JP4761732B2 - Bone defect filler and manufacturing method thereof - Google Patents

Description

  The present invention relates to a bone defect filling material for filling a bone defect and a bone resorption part, particularly an extraction fossa, and promoting its healing in the medical field, and a method for producing the same.

  Conventionally, as a method of removing bone tissue for a lesion such as a tumor or necrosis, or a treatment method for a bone tissue defect site due to trauma, natural healing or filling with a biologically inert implant material has been performed.

  However, in recent years, regenerative medical techniques have come to be used, and treatment by regeneration of self-living tissue has been attempted. In addition, for wounds (extraction fossa) caused by extraction, conventionally, only local compression and hemostasis with absorbent cotton was used, and then left to natural healing. However, in recent years, some materials are filled to protect the extraction wound. Treatment is often done.

  That is, in the treatment with simple hemostasis, the blood in the extraction socket coagulates to form a blood clot, and has an action of protecting the wound part from external contamination. Mouth washing and oral movements have weakened clot protection and may cause bacterial infections. In order to prevent this, a method has been proposed in which an antibiotic tablet is inserted and then hemostasis is performed with absorbent cotton. However, such a tablet remains completely undissolved by the blood, and heals the wound. There is a problem of delay.

  Several new extraction cavity filling materials (extraction tooth protecting materials) have been proposed for such problems. Examples of commercially available products include calcium phosphate granules such as apatite and collagen sponge. New technologies include the following. For example, Patent Document 1 discloses a calcium phosphate granule cement that can be easily filled in an extraction fossa or the like and is converted into apatite early after curing.

  Further, Patent Document 2 discloses a wound protective material made of powdered sodium polyacrylate, which is useful when applied in an extraction cavity, preventing the bacteria from entering the wound deep into the wound. Alternatively, Patent Document 3 discloses a hemostatic composition comprising a composite material carrying hydroxyapatite or calcium carbonate having an average particle size of 50 μm or less in a biocompatible polymer matrix as an active ingredient, and the periodontal portion at the time of tooth extraction It is said that it is effective for wound hemostasis.

  Further, an absorbent biomaterial made of thermally cross-linked chitin or a derivative thereof and a method for producing an absorbent biomaterial mainly characterized by performing a vacuum thermal cross-linking treatment are known (see Patent Document 4).

  According to this, it is possible to provide a biomaterial as a resorbable material that is relatively easily in vivo and to provide an environment that promotes bone formation by using the biomaterial.

  There are commercially available calcium phosphate granules, calcium phosphate granule cements disclosed in Patent Document 1, and compositions for hemostasis comprising a composite material supporting hydroxyapatite or calcium carbonate in a polymer matrix of Patent Document 3 as active ingredients. In order to inhibit clot formation in the extraction fossa, there is a problem that the effect of protecting the wound portion from contamination from the outside is impaired and infection is likely to occur.

  Further, a commercially available collagen sponge, a wound protective material made of powdered sodium polyacrylate of Patent Document 2, and an absorbable biomaterial made of thermally cross-linked chitin or a derivative thereof of Patent Document 4 do not inhibit clot formation. In addition, since it does not have osteoconductivity, there is a disadvantage that the gingival tissue enters the portion where the alveolar bone tissue should be regenerated and the bone morphology cannot be maintained.

  In addition, since polyacrylic acid is not subject to degradation by in vivo enzymes, it may remain for a long period of time, and the same concern may be caused by chitin derivatives whose dissolution rate is not controlled.

In view of such problems of the prior art, the bone defect filling material and the method for producing the same of the present invention can prevent infection even when the bone defect is filled in a site where there is a risk of infection, such as in the oral cavity. The purpose is to enable superior bone regeneration that is superior to natural healing.
Japanese Patent Laid-Open No. 5-116999 JP-A-6-39028 JP 2004-26653 A JP-A-7-116241

The bone defect porous bone repair material of the present invention for solving the problems is to obtain a chitin or precipitates of chitin or derivatives thereof are obtained by mixing a water-soluble organic solvent in an aqueous solution containing a derivative thereof, wherein A step of cross-linking the precipitate at 130 ° C. to 160 ° C. in vacuum to obtain a cross-linked fiber, and a step of mixing the cross-linked fiber, calcium phosphate granules, and water and freeze-drying
A bone defect filler obtained by including the above-mentioned bone defect filler was immersed in a phosphate buffered saline containing 10 micrograms of lysozyme in 1 ml at a temperature of 37 ° C. for 120 hours. Sometimes, 30% to 85% of the chitin or its chitin derivative component is eluted.

The manufacturing method of the present invention includes the steps of obtaining a chitin or precipitates a derivative thereof in an aqueous solution containing chitin or its derivatives as a mixture of water-soluble organic solvent, the precipitate is 130 ° C. to 160 ° C. in vacuo wherein the step of cross-linking, forming a substantially uniform mixture by mixing crosslinking fibers with water retention capacity less water calcium phosphate granules and crosslinked fibers, that and a step you lyophilized the mixture And

  In the bone defect filling material, chitin or a derivative thereof is a material that serves as a scaffold in a regenerative medical technique, and calcium phosphate granules are a material having osteoconductivity.

  In the present invention, chitin or a derivative thereof is an aggregate of fibers for the following reason.

  That is, by adopting a configuration in which the calcium phosphate granules are supported between the fibers of the aggregate of fibers, it is possible to prevent the granules from being settled and causing uneven distribution of the granules during the preparation of the bone defect filler. In addition, since the fiber aggregate is almost completely provided with continuous pores, it is possible to actively invade bone cells. Furthermore, since the calcium phosphate granules are exposed in the continuous pores, the osteoconductivity is sufficiently exerted, resulting in early bone tissue regeneration.

      As chitin or a derivative thereof, carboxymethyl chitin (sodium salt) that is soluble in neutral water or water-soluble chitosan is preferable in the production process. In addition, water-soluble chitin derivatives such as hydroxyethyl chitin (glycol chitin) and glycol chitosan may be used.

  Here, the derivative refers to a product obtained by chemically modifying chitin existing in nature.

  As the calcium phosphate granules, apatite, β or α tricalcium phosphate, OCP, bioglass, AW glass or the like can be used alone or in combination.

  The size of calcium phosphate granule can be applied if it is 2000 micrometers or less, and it is the optimum size according to the bone reactivity (cortical bone, cancellous bone) of the application site and the fluctuation of bone reactivity depending on the site. Can be selected.

  In the present invention, the porous body of the fibrous chitin derivative is used for the following reason. In other words, since the fiber assembly is almost completely equipped with continuous pores, the invasion of blood and blood cells, as well as osteoblast progenitor cells (interstitial stem cells) existing in the bone marrow, should be actively invaded. Can do. The blood infiltrated into the material forms a blood clot and has an action of protecting the wound portion from external contamination.

  In the porous body of fibrous chitin derivative, the supported calcium phosphate granules are exposed in the continuous pores, so that the osteoconductivity of the material is fully exerted and promotes the formation of bone matrix by osteoblasts And lead to early bone tissue regeneration.

  In addition, the bone defect filler can achieve bone regeneration that is much superior to natural healing by providing predetermined elution characteristics.

  Specifically, as elution characteristics, when immersed in a phosphate buffered saline containing 10 micrograms of lysozyme in a temperature of 37 ° C. for 120 hours, 30% to 85% of the chitin or its chitin derivative component % To elute.

  That is, the elution characteristics in phosphate buffered saline containing lysozyme, which is one of the chitin degrading enzymes, was evaluated, and the measurement method was as follows: phosphate buffered saline containing 10 micrograms of lysozyme in 1 ml. Thus, those that exhibit the elution rate when immersed for 120 hours under a temperature condition of 37 ° C. under stirring (or under shaking) can realize bone regeneration that is much superior to natural healing. When the elution rate is less than 30% and more than 85%, bone regeneration can be as excellent as natural healing, but it is difficult to make it more.

  Next, as a method of forming a fiber of a water-soluble chitin derivative, an aqueous solution of the material is mixed with a water-soluble organic solvent such as ethanol or acetone, and a short fiber-like precipitate is precipitated by rapid stirring. The fiber can be obtained by collecting and washing the precipitate and then drying to remove the solvent. The obtained fiber is water-soluble, but can be insolubilized by using a thermal crosslinking or crosslinking agent, and the elution characteristics in vivo can be adjusted depending on the degree of crosslinking. In the crosslinking treatment, the above elution characteristics can be obtained by performing thermal crosslinking by heating at 130 ° C. to 160 ° C. under a reduced pressure of about 2 kPa.

  In mixing the crosslinked fiber and the calcium phosphate granule, first, water equal to or less than the water retention amount of the crosslinked fiber is added. Here, the water retention amount of the cross-linked fiber refers to the amount of water that the fiber assembly takes in and holds between the fibers when the fiber assembly is pulled up after being immersed in a large amount of water. When water exceeding the water retention amount is added, when mixing with the calcium phosphate granules, the excess water has excessive fluidity, so that the granules are settled and aggregated due to gravity, and the dispersion becomes uneven. When the amount is less than the water retention amount, the moisture retained by the fiber assembly is difficult to flow, and aggregation and sedimentation of calcium phosphate granules are suppressed, resulting in the formation of a homogeneous composite.

  The mixing ratio of the crosslinked fiber and the calcium phosphate granule can be made from about 1: 0 to about 1:10.

  In order to make this mixture into a usable form, the mixture is freeze-dried. Freeze-drying is a process commonly used in the manufacture of pharmaceuticals and dry foods. Keep the inside of the freeze-dryer at a temperature below the freezing point (0 ° C or below) of the water contained in the mixture, and a vacuum pump This is achieved by reducing the pressure using a method such as sublimation and evaporating water. The lyophilization conditions are not particularly limited, such as temperature, degree of vacuum, and drying time.

  When this material is applied to the oral cavity such as a tooth extraction socket, these effects are combined to enable early bone repair without infection.

  According to the bone defect filling material of the present invention, by using a porous material, the formation of blood clots by intrusion into the blood is promoted, and the immunoreactive ability of chitin derivatives allows infection such as tooth extraction fossa. It can be used without any problem even in application to a site where there is a high risk. Further, by adopting a configuration in which calcium phosphate granules are supported between the fibers of the fiber assembly, it is possible to prevent the granules from being settled and causing uneven distribution of the granules during the preparation of the bone repair material. Therefore, it can implement | achieve without intrusion into the inside of a bone cell. In addition, since the fiber aggregate is almost completely provided with continuous pores, it is possible to actively invade bone cells. Furthermore, since the calcium phosphate granules are exposed in the continuous pores, the osteoconductivity is sufficiently exhibited, promotes the formation of bone matrix by osteoblasts, and leads to early bone tissue regeneration. And, by appropriately controlling the elution characteristics, bone regeneration much superior to natural healing became possible.

  In addition, according to the production method of the present invention, a step of forming a fiber of chitin or a derivative thereof, a step of crosslinking the fiber at 130 ° C. to 160 ° C. in vacuum, the calcium phosphate granules and the crosslinked fiber The bone defect filler can be manufactured from a step of mixing with water having a water retention amount or less to form a substantially uniform mixture and a step of freeze-drying the mixture to form a porous material. Examples are shown below.

  Examples are shown below, but the scope of the present invention is not limited to the following examples.

  3 g of CM chitin raw material was precisely weighed, added to a glass container together with 72 g of pure water (Milli Q water), stirred and dissolved with a magnetic stirrer to prepare a 4 wt% CM chitin aqueous solution. In order to accelerate the dissolution of CM chitin, the container was heated to 30 to 50 ° C. in a water bath and stirred for 3 to 5 hours to obtain a homogeneous solution on the visual level.

  The solution was poured into a beaker containing 150 ml of ethanol and rapidly stirred with a glass rod. By this operation, a short fibrous CM chitin precipitate has been deposited. The precipitate was transferred to another container and further washed several times with 100 ml of ethanol. After removing as much ethanol as possible, the precipitate was placed in a dryer at 40 ° C. for 12 hours or more to evaporate ethanol and residual moisture. Then, it processed for 12 hours at the predetermined temperature of 120-170 degreeC under pressure reduction (about 2 kPa or less) with the vacuum heat processing apparatus.

The above-mentioned CM chitin, β-TCP granules (particle size: 50 to 150 microns) and pure water were weighed at a weight ratio of 1: 5: 32, taken in a petri dish, and stirred and mixed with a spatula. The mixture was filled into a polystyrene cylinder of a size suitable for the subsequent experiments, and both ends of the cylinder were sealed with a PET film, and then immediately put in a freezer at −80 ° C. After 1 hour or more had elapsed, the mixture was completely frozen, and then the PET film on both ends was removed and the mixture was transferred to a lyophilizer to evaporate the water in the frozen state.
Thereafter, the specimen was taken out from the polystyrene cylinder and subjected to an extraction socket insertion test after γ-ray sterilization.

  In addition, a type not containing β-TCP granules was prepared by the same method for dissolution measurement.

  As a comparative control, a chitosan porous body and a chitin porous body were measured.

  For the chitosan porous material, a commercially available chitosan powder (deacetylation degree of 95% or more) was used, and a 4% concentration 0.13 mol / L formic acid solution of chitosan was prepared. (TCP / chitosan = 5/1 weight ratio), and the mixture was stirred near the freezing point, filled into a polystyrene cylinder, and freeze-dried. Then, in order to remove formic acid, after drying at 120 degreeC, it sterilized with the gamma ray and used for the extraction socket insertion test. In addition, for the dissolution rate measurement, a type not containing β-TCP granules was prepared by the same method.

  The production method of the chitin porous body is as follows. Commercially available chitin powder was used and dissolved in a calcium chloride dihydrate saturated methanol solution at a concentration of 1 wt%. Thereafter, the solution was dropped into pure water to precipitate fine crystals of chitin. The solvent is removed by several centrifugations, and pure water is 32 in weight ratio with respect to chitin (pure water / chitin = 32/1 weight ratio), and β-TCP granule is in weight ratio with respect to chitin 5 (TCP / chitin = 5/1), filled into a polystyrene cylinder and freeze-dried. Then, it sterilized with γ-rays and used for the extraction socket insertion test. In addition, for the dissolution rate measurement, a type not containing β-TCP granules was prepared by the same method.

[Elution experiment]
CM chitin without granule (vacuum heat treatment temperature 170 ℃, 160 ℃, 140 ℃, 130 ℃)
And chitin and chitosan specimens were used. A 10 mg sample (1.8 cm x 1.8 cm x 1.8 cm in size) was precisely weighed and eluted in an egg white lysozyme phosphate buffered saline (10 µg / ml) with stirring at 37 ° C for 120 hours. .

   Thereafter, the test solution was filtered, and the residue remaining on the filter was dried and weighed. The weight of the residue was subtracted from the weight before the test and divided by the weight before the test to obtain the dissolution rate (%).

When measuring the dissolution rate from a product, cut a 1.8 cm × 1.8 cm × 1.8 cm block from the product and weigh the weight (W ₁ g). Thereafter, elution was carried out under the above elution conditions, and the weight (W ₂ g) of the dried product remaining on the filter was weighed. The dried product was burned and the residue weight (W ₃ g) was weighed.

The elution rate of CM chitin can be determined by the following formula.

Dissolution rate (%) = {(W ₁ −W ₂ ) / (W ₁ −W ₃ )} × 100

[Embedded experiment in dog extraction cavity]
The experimental animals were beagle dogs (adult dogs, approximately 11 kg in weight), and the experimental sites were bimaxillary premolars (I1-I3). After tooth extraction under general anesthesia, the test body buried group was taken as the experimental group. The control group was a site where only the extraction of the same individual was performed, and the experimental period was 4 weeks.

   During the experimental period, the surgical site was observed every few days to check for the presence of suppuration and inflammation due to infection, and if infection was observed, it was excluded from the subsequent tests. At the end of the experimental period, the observed site was collected, and after tissue embedding, a tissue section was prepared and stained with toluidine blue, and the amount of new bone was measured. The bone mass was expressed as a ratio when the group in which only tooth extraction was performed was taken as 100.

In addition, as a comparison with the prior art, commercially available collagen sponge, hydroxyapatite granules and β-TCP granules were similarly embedded and evaluated.

   As is clear from Table 1, the bone defect filling material using CM chitin with an elution rate of 30% or higher had a higher bone defect repair rate than natural healing, and no infection was observed.

Although CM chitin, chitosan, and chitin porous bodies with an elution rate of 20% were able to suppress infection, the residual bone formation of the material in vivo was inhibited, resulting in inferior results to natural healing. In addition, bone formation with commercially available collagen sponge and apatite granules was similar to natural healing. Apatite granules tended to increase infection. Although β-TCP granules outperformed the bone repair rate, there was a clear tendency for infection to increase.

Claims (4)

Obtaining a chitin or precipitates of chitin or a derivative thereof obtained by mixing an aqueous solution of a water soluble organic solvent containing a derivative thereof,
Crosslinking the precipitate in vacuum at 130 ° C. to 160 ° C. to obtain a crosslinked fiber; and
A step of mixing the cross-linked fiber, calcium phosphate granules, and water and freeze-drying
A bone defect filler obtained by including
The bone defect filler, lysozyme in phosphate-buffered saline for 10 micrograms contained in 1 ml, temperature of 37 ° C., 120 hours, when immersed, 30% of the chitin or chitin derivatives component - Bone defect filler characterized in that 85% is eluted.   The bone defect filler according to claim 1, wherein the chitin or a derivative thereof is carboxymethyl chitin or chitosan.   The bone defect filler according to claim 1, which is used for a bone defect portion in an oral cavity. Obtaining a chitin or precipitate derivative thereof, an aqueous solution containing chitin or its derivatives are mixed with a water-soluble organic solvent,
Obtaining a crosslinked fibers by the precipitate was crosslinked at 130 ° C. to 160 ° C. in vacuo,
Mixing the crosslinked fibers with calcium phosphate granules and water below the water retention capacity of the crosslinked fibers to form a mixture;
Method for producing a bone defect filling material according to any one of claims 1 to 3, characterized in that it comprises a step of lyophilized the mixture.