JPH01179689A - Stabilizer for organism catalyst - Google Patents
Stabilizer for organism catalystInfo
- Publication number
- JPH01179689A JPH01179689A JP204888A JP204888A JPH01179689A JP H01179689 A JPH01179689 A JP H01179689A JP 204888 A JP204888 A JP 204888A JP 204888 A JP204888 A JP 204888A JP H01179689 A JPH01179689 A JP H01179689A
- Authority
- JP
- Japan
- Prior art keywords
- stabilizer
- heavy water
- biocatalyst
- organism
- catalyst
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000003381 stabilizer Substances 0.000 title claims abstract description 25
- 239000003054 catalyst Substances 0.000 title abstract 9
- XLYOFNOQVPJJNP-ZSJDYOACSA-N Heavy water Chemical compound [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims abstract description 62
- 108090000790 Enzymes Proteins 0.000 claims abstract description 36
- 102000004190 Enzymes Human genes 0.000 claims abstract description 36
- 238000006243 chemical reaction Methods 0.000 claims abstract description 7
- 239000011942 biocatalyst Substances 0.000 claims description 27
- 230000001580 bacterial effect Effects 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 10
- 108010093096 Immobilized Enzymes Proteins 0.000 claims description 3
- 230000006641 stabilisation Effects 0.000 claims description 3
- 238000011105 stabilization Methods 0.000 claims description 3
- 230000000087 stabilizing effect Effects 0.000 claims 1
- 239000000758 substrate Substances 0.000 abstract description 7
- 244000005700 microbiome Species 0.000 abstract description 6
- 230000006866 deterioration Effects 0.000 abstract description 5
- 239000000654 additive Substances 0.000 abstract description 3
- 238000002156 mixing Methods 0.000 abstract description 3
- 238000004321 preservation Methods 0.000 abstract description 3
- 239000002904 solvent Substances 0.000 abstract description 3
- 230000000996 additive effect Effects 0.000 abstract 1
- 230000003139 buffering effect Effects 0.000 abstract 1
- 230000002265 prevention Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 10
- 230000000694 effects Effects 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 235000011073 invertase Nutrition 0.000 description 3
- 239000001573 invertase Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000008351 acetate buffer Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- 241000589220 Acetobacter Species 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108010059881 Lactase Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 108700040099 Xylose isomerases Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- -1 ammonium sulfate Chemical class 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 239000002360 explosive Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 108010046301 glucose peroxidase Proteins 0.000 description 1
- JEGUKCSWCFPDGT-UHFFFAOYSA-N h2o hydrate Chemical compound O.O JEGUKCSWCFPDGT-UHFFFAOYSA-N 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- JUINSXZKUKVTMD-UHFFFAOYSA-N hydrogen azide Chemical compound N=[N+]=[N-] JUINSXZKUKVTMD-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 229940116108 lactase Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000008239 natural water Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000006174 pH buffer Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 238000001223 reverse osmosis Methods 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、生体触媒の安定化剤に関し、更に詳しくは、
保存時及び使用時に生体触媒の活性を長期間安定的に維
持できる生体触媒の安定化剤に関する。[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a stabilizer for biocatalysts, and more specifically,
The present invention relates to a stabilizer for biocatalysts that can stably maintain the activity of biocatalysts for long periods of time during storage and use.
生体触媒は、常温常圧で使用できることから、食品工業
や化学工業界で広く用いられている。特に再使用が可能
なためコスト面で有利な固定化酵素や、やはりコストの
低減を図る目的から酵素抽出の手間が省ける固定化菌体
が実用化されている。Biocatalysts are widely used in the food and chemical industries because they can be used at room temperature and pressure. In particular, immobilized enzymes, which are advantageous in terms of cost because they can be reused, and immobilized bacterial cells, which can save the labor of extracting enzymes, have been put into practical use in order to reduce costs.
バイオリアクターとして用いられている酵素または菌体
としては、例えば、異性化糖製造用グルコースイソメラ
ーゼ、低乳糖牛乳製造用ラクターゼ及び酢酸製造用アセ
トバクターアセチ（八ｃｅｔｏｂａｃｔｅｒ ａｃｅｔ
ｉ）等がある。又、バイオセンサーとしては、ぶどう糖
測定用グルコースオキシダーゼ・パーオキシダーゼ等が
ある。さらにＢＯＤの測定にも微生物固定化膜を用いた
センサーが使用されている。Examples of enzymes or bacterial cells used in bioreactors include glucose isomerase for producing high-fructose sugar, lactase for producing low-lactose milk, and acetobacter acetase for producing acetic acid.
i) etc. Furthermore, examples of biosensors include glucose oxidase and peroxidase for measuring glucose. Furthermore, sensors using microorganism-immobilized membranes are also used to measure BOD.
上記のように生体触媒はますますその応用範囲の拡大が
期待される。しかるに、以下に示す大別して２つの問題
点がある。As mentioned above, biocatalysts are expected to further expand their range of applications. However, there are two main problems as shown below.
（１）第１の問題点は、長時間の使用または再使用のた
めの保存中にその活性が劣化することである。(1) The first problem is that the activity deteriorates during long-term use or storage for reuse.
それに対して従来から劣化防止対策として種々の方法が
用いられている。例えば、硫酸アンモニウム等の塩によ
る塩析状態での保存、有機溶媒によって沈殿させてトル
エン等に！ｇ濁させる方法などがある。しかしこれらの
方法は、固定化生体触媒には利用できないという欠点が
あった。そこで固定化生体触媒の保存方法としては、基
質を添加する方法や、−２０℃程度の低温下で保存する
方法等が用いられている。しかし前者は基質の分解が生
じ、後者は、凍結融解時に生体触媒が失活することがあ
り、多量の生体触媒を用いる実プラントにおいては大規
模な設備が必要であり、かつ電気使用料の点から有利な
方法とはいえない。In response to this, various methods have been used to prevent deterioration. For example, storage in a salted-out state with a salt such as ammonium sulfate, precipitation with an organic solvent, and toluene, etc. There are ways to make it cloudy. However, these methods have the disadvantage that they cannot be used for immobilized biocatalysts. Therefore, as methods for preserving the immobilized biocatalyst, methods such as adding a substrate or preserving it at a low temperature of about -20° C. are used. However, in the former case, the substrate decomposes, and in the latter case, the biocatalyst may be deactivated during freezing and thawing.Actual plants that use a large amount of biocatalysts require large-scale equipment, and there are also concerns about electricity usage costs. Therefore, it cannot be said to be an advantageous method.
（２）第２の問題点は、保存中に、保存液中に微生物が
混入し、生体触媒の劣化がおこることである。(2) The second problem is that during storage, microorganisms get mixed into the storage solution, causing deterioration of the biocatalyst.
それに対する防止対策としては、保存液中にアジ化ナト
リウム等を添加することが多い。ところがアジ化ナトリ
ウムは、酸と反応して有毒なアジ化水素を発生し、又、
重金属とは爆発性の塩を形成し易い等、取扱いが容易で
ないという問題点があった。As a preventive measure against this, sodium azide or the like is often added to the storage solution. However, sodium azide reacts with acids to generate toxic hydrogen azide, and
Heavy metals have the problem of not being easy to handle, such as the tendency to form explosive salts.
本発明者らは、上記問題点を解決すべく検討した結果、
重水を用いることにより生体触媒の反応中及び保存中に
その劣化を防止することができ、かつ微生物の生育を抑
制することができることを見出し本発明を完成するに至
った。さらに、重水の使用は、他の安定化法、保存法と
併用でき、かつ再使用が可能であるとの利点をも有する
。As a result of studies to solve the above problems, the present inventors found that
The present invention was completed based on the discovery that by using heavy water, deterioration of biocatalysts can be prevented during reaction and storage, and growth of microorganisms can be suppressed. Furthermore, the use of heavy water has the advantage that it can be used in conjunction with other stabilization and preservation methods, and that it can be reused.
本発明は、重水を含む生体触媒安定化剤に関する。 The present invention relates to a biocatalyst stabilizer containing heavy water.
以下本発明について説明する。The present invention will be explained below.
本発明の安定化剤は重水を有効成分として含有し、その
含有率は約３０〜１００％、好ましくは約５０〜１００
％であることが適当である。又重水以外の成分としては
用途に応じて例えば水等の溶媒及びその他の添加剤（例
えば基質）を含有させることができる。The stabilizer of the present invention contains heavy water as an active ingredient, and its content is about 30 to 100%, preferably about 50 to 100%.
% is appropriate. In addition, as components other than heavy water, solvents such as water and other additives (eg, substrates) can be included depending on the purpose.
本発明の安定化剤は、重水をそのまま用いても、あるい
は重水と上記溶媒及び添加剤を適宜混合して得ることも
できる。さらに本発明の安定化剤としては、重水を得る
ための濃縮工程で得られる重水（Ｄ２０）及び水（Ｈ２
Ｏ）の混合物をそのまま用いることもできる。The stabilizer of the present invention can be obtained by using heavy water as it is, or by appropriately mixing heavy water with the above solvents and additives. Further, as the stabilizer of the present invention, heavy water (D20) obtained in the concentration step for obtaining heavy water and water (H2
A mixture of O) can also be used as is.
本発明の安定化剤は、そのまま生体触媒の保存液として
該生体触媒の安定条件（ｐＨ，温度等）で用いることが
できる。又、本発明の安定化剤は、生体触媒の基質を含
有する溶液に重水濃度３０％以上、好ましくは５０％以
上となるように添加混合することによって、あるいは該
安定化剤に基質及びｐＨ緩衝剤等を添加することにより
生体触媒の反応液として用いることもできる。The stabilizer of the present invention can be used as it is as a storage solution for the biocatalyst under stable conditions (pH, temperature, etc.) for the biocatalyst. Furthermore, the stabilizer of the present invention can be added to a solution containing a substrate for a biocatalyst at a heavy water concentration of 30% or more, preferably 50% or more, or mixed with the substrate and a pH buffer. It can also be used as a reaction solution for biocatalysts by adding agents and the like.
本発明の安定化剤を適用し得る生体触媒としては、例え
ば酵素及び菌体を挙げることができ、酵素は固定化酵素
も含み、菌体には固定化菌体及び休止菌体も含む。Biocatalysts to which the stabilizer of the present invention can be applied include, for example, enzymes and bacterial cells; enzymes include immobilized enzymes, and bacterial cells also include immobilized bacterial cells and resting bacterial cells.
前記生体触媒の保存液または反応液として用いた安定化
剤は、逆浸透等の処理をすることによって再生使用する
ことができる。The stabilizer used as the storage solution or reaction solution for the biocatalyst can be reused by performing treatment such as reverse osmosis.
重水は水の同位体の一種であり、天然水中０．０１〜０
．０１５％の割合で含まれている。通常重水は、重水を
含む水を水酸化すＩ−ＩＪウム等のアルカリ溶液として
電気分解することにより、濃縮、純粋化して得られる。Heavy water is a type of water isotope, and is present in natural water from 0.01 to 0.
．． It is included at a rate of 0.015%. Usually, heavy water is obtained by concentrating and purifying water containing heavy water by electrolyzing it as an alkaline solution such as I-IJium hydroxide.
現在重水は低価格とはいえない。Currently, heavy water is not cheap.
この点からは、本発明の安定化剤は、特に小型かつ高価
格のバイオセンサーの保存液として優れている。しかし
、前記のように本発明の安定化剤は再使用が可能である
。従って、重水が比較的高価格であっても、小型のバイ
オセンサーの保存液以外の目的で工業的に使用すること
も可能である。From this point of view, the stabilizer of the present invention is particularly excellent as a storage solution for small and expensive biosensors. However, as mentioned above, the stabilizer of the present invention can be reused. Therefore, even though heavy water is relatively expensive, it can be used industrially for purposes other than as a storage solution for small biosensors.
さらに、本発明には、低い濃縮率の重水そのまま用いる
こともできコストの低減が可能である。また、微生物を
用いた重水濃縮法も発明され（特開昭５０−１３２４０
０号）、重水の価格の低減化が期待される。Furthermore, in the present invention, heavy water with a low concentration ratio can be used as it is, and costs can be reduced. In addition, a heavy water concentration method using microorganisms was invented (Japanese Patent Application Laid-Open No. 13240-1980).
0), it is expected that the price of heavy water will be reduced.
以下本発明を実施例によりさらに説明する。The present invention will be further explained below with reference to Examples.
実施例１
市販のインベルターゼを用い、その熱安定性を調べるた
め、加速試験を行った。すなわち、インベルターゼをｐ
Ｈ５，５の酢酸緩衝液にて溶解した後、水または重水で
希釈した。この時点での重水濃度は、０．３０．５０及
び８０％とした。各溶液を、６５℃中に１０分間保持し
、残存活性を比較した。Example 1 An accelerated test was conducted using commercially available invertase to examine its thermal stability. That is, invertase is p
After dissolving in H5.5 acetate buffer, it was diluted with water or heavy water. The heavy water concentration at this point was 0.30.50 and 80%. Each solution was kept at 65° C. for 10 minutes and the remaining activity was compared.
残存活性の相対値を表１に示す。Table 1 shows the relative values of residual activity.
表 １
実施例２
酵母をＹＰＤ培地で４８時間培養後、その菌体を遠心分
離法によって集め、常法により、アルギン酸ナトリウム
に固定化した。この固定化菌体を、重水濃度Ｏ及び８０
％の酢酸緩衝液（ｐＨ５，５）中にいれ、７０℃中に１
０〜３０分間保持した後、固定化菌体中のインベルター
ゼの残存活性を測定した。残存活性の相対値を表２に示
す。Table 1 Example 2 After culturing yeast in YPD medium for 48 hours, the cells were collected by centrifugation and immobilized on sodium alginate by a conventional method. The immobilized bacterial cells were mixed with heavy water at a concentration of O and 80
% acetate buffer (pH 5,5) and at 70°C.
After holding for 0 to 30 minutes, the residual activity of invertase in the immobilized bacterial cells was measured. The relative values of residual activity are shown in Table 2.
表 ２
実施例３
重水濃度０及び８０％の培地中での大腸菌及び酵母の生
育速度を調べた。大腸菌にはＬＢ培地を、酵母にはＹＰ
Ｄ培地を用い共に３０℃で培養した。Table 2 Example 3 The growth rates of Escherichia coli and yeast in medium with heavy water concentrations of 0 and 80% were investigated. LB medium for E. coli, YP for yeast
Both were cultured at 30°C using D medium.
培養経過を第１図（大腸菌）及び第２図（酵母）に示す
。重水の存在により微生物（大腸菌及び酵母）の生育が
抑制されることがわかる。The progress of the culture is shown in Figure 1 (E. coli) and Figure 2 (yeast). It can be seen that the presence of heavy water suppresses the growth of microorganisms (E. coli and yeast).
本発明の生体触媒安定化剤は、生体触媒の反応中及び保
存中にその劣化を防止でき、かつ生体触媒保存中の微生
物の生育を抑制することもできる。The biocatalyst stabilizer of the present invention can prevent deterioration of the biocatalyst during reaction and storage, and can also suppress the growth of microorganisms during the storage of the biocatalyst.
第１図は大腸菌の培養結果を示し、第２図は酵素の培養
結果を示す。FIG. 1 shows the results of culturing E. coli, and FIG. 2 shows the results of culturing enzymes.
Claims (9)
安定化剤。(2) The stabilizer according to claim 1, containing 30 to 100% by weight of heavy water.
安定化剤。(3) The stabilizer according to claim 1, wherein the biocatalyst is an enzyme or a bacterial cell.
剤。(4) The stabilizer according to claim 3, wherein the enzyme is an immobilized enzyme.
。(5) The stabilizer according to claim 3, wherein the bacterial cells are dormant bacterial cells.
剤。(6) The stabilizer according to claim 3, wherein the bacterial cells are immobilized bacterial cells.
体触媒の安定化法。(7) A method for stabilizing a biocatalyst, characterized by allowing the biocatalyst to coexist with heavy water.
７に記載の安定化法。(8) The stabilization method according to claim 7, wherein the biocatalyst is stored in a storage solution containing heavy water.
７に記載の安定化法。(9) The stabilization method according to claim 7, wherein the biocatalyst is used in a reaction solution containing heavy water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP204888A JPH01179689A (en) | 1988-01-08 | 1988-01-08 | Stabilizer for organism catalyst |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP204888A JPH01179689A (en) | 1988-01-08 | 1988-01-08 | Stabilizer for organism catalyst |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01179689A true JPH01179689A (en) | 1989-07-17 |
Family
ID=11518445
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP204888A Pending JPH01179689A (en) | 1988-01-08 | 1988-01-08 | Stabilizer for organism catalyst |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01179689A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003018614A1 (en) * | 2001-08-24 | 2003-03-06 | Wako Pure Chemical Industries, Ltd. | Method of stabilizing substance altering in aqueous medium |
| US8911759B2 (en) | 2005-11-01 | 2014-12-16 | Warsaw Orthopedic, Inc. | Bone matrix compositions and methods |
| US9011537B2 (en) | 2009-02-12 | 2015-04-21 | Warsaw Orthopedic, Inc. | Delivery system cartridge |
| US9034358B2 (en) | 2003-12-31 | 2015-05-19 | Warsaw Orthopedic, Inc. | Bone matrix compositions and methods |
| US9333082B2 (en) | 2007-07-10 | 2016-05-10 | Warsaw Orthopedic, Inc. | Delivery system attachment |
| US9415136B2 (en) | 2003-12-31 | 2016-08-16 | Warsaw Orthopedic, Inc. | Osteoinductive demineralized cancellous bone |
| US9554920B2 (en) | 2007-06-15 | 2017-01-31 | Warsaw Orthopedic, Inc. | Bone matrix compositions having nanoscale textured surfaces |
| US9717822B2 (en) | 2007-06-15 | 2017-08-01 | Warsaw Orthopedic, Inc. | Bone matrix compositions and methods |
-
1988
- 1988-01-08 JP JP204888A patent/JPH01179689A/en active Pending
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003018614A1 (en) * | 2001-08-24 | 2003-03-06 | Wako Pure Chemical Industries, Ltd. | Method of stabilizing substance altering in aqueous medium |
| US9034358B2 (en) | 2003-12-31 | 2015-05-19 | Warsaw Orthopedic, Inc. | Bone matrix compositions and methods |
| US9415136B2 (en) | 2003-12-31 | 2016-08-16 | Warsaw Orthopedic, Inc. | Osteoinductive demineralized cancellous bone |
| US8911759B2 (en) | 2005-11-01 | 2014-12-16 | Warsaw Orthopedic, Inc. | Bone matrix compositions and methods |
| US8992965B2 (en) | 2005-11-01 | 2015-03-31 | Warsaw Orthopedic, Inc. | Bone matrix compositions and methods |
| US10328179B2 (en) | 2005-11-01 | 2019-06-25 | Warsaw Orthopedic, Inc. | Bone matrix compositions and methods |
| US9717822B2 (en) | 2007-06-15 | 2017-08-01 | Warsaw Orthopedic, Inc. | Bone matrix compositions and methods |
| US10357511B2 (en) | 2007-06-15 | 2019-07-23 | Warsaw Orthopedic, Inc. | Bone matrix compositions and methods |
| US10220115B2 (en) | 2007-06-15 | 2019-03-05 | Warsaw Orthopedic, Inc. | Bone matrix compositions having nanoscale textured surfaces |
| US9554920B2 (en) | 2007-06-15 | 2017-01-31 | Warsaw Orthopedic, Inc. | Bone matrix compositions having nanoscale textured surfaces |
| US9492278B2 (en) | 2007-07-10 | 2016-11-15 | Warsaw Orthopedic, Inc. | Delivery system |
| US10028837B2 (en) | 2007-07-10 | 2018-07-24 | Warsaw Orthopedic, Inc. | Delivery system attachment |
| US9333082B2 (en) | 2007-07-10 | 2016-05-10 | Warsaw Orthopedic, Inc. | Delivery system attachment |
| US10098681B2 (en) | 2009-02-12 | 2018-10-16 | Warsaw Orthopedic, Inc. | Segmented delivery system |
| US9220598B2 (en) | 2009-02-12 | 2015-12-29 | Warsaw Orthopedic, Inc. | Delivery systems, tools, and methods of use |
| US9011537B2 (en) | 2009-02-12 | 2015-04-21 | Warsaw Orthopedic, Inc. | Delivery system cartridge |
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