials, which are usually conventional in the art, and can reduce immunogenicity of bone graft materials, for example, from allogeneic or xenogeneic bone after decalcification.

The decalcified bone matrix material can be a decalcified bone matrix material (DBM) prepared by adopting a method of partially decalcified bone biological derived bone scaffold mentioned in Chinese patent application No. 00132082.3. DBM prepared by this method, i.e. retaining the appropriate amount of Ca²⁺The material degradation and absorption rate can be further delayed, and the collapse after the rhinoplasty can be prevented. However, the overall material of bDBM is hard, and the residual Ca needs to be strictly controlled²⁺In order not to affect the engravability of the material.

Further, the Ca content of the DBM of the present invention is between 5 and 20% (mass percent), such as 5%, 10%, 15%, or 20%.

The decalcified bone matrix of the present invention is especially one kind of allogeneic decalcified bone.

Further, the glycosaminoglycan can be selected from one or more of hyaluronic acid, chondroitin sulfate and heparin.

Further, the temperature of the thermal crosslinking reaction is 80-140 ℃, preferably 100-120 ℃, and more preferably 120 ℃. The decalcified bone matrix can strengthen the mechanical strength and hardness of the material after the cross-linking treatment. In order to improve the suturable performance, the invention further prepares a plurality of rows of small holes on the surface of the material for suturing and fixing the material in operation (as shown in figure 1). Furthermore, the aperture of the holes is preferably 0.4-0.8 mm, and the distance between the holes is 3-6 mm.

The present invention also provides a method for preparing a bone graft implant, comprising:

placing the decalcified bone matrix in a glycosaminoglycan solution for soaking treatment;

taking out, freeze drying, and then carrying out thermal crosslinking reaction under vacuum condition.

Wherein the decalcified bone matrix and glycosaminoglycan have the same meanings as described above.

The solvent of the glycosaminoglycan solution is preferably purified water, and the concentration thereof is preferably 0.1 to 0.5% by mass.

Further, the soaking treatment time is 4-24 hours.

Lyophilization may be carried out by conventional methods.

The vacuum pressure of the vacuum condition is preferably 150-300Pa, and more preferably 150-200 Pa. The thermal crosslinking reaction performed under vacuum conditions has an advantage in that oxygen can be removed and the oxidation of the matrix in the DBM in a high temperature environment can be prevented.

Further, the temperature of the thermal crosslinking reaction is 80-140 ℃, preferably 100-120 ℃, and more preferably 120 ℃.

Further, the thermal crosslinking reaction time is 4 to 48 hours, preferably 8 to 16 hours.

In some embodiments of the present invention, the thermal crosslinking reaction is performed at a temperature of 100 ℃ to 120 ℃ for a period of 8 to 16 hours.

Furthermore, the preparation method also comprises the step of arranging a plurality of rows of small holes on the surface of the decalcified bone matrix after the thermal crosslinking treatment so as to suture and fix the materials in the operation.

The invention improves the mechanical strength and the degradation period of the decalcified bone matrix after the decalcified bone matrix is subjected to thermal crosslinking reaction, is more favorable for promoting the transformation of chondrocytes, can be used as a bone transplantation implant for operations such as nasal plastic and the like, and can replace autologous cartilage tissue to be used for the comprehensive plastic of the nose under the condition that a patient does not want to take the autologous cartilage tissue or the autologous cartilage tissue is not applicable.

Drawings

FIG. 1 is a schematic view of the structure of a bone graft implant according to the present invention; wherein 1 denotes a body of the bone graft implant, and 2 denotes a plurality of rows of prepared holes provided on the body for intraoperative suture threading.

Fig. 2 and fig. 3 show the chondrocyte formation results of the non-crosslinked control group and the DHT-crosslinked group, respectively, in the experiment for promoting chondrocyte transformation with the material in the experimental example.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

Example 1

Preparation of completely demineralized bone matrix and partially demineralized bone matrix: taking cortical bone or cancellous cortical mixed bone of the allogenic bone limb bone skeleton part frozen at a deep low temperature of (-80 ℃ for more than 6 months), removing soft tissues, central bone marrow and cartilage parts attached to the cortical bone or cancellous mixed bone, mechanically cutting the cortical bone or cancellous mixed bone into a prefabricated shape by adopting a numerical control machine tool, accurately drilling a plurality of rows of small holes, degreasing the mixture in 30 percent hydrogen peroxide at 38 ℃ for 72 hours, and changing the liquid once every 24 hours; cleaning the injection with water, removing residual hydrogen peroxide in 75% ethanol for 24 hr, and cleaning the injection with water; chloroform: methanol 3:1, chloroform: partially deproteinizing in methanol (3:1) solution for 4 hr, and soaking and washing with water for injection; the partially demineralized bone matrix was prepared by decalcification with 0.6M hydrochloric acid for 12-48 hours. The Ca content in DBM is between 5 and 20 percent by adopting a plasma emission spectrum analysis method.

Example 2

Performing crosslinking reaction on DBM and glycosaminoglycan DHT, preparing 0.1% hyaluronic acid solution, fully stirring and dissolving, immersing DBM in the solution, performing oscillation incubation at 80-10rpm for 8 hours at room temperature, removing water by using a freeze drying method, determining that the residual water content is lower than 15% by using a drying weight loss method, placing the DBM in a vacuum drier for pre-vacuumizing to 100Pa, starting heating and raising the temperature, starting timing after the temperature is raised to 100 ℃ and maintaining the reaction temperature for 8 hours, taking out a sample, and performing 25kGy radiation sterilization.

Multiple rows of small holes are further prefabricated on the surface of the material for suturing and fixing the material in operation, and the structural schematic diagram is shown in fig. 1.

Example 3

The only difference from example 2 is: the temperature of the thermal crosslinking reaction was 120 ℃ and the reaction temperature was maintained for 8 hours.

Example 4

The only difference from example 1 is: the temperature of the thermal crosslinking reaction was 140 ℃ and the reaction temperature was maintained for 12 hours.

Examples of the experiments

1) Mechanical strength of material

The DHT-crosslinked material has a higher flexural strength than the non-crosslinked material, and therefore, the crosslinked material can maintain a better shape of the orthopedic repair site. By comparing the three-point bending test of the non-crosslinked group and the DHT-crosslinked group treated at different temperatures, the results demonstrate that the DHT-crosslinked group has higher bending resistance than the non-crosslinked group, and the material has the highest bending resistance at a reaction temperature of 120 ℃, as shown in table 1 below:

TABLE 1 three point bend test (Unit: N) of bDBM for different treatment methods

2) Time of material degradation

The DHT crosslinking treatment improves the degradation period of the material, so that the material can keep the shape for a long time in the plastic repair application. The percent weight loss after degradation was analyzed for groups of different treated materials by in vitro collagenase degradation, the main experimental conditions being that each group was soaked in 75 μ g/ml of type IA bacterial collagenase solution in 4.4mM Tris buffer (pH7.4, containing 10mM CaCl)₂) And oscillating and incubating at 37 ℃, replacing collagenase solution once a day, testing the percent degradation weight loss of each group after 7 and 14 days, wherein the degradation weight loss rate of the non-crosslinked DBM at any observation time point is higher than that of the DHT crosslinked DBM material. The results are shown in Table 2.

TABLE 2

3) Material for promoting chondrocyte transformation

The DHT crosslinking treatment cross-links and complexes glycosaminoglycan with collagen molecules in the DBM to promote chondrocyte formation, which in turn promotes cartilage formation in the repair area. The material prepared in example 3 (i.e., DBM material crosslinked at 120 ℃ for 8 hours with DHT) was ground to about 1mm small particles, filled into sterile capsules of size 5, and implanted into mouse muscle tissue, and the control used uncrosslinked DBM matrix material. The DHT-crosslinked group was seen to have a large number of chondrocyte formation 10 days after implantation (see fig. 2), whereas the control group had little chondrocyte or chondrocyte-like formation (see fig. 3).

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A bone graft implant, comprising:

decalcified bone matrix as a skeleton; and

a glycosaminoglycan; the glycosaminoglycan is hyaluronic acid;

wherein collagen in the decalcified bone matrix is polymerized by a thermal crosslinking reaction; and collagen in the decalcified bone matrix and the glycosaminoglycan are polymerized by a thermal crosslinking reaction;

the preparation method of the bone graft implant comprises the following steps:

placing the decalcified bone matrix into a glycosaminoglycan solution with the mass percentage concentration of 0.1-0.5% for soaking treatment for 4-24 hours;

taking out, freeze-drying, and then carrying out thermal crosslinking reaction under the vacuum condition; the temperature of the thermal crosslinking reaction is 100-140 ℃, and the time is 8-16 hours.

2. The bone graft implant of claim 1, wherein the demineralized bone matrix is an allogeneic demineralized bone matrix or a xenogeneic demineralized bone matrix.

3. The bone graft implant according to claim 1 or 2, wherein the Ca content in the decalcified bone matrix is 5-20%.

4. The bone graft implant according to claim 1 or 2, wherein the temperature of the thermal crosslinking reaction is 100-120 ℃.

5. Bone graft implant according to claim 1 or 2, characterized in that the temperature of the thermal crosslinking reaction is 120 ℃.

6. The bone graft implant according to claim 1 or 2, wherein the surface of the bone graft implant is prefabricated with rows of small holes for suture fixation of the intraoperative material.

7. The bone graft implant according to claim 6, wherein the small holes have a hole diameter of 0.4 to 0.8mm and a hole pitch of 3 to 6 mm.

8. A method of preparing a bone graft implant, comprising:

placing the decalcified bone matrix into a glycosaminoglycan solution with the mass percentage concentration of 0.1-0.5% for soaking treatment for 4-24 hours; the glycosaminoglycan is hyaluronic acid;

9. The method for preparing a demineralized bone matrix according to claim 8, characterized in that said demineralized bone matrix is identical to the demineralized bone matrix according to claim 2 or 3.

10. The method according to claim 8 or 9, wherein the vacuum pressure of the vacuum condition is 150-300 Pa; and/or the presence of a gas in the gas,

the temperature of the thermal crosslinking reaction is 100-120 ℃.

11. The method according to claim 8 or 9, wherein the vacuum pressure of the vacuum condition is 150-200 Pa; and/or the presence of a gas in the gas,

the temperature of the thermal crosslinking reaction was 120 ℃.

12. The method for preparing a calcium-removed bone matrix according to claim 8 or 9, further comprising the step of providing a plurality of rows of pores on the surface of the thermally crosslinked calcium-removed bone matrix for suture fixation of the intra-operative material.