JPS6162459A - Repairing of bone using collagen - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed Description of the Invention] [Technical Field] The present invention is based on the invention! #Related to the repair of bones, especially of mammals and humans. More particularly, the present invention relates to a method of bone repair using collagen-based implant materials to promote bone conduction repair. This collagen material is highly purified, non-immune and may, if desired, belong to a different species than the recipient.
Reconstituted material from skin or bone-derived materials or mixtures thereof.

[Technical Background] Repair of defective bone is a problem that has plagued humanity for centuries. Until relatively recently, the only practical method was to fix broken bones and rely on the natural regrowth of bone tissue in the injured area. However, advances in surgical techniques have made it possible to use grafted bone substitutes not only to replace damaged or diseased bone structures, but also to repair congenital and acquired defects in the skeletal structure. Became.

Since then, a wide variety of materials have been utilized, and sophisticated methods have been disclosed for replacing entire canals, such as hip joints (U.S. Pat. No. 3,820,187) or teeth (U.S. Pat. No. 4,820,488). has been done. Among the materials used were titanium (EP publication 0071242. February 9, 1983:
metals such as U.S. Pat. No. 3,918,100), ceramics such as aluminum oxide (U.S. Pat. No. 3,919,723), molded or treated bone (U.S. Pat.
318,774), and various bone preparations, such as bone powder compacted into a flexible mat (US Pat. No. 2,821,145).

It is well known that skeletal tissue contains both inorganic and organic components. Most of the inorganic components that are probably responsible for the strength and hardness of the bone structure exist in the form of calcium phosphate, or hydroxyapatatite. The organic component is also primarily composed of a single type of protein, collagen, which maintains elasticity and prevents the bone structure from becoming unduly brittle. Of course, since skeletal tissue is living, additional metabolically active organic components must be present in the bone structure, and these bone cells and their active metabolites participate in the natural healing process and in the maintenance of treatment. Contributing.

However, since the main components of bone are collagen and ceramic in quantity, implants have also been used in a variety of reconstructions, including similar or different ceramic materials as well as various types of collagen preparations. For example, Hayashi, K.
,,et al,Arc OrthopTraum
at Surg (1982) 99:265. and U.S. Pat. No. 4,314,380).

It is known that bone repair occurs by one of the following two mechanisms or a 4y1 mechanism that is a combination of these mechanisms.

That is, conductive repair
In r), cells that already have characteristics as bone cells (
ischial cells) migrate from adjacent bones into the defect space and directly form bone. In this case (other than non-specific nutrients)
No special factors are required. However, in induction repair, this process is preceded by the conversion of undifferentiated cells with multiple potentials into ischial cells. The ischial cells first form cartilage, which is calcified and transformed into bone. The ability to do this requires specific protein factors. The nature of these factors is currently not understood. For both conductive and guided repair, a quantitative amount of skeletal tissue must be provided from the host's living tissue.

Therefore, implants that mediate these processes do not act as replacements for defective or removed bone, but rather as stromal supports to activate regeneration of the missing tissue. .

It is therefore precisely for this purpose that attempts have been made to devise implants for defective skeletal tissue or for bone or dental lesions. These implants do not mimic the composition of the missing bone, but rather provide 411 structural supports,
and ultimately act as a guiding matrix for gradual invasion of bone deposits arising from adjacent fresh bone, such supports merely serve the function of matrix supports (i.e.
mediate conduction repair) as well as currently 'bone morphogenetic factors'
Collagen may also contain factors that promote the differentiation of undifferentiated cells into rebellious cells by adding what is known as bone morphogenetic protein J (OF) or bone morphogenetic protein J (BMP). Attempts have been made to use implants consisting largely of collagen for the purpose of both inductive and conductive bone repair, as it is already a well-known substance in the metabolically viable cells associated with collagen. ing.

For implants useful in guided repair, see U.S. Pat.
No. 94,753 discloses a method for producing bone morphogenetic protein. A method of purifying OF, perhaps different from the EMP of the aforementioned patent, is described in U.S. Pat. No. 4,434,094 (1984).
Published on February 28, 2017). These factors are used in bone as well as in the manufacture of implants, presumably to release the factors in an effective manner. Naturally present in demineralized bone particle preparations.

These factors are used in many implants in purified and unpurified form.

They also disclosed an attempt to utilize only a collagen preparation as a substrate for conductive bone repair activity, i.e., a preparation that does not contain factors for the maturation of protocells into osteogenic cells and thus mediates conductive repair. There are also researchers.

Krekeler (V, G, etc.)
al, J.

0ral Surg (1981) 10: 5u
pp1. 1:151) compared the effectiveness of autologous cancellous tissue, a collagen preparation (Collagen Fleece; trade name, Pentafirm), and bonded gelatin as fillers for trout defects in pegle dogs. These preparations were simply filled into artificially created bone cavities, and the healing process was followed by sequential polychrome labeling. Although collagen fleece was found to mediate adhesions.

The effect was smaller compared to autologous cancellous grafts.

The collagen fleece used in the above formulation was made from pig skin according to the method disclosed in US Pat. No. 4,086,083. Pig skin is cut into small pieces, degreased and washed with detergent, then digested with pepsin to form a viscous suspension, and saturated salt solution is added to precipitate collagen. This precipitate is suspended in chains and then reprecipitated as a fibrous white precipitate in a salt solution. The precipitate is washed as many times as necessary and then washed with alcohol to desalt. Purified collagen is suspended in an acid solution and freeze-dried. This is sterilized by γ-ray irradiation, but the preparation may deteriorate or crosslink during this process. This preparation is made from collagen fleece (C:ollagen).
fleece) and has been used in several other bone repair studies.

Joos, U, et al, Bi
materials (+980) 1: 23-2
G) Using collagen fleece as a graft for artificially injured rabbit mandibles, we found that the defect was filled with mesh-like bone powder after 2 weeks, and completely ossified after 4 weeks. Ze Lzrnan et al.
et al, 5chveiz MschrSabn
Helik (1982) 92:119) also succeeded in bone regeneration in facial surgery using Lage 77 Ris as a graft. Spring Rum etc.
orum.

H, W,, et at, Z, 0rthop (1
977) 115:88B) obtained similar results using collagen fleece in defects in the cortical layer.

Jaffee et al.
Arches Oral Biol (1878) 23
: 415; 1 bid, (1f382) 27 :
999) is extracted from dog skin with acetic acid and trichloroacetic acid/
We report the successful fixation of acrylic acid resin tooth grafts in dogs using a collagen solution purified with ethanol. Grafts that were successfully fixed remained intact after 1 year.

Cucin 9 (Cucin, R, L, et at,
Nav York State Journal of
Medicine (1979) 185B) is
Gamma-irradiated atelopeptide collagen from calf skin was used to repair rabbit ribs.

It has also been used to repair holes in the skull of dogs, supported by gelatinous sponge material or autologous bone powder.

Filling the dental pulp cavity with a collagen preparation cross-linked by γ-ray irradiation, while probably still containing telopeptide, and subcutaneously [for ossification and for use as a graft, respectively, EP publication 00124
43 (June 25, 1980) and EP release 0012
No. 959 (July 9, 1980).

[Problems to be resolved] None of the above-mentioned collagen repair methods have been completely successful, and may cause inflammation or incomplete adhesion (especially when collagen of a different type to the transplanted area is used). The basic objective of the present invention is to overcome the above-mentioned drawbacks, and it is an object of the present invention to provide an implantable collagen preparation capable of transmitting ingrown bone repair tissue from predetermined bone cells to the defect desired to be repaired. shall be.

[Disclosure of the Invention] Since the present invention can use collagen of a different type from the transplanted area, it can be obtained in large quantities and has an extremely wide range of applicability. It is extremely applicable to various applications.

In the present invention, defects obtained from an organism foreign to 111 are repaired (if necessary). Provided are methods for repairing bone defects and regenerating skeletal matrix in 1f+ mammals (particularly humans) by transplanting purified non-immunogenic collagen. Accordingly, the present invention also provides a method of mediating the natural repair mechanisms of bone defects in living tissues using highly purified, versatile collagen formulations that can provide a matrix for new bone growth.

In the method of the present invention, living talar cells (ost
Fresh bone containing aoprogenitor cells is exposed to the defect and contacted with a collagen preparation, a composition obtained from either or both bone and skin.

Collagen originating from bone is bone (DMB) from which inorganic components have been removed.
and is substantially composed of Type I from which the telopeptide has been substantially removed. It is produced by treating DMB with a non-collagen-degrading protease, such as trypsin, which destroys factors mediating guided repair and removes telopeptides.

This material is obtained in powder form and is a bone collagen powder (B C
Collagen of skin origin, referred to as P), is primarily type 1 collagen with small amounts of type m and is typically obtained from calf skin. This type of collagen is called Zyderm (Zyde
It is commercially available under the name Collagen Implant (RN, trade name). This collagen preparation, typified by ZCR, is reconstituted microfibrillar atelopeptide collagen.

Furthermore, collagen preparations of dermal origin can be lyophilized before implantation.

By varying the ratio of BCP to ZCI in the mixture of these peptides, the physical properties of the repair material can be adjusted to adapt to the specific requirements of the defect and the surrounding environment.

In one aspect, the present invention includes (a) exposing a fresh bone surface with living talar cells to the defect;
b) A method for repairing bone defects, comprising filling said defect with a collagen preparation and bringing it into contact with fresh bone surface. The collagen preparation is purified reconstituted atelopeptide microfibrous dermal collagen or bone collagen powder (as defined above), or a mixture thereof.

The purified reconstituted microfibrous collagen may be freeze-dried and cooked.
This lyophilized preparation (lyophilized collagen gel or LOG
) can be easily handled by rolling or casting into a plate shape, and this can be easily used as an implant (piece).

Collagen of skin IG origin is typically produced from the skin through a process of dissociation into a solubilized form, sterilization by filtration, and reconstitution into a fibrillar form after telopeptide removal.
A representative formulation has been described as useful for soft tissue repair and is commercially available for this purpose under the name Zydermu Collagen Implant (ZCI). The method for making this is described in detail in US Pat. No. 3.94J073.

Bone nilagen powder is obtained from demineralized bone (DMB) and consists essentially of type I collagen from which telopeptides have been removed. This is produced by treating DMB with trypsin. Trypsin destroys both bone formation and telopeptide related factors. The bone collagen powder (B CP) obtained by this method - together with the above-mentioned preferred properties rj also has stress resistance and becomes a load support for skeletal defects.

In other aspects, the present invention relates to collagen preparations useful in the present invention and methods for producing the same.

EMBODIMENTS OF THE INVENTION A. Definitions As used herein, "conduction" of bone defects
tive) repair refers to the process in which missing bone is replenished or desired new bone grows, and it is the process by which the metabolism of osteoprogenitor cells (osteoprogenitor cells), which were originally instructed to be These cells can generate cartilage and/or bone without induction by protein factors, commonly known as osteogenic or morphogenetic protein factors. The production of differentiated cells does not require the addition of protein factors.

It is understood that the transformation of undifferentiated cells into ischial cells can also be carried out by proteins specific to the body. However, "conductive" bone repair, as defined herein, is mediated only by an externally supplied supporting matrix, and not by providing xenobiotic tissue or non-endogenous osteogenic factors.

A "bone defect" is a gap in skeletal tissue to which bone attachment is desired, and this gap is caused by injury to the living body or congenital abnormality or deterioration of the skeletal tissue of the living body. These defects may occur as a result of a simple fracture, breakdown of bone tissue due to disease, surgical removal of diseased bone tissue or undesirable congenital anomalies, or Mochio surgery or cosmetic surgery.

"Fresh bone" refers to bone in the skeletal system of living tissue that is in a healthy state and is processed by making an incision to expose the living tissue. It has been found that fresh bone needs to be brought into contact with the defect in order to provide living cells for regeneration.

"Collagen preparation" refers to a composition containing a certain type of collagen protein as a main component.

"Xenogeneic" refers to a species that is different from the species in the organism being treated.

“Contains no impurities” or “purified” means in its natural state.
This refers to the absence of impurities normally associated with preparations such as collagen. In other words, collagen prepared from calf skin will not contain any impurities if components other than calf skin I+W are substantially removed, and collagen preparations from bones will not contain any impurities if components other than the bones are removed. It will not contain any impurities.

"Reconstituted J-collagen is a three-helical molecule that is separated into a solution with or without the tecopeptide extensions, introduced into a solution, and then reconstituted into fibrillars. In this form, microfibrils are composed of long and thin collagen molecules that overlap each other in a zigzag shape and have a width that is about 1/4 of the length.As a result, they form a band-like structure and are further aggregated. can become fibrous.

"Substantially free of crosslinks" refers to collagen formulations that have telopeptides removed and lack the inherent ability to form crosslinks.Such formulations are treated with glutaraldehyde, for example, or are treated with simulative forms of such bonds. It is not substantially crosslinked unless it is intentionally crosslinked by a treatment to form it. Additional crosslinks are formed between the helical molecules due to treatments that are often performed for sterilization purposes, such as high temperatures or γ-ray irradiation.In the formulation used in the present invention, skin-derived collagen is dispersed in a solution. Fflicrofiltration
and sterilization followed by processing under sterile conditions, thereby avoiding the formation of undesirable crosslinks.

"Bone Collagen Powder J (BCP) is a purified atelopeptide preparation of bone-derived collagen from which inorganic components have been removed. This preparation consists essentially of collagen itself and does not contain metabolically active proteins. Because it is derived from bone, 1
It is composed of type collagen and has a structure very similar to the unique three-dimensional structure found in bone.

B. Detailed explanation B1. Applications The bone defects to which the present invention is applied include voids in the bone tissue that need to be filled in order to complete the skeletal tissue. Medical or veterinary methods include orthopedic surgery, removal of diseased bone tissue and replacement with overlays or prostheses, fixation of loose teeth, replacement of teeth, repair of trauma, etc. The exact method of applying the collagen preparation will vary depending on the nature of the bone defect as well as the treatment chosen, as will be understood by those skilled in the art. Generally, however, the composition of the present invention is in the form of a paste or gel, and the collagen preparation of the present invention can also be shaped into the shape of the defect by surgically filling the paste into the defect. If mixed to form a more dilute suspension, it can be injected into the defect. However, in either case, the defect to be treated must first be cleaned to expose a fresh bone surface and allow living bone cells to communicate with the defect. It is necessary to obtain the canal thick cells required for the synthesis of the bone structure.

B2. Collagen preparationsNatural collagen consists mostly of triple helical structures, which contain repeating triplet sequences in which glycine is linked to two amino acids (usually proline and hydroxyproline), so there are three glycines in the chain. It appears in every eye. Furthermore, at each end of every collagen chain there is a non-helical region containing a glycine triplet sequence. These areas are believed to be relevant to the immunogenicity associated with most collagen preparations. Immunogenicity can be largely eliminated by removing these regions to create "atelopeptide" collagen. This can be accomplished by digestion with proteolytic enzymes such as trypsin or pepsin. These differences in the specificity of proteolytic enzymes result in differences in the completeness of telopeptide removal. Therefore, certain proteolytic enzymes that are capable of the most complete removal are preferred; such enzymes include trypsin, which is capable of removing substantially all of the telopeptide moieties.

The non-helical telopeptide region forms cross-links and
It is also necessary to stabilize the fiber structure.

These regions contain aldehydes that can be crosslinked to lysine residues. If necessary, the atelopeptide collagen must be artificially crosslinked.

Although all collagens share the above-mentioned properties, collagens are classified into approximately 10 types based on the exact order of amino acids in the individual chains of the triple helix, carbohydrate content, and the presence or absence of disulfide bridges. The common subtypes are type I and type II, and type I exists in the skin, calf and bone and is formed from fibroblasts. In addition, type ■ is mainly present in the skin. Other types exist in specialized membranes or cartilage, or on cell surfaces. Type I and Type II contain several amino acids in their helices, but include two cysteines close to the C-terminus of the triple helix that can be internally crosslinked, whereas Type II does not.

111, l collagen has one α2(I)m and two α trees
1 (I) chain, each of which has 101 chains in its triplex region.
Contains 4 amino acids. Also, on each brocade is the number I + I
n):4. ! k lk ++ n b 4)
h()i presu m J[++ 1
-1 - )t' ・ノJ斗αl (III)
(3 chains), which contain 1026 residues in their triple helical region; internal crosslinks are stabilized by a pair of vicinal cysteines at the carboxy terminus of the triple region of type m collagen, as described above. be converted into Both collagens contain short non-triple helical ends (telopeptides). The reconstituted microfibrous atelopeptide collagen used in the present invention contains both type 1 and type II atelopeptide forms, whereas bone collagen powder consists exclusively of type 1 atelopeptide form.

Two-layer one-source collagen Representative atelopeptide reconstituted microfibrous dermal collagen preparations useful as the composition of the present invention (1) Commonly sold under the trade name Zyderm (ZY [IER'M) and Collagen Implant (ZCI)] has been done. There is a reconstituted fine fibrous suspension derived from purified calf skin atelopeptide collagen.

This and other skin measures Origin-purified atelopeptide reconstituted microorganism 1
B Male collagen is well known to those skilled in the art. ZC
I is widely used for soft tissues, including wrinkle removal and repair of dented injury areas by injecting the preparation directly into the skin. However, such formulations have not been used for bone repair, except for their use in conjunction with support materials that also contain OF and are intended for guided bone repair; such applications are described in U.S. Patent Application No. 348,414 (February 1, 1882
(filed on the 2nd).

This collagen preparation is obtained from calf skin,
Atelopeptide collagen, which mainly contains type 1 collagen and approximately 1-5% type II collagen, is sterilized using appropriate filtration techniques to remain at 8% in solution, so no deterioration or cross-linking occurs. is reconstituted into fine fibers and packaged under sterile conditions.

(Kojin Shimokanedai) ``Collage・Go1゛15...1 Collagen When this collagen is freeze-dried to make freeze-dried collagen gel (LCG), it is firmly fixed in the transplanted or filled cavity and has excellent properties. In addition to exhibiting the ability to resist migration from a desired location through structural retention, LOG matrices also exhibit the desirable and necessary property of supporting the union and regrowth of bone tissue into the implanted area. As explained in more detail later, the LCG9 agent of the present invention can obtain a wide range of physical properties by controlling the pH 1 freezing rate and the concentration of the suspension upon freezing. Regardless of the desired properties, part of the characteristics of the LOG formulations of the present invention is that they consist of substantially impurity-free atelopeptide-reconstituted micro-yang male collagen. Even when this formulation is sterilized by microfiltration and processed under sterile conditions, there is still virtually no cross-linking; additional freeze-drying results in a mat-like form that can be easily cut with scissors or a sharp knife. When wet, this mat yields a putty-like material that can be formed into any shape and inserted into the defect. .

Other forms of medical lyophilized collagen have also been disclosed, but these are different from the collagen formulations of the present invention. Battista, U.S. Pat. No. 3,47
No. 1,598) discloses a lyophilized collagen preparation in an intermediate microcrystalline form obtained by treating bovine skin with saline to swell and then separating the collagen fibers. This collagen has not been purified and the telepeptides have not been removed. The material produced in this way is suitable for use as a water-absorbing sponge, but it is clearly not suitable for direct medical use as it contains impurities and is immunogenic. is not suitable. Furthermore, the acid used to produce this intermediate texture remains in the sponge. Kunz et al. (
Kuntz et al, U.S. Patent No. 3°368.9
No. 11) disclosed a method for producing a water-absorbing collagen sponge in which carboxylic acid is used instead of the soft, non-volatile acid used by Batista so that no acid remains. This formulation discloses the use of substantially pure collagen fibers, but does not remove telepeptides, Miyata (
Jiyata, US Pat. No. 4,294,241) discloses lyophilized flagen sheets for skin dressings.

This sheet is manufactured from atelopeptide collagen reconstituted into fine fibers, but differs from the LOG of the present invention in that they are artificially crosslinked. Rie
s, U.S. Pat. No. 4,066.083) is an Ichisaka product for the treatment of wounds, collagen fleece (fl:olage
n fleece, trade name) is disclosed. Luck and Daniels
, US Pat. No. 4,233,360) describes a freeze-explosion formulation, which results in a foam rather than a mat as in the present invention.

The LCG of the present invention is typically purified reconstituted microfibrillar atelopeptide collagen, such as Zygoome (Zyder resistant).
It is manufactured from Ecollagen Implants or Zyderm II Collagen Implants (both trade names for reconstituted collagen preparations manufactured by Bovine Branch').

Reconstitution purified skin collagen raw material 1 For example, skin elasticity of other mammals can also be used. In either case, however, the starting material must be prepared from these raw materials by a process that reconstitutes substantially pure collagen microfibrils from a solubilized form. Furthermore, the telopeptide must be removed by appropriate steps, such as tryptic digestion. Typically, such purified preparations involve finely dividing the starting material and then adding enzymes and/or a suitable pH.
The collagen is then treated with digestive enzymes such as trypsin to remove telopeptides, sterilized by microfiltration, and the pH and salt concentration adjusted appropriately ( In other words, it is produced by reconstituting collagen microfibrils (from acidic to neutral). Such formulations are known to those skilled in the art and, in fact, products using them are commercially available.

The use of reconstituted collagen in the production of freeze-dried forms is also important from the standpoint of preventing deterioration during the sterilization process. Collagen takes a solubilized form at one stage of the manufacturing process, so microfiltration is possible, and this process is completely neutral, so the structure does not change due to WJ.
No significant chemical or physical changes occur. 7. Perform subsequent steps in a sterile state. ! ? If you do this, north of this will be &1. Because no bacteria are required, there is no degradation or cross-linking that would occur if the product were subjected to harsher sterilization conditions such as heating or gamma irradiation.

In producing the LCG of the present invention, a suitable collagen suspension solution is cast at a temperature of about 20-100 mH/m or extruded into a sheet. These molded bodies were then frozen at approximately -10 to -50°C and then cooled to a condenser temperature J.
Lyophilize at about 10-100 ml 11-tarr with R about -45 to -55°C and a shelf temperature of about -30°C. After freeze-drying for 1-7 days, the shell temperature is raised to 15-25°C, and after another 24 hours, the LOG is removed from the freeze dryer.

Alternatively, purified collagen in solubilized form can be used. Such a solution is prepared by dissolving purified atelopeptide collagen in an aqueous solution at about pH 2-3, optionally sterilizing it by microfiltration, and then bringing the pH to approximately neutral using, for example, a phosphate buffer to precipitate the reconstituted form. Manufacture. The resulting precipitated S-collagen is collected by centrifugation, and the pelleted collagen is resuspended in a neutral buffer and processed as described above.

There are other forms of lyophilization that represent improvements to the previously described methods and provide better results in certain applications. Culture plate (e.g. 35 mm) containing sterilized collagen suspension
6. Then, freeze the cast body at about -100 to -50°C, and smooth the obtained cast body with a spatula etc.6.
Preferably, it is kept at about -80°C for 1 hour and then freeze-dried.

It is freeze-dried for about 24 hours at about 10 to 100 millitorr to obtain a porous mat with a thickness of several mm.

Green and Bunjij! Bone collagen powder (BCP) used in the present invention is obtained from bone from which inorganic components have been removed, and therefore its collagen component is exclusively type I collagen.

This collagen preparation is a new material with physical and chemical properties different from conventionally produced forms of collagen, and can be conveniently stored in the form of a dry powder. After washing, freezing, and crushing the bones of mammals such as pigs, cows, and pigs, preferably dense bones, inorganic substances are removed using a suitable acid such as hydrochloric acid using a conventional method. Residual organic matter is separated and digested using proteolytic enzymes, either sequentially or in combination. Oliver and Grant (Oliver, Grant, British Patent, GB
Neutral proteolytic enzymes such as trypsin, which selectively degrade non-collagen proteins, are preferred, as described by 1565340A). Non-proteinaceous bone components, which are undesirable because certain acidic proteolytic enzymes such as pepsin partially digest collagen, can also be removed using appropriate enzymes such as chondroitinase, hyaluronidase, and various nucleases. Of course, the use of collagenase is inappropriate. Appropriate I! The insoluble material after treatment with f-prime consists of purified atelopeptide in BCP form. This is γ
It can be used after sterilization by known methods such as irradiation or heating. The presence of crosslinks in this type of collagen is often preferred, as any residual antigenic effects remaining after trypsin treatment are alleviated.

The BCP obtained in this way is new.

It is woody and consists of type 1 collagen, which apparently retains the original molecular structure of bone collagen, but does not contain telopeptides.

The BCP is resuspended in physiological buffer for use by injecting into appropriately prepared bone defects.

When a mixture is used to produce the implant or pasted restorative material of the present invention, the material should be prepared according to the material's handling needs and the physical properties necessary to cope with the stresses expected to be placed on the repair site. A suspension of reconstituted microfibrillar atelopeptide collagen such as ZCI and BCP are used in the ratio range.

Increasing the amount of BCP can impart weight and stress resistance to the composition, while decreasing the ratio of BCP to ZCI increases flexibility and applicability. Therefore, the method of the present invention is extremely flexible in repairing biological defects, depending on its nature, compared to conventional methods.

The range of ratios is of course large enough that either component can be used alone. The suitable mixture depends on the nature of the defect, so
A single generally preferred range cannot be specified, but generally about 50% ZC for a defective area bearing 1 weight.
I750%BCP ~ approx. 10%ZCI/90%BCP (
% by weight) is preferred, and for periodontal ligament or surface defects, from about 90% ZCI/l O%BCP to about 50% Z
CI 150% BCP (wt%) is preferred (the above ratio assumes a 35 mg/m suspension of ZCI, weight refers to the weight of the total suspension), c.
t+ The present invention will be described below, but this is not intended to limit the invention.

C, I Z start-up lj Zyderm collagen implant (Col jagan
Corporation (purchased from Palo Alto, Calif.) was used as the reconstituted microfibrillar atelopeptide component. Reconstituted natural collagen micro 1am was used at a concentration of 35mg/mu. This formulation was filtered and sterilized during processing, so processes that would cause deterioration such as heating or irradiation were not required.

C. 2L and 1 concentration A. Pepsin-cho solubilized cow skin collagen aqueous solution of approximately pH 2 to 3, sterilized by microfiltration, is mixed with phosphorus #salt buffer to pH 7.4.
To: AfrAshita. After collecting the forged collagen by centrifugation, it was collected at a concentration of 35 mg in a phosphate buffer solution with a pH of 7.4.
/mu. The suspension liquid was cast into a sheet, and this sheet was frozen at -25°C.
Freeze-dried at 25 milli-tarr. After storing the lyophilized sheets at -30"C for 5 days, the temperature was raised again to 20"C and held for 24 hours immediately before use.

B. As an alternative (or improved method), approximately 5 cc of Zyderm collagen implants were sterilized.
Cast into 5 mm tissue culture plates.

Smoothed with a spatula. This collagen gel was frozen and kept at -80°C for about 1 hour. Next, freeze-dry the collagen gel for about 24 hours until it dries, and then dry it directly (￥35
A porous white disk with a thickness of 3-5 mm was obtained.

C, 3×322 m5 A- The bovine femur was hand-washed to remove attached soft tissue, and the articular end of the bone was removed near the epiphyseal plate to obtain essentially dense bone. The diaphysis was cooled with liquid nitrogen (T-N2), split axially, and the bone marrow was removed. 6 Compact bone fragments were crushed into fragments with a particle size of 5 mm or less using a show crusher at LN2 temperature. This bone fragment was ground using a hammer mill, also at LN temperature.

Sift the bone meal under running water to 125-250pm and 250pm.
Divide into two particle size portions of ~450 pm. These two types of grain size portions were processed separately in parallel.

Using 0.5M HCl (HCuC for 1g of bone powder
Inorganic components were removed from the bone powder over a period of 3 hours at room temperature by exchanging the u solution with 2 teric acid. At the end of the collector component removal process, the bone meal was washed three times with water to remove acid-soluble inorganic substances. Bone powder from which inorganic matter has been removed is
Prior to proteolytic treatment, the mackerel was freeze-dried.

Freeze-dried bone meal was digested with trypsin (0.5 mg
/m sentence 0.1M Na7 HPO41m including NaN3
50 mg bone meal and 2 mg trypsin per J1; ie trypsin: bone meal = 125). Enzymatic digestion at 15°
I went to C for 10 mouths. The digestion supernatant was analyzed for thermogenic proteins by the Buret method to monitor the progress of digestion.

To remove trypsin digestion products, the bone meal was sedimented, the supernatant liquid was separated, and a 4M NaCJ1 solution (1
0:l/volume duplex) was added. The bone meal was stirred at room temperature for 1 hour, at which point the powder was allowed to settle and was decanted. Extraction with 4M NaCM was repeated a total of three times.

To remove sodium chloride, it was washed six times in 10 times the volume of Water for Injection (WFI), United States Pharmacopoeia, with stirring for 10 minutes as described for the NaCl extraction.

The bone meal was then extracted twice with 10 times the volume of acetone to remove any lipids present. After acetone extraction, the bone meal is subjected to WFI
Bone collagen powder (B CP) was obtained by washing 5 times with water, vacuum filtration, and freeze-drying.

BCP samples were mixed with lipids, glycosaminoglycans (41yc
osamino-glycan), and chemically about the amino acid composition, residual trypsin, stub blood cells,
and bovine collagen telopeptide (an antigenic terminal of collagen) were analyzed immunologically and ultrastructurally by electron microscopy. As the trypsin treatment progresses, preferably within 4 to 10 days, the BCP gradually becomes pure engineered bovine collagen containing almost no telopeptides. Using an electronic microscope, characteristic band-shaped collagen microfibers and bovine bone tissue were observed. In addition, it was determined from the amino acid composition that it was chemically pure collagen. BCP produced in this way has two types of particle sizes.
Furthermore, as shown in Table 1, there were samples with different degrees of trypsin treatment. All samples were sterilized by gamma irradiation at 1.5 Mrad.

Table 1 Summary of BCP samples 22, 125-250 02A
125-250 12B 1
25-250 42C125-25010 3Z 250-425 03A
250-425 13B 25
0-425 43C250-425t.

Bone collagen powder (B
CP) at a ratio of 55745 (weight ratio) and 1.5Mr
It was sterilized by γ-ray irradiation with ad.

2 patches of BCP/ZC using this sterile BCP mixture
I mixture was prepared. i.e. dry BCP 0.33
A 33% BCP mixture of ZCI was prepared by mixing 0.67 g of ZCI (35 mg/m suspension, i.e., solids: approximately 0.87 g x O, 035 g/m). In addition, 17 g of BCPO was added to ZCI (3
A second mixture was prepared by mixing with 0.83 g of 5 mg/m suspension). Each of these materials was thoroughly mixed and filled into a 1-Vicc syringe, which was used in the following procedure.

Forty-five rats aged 6 to 8 weeks were anesthetized, the scalp was inverted, and the pericranium was removed. A 3 x 7 mm penetrating defect was made symmetrically in the cranial lateral wall bone using a dental drill.

The injectable collagen gel described in Section 0.1 was filled into the defect using a small spatula, and the scalp was replaced and sutured. Intra-defect healing rates after implantation of 2, 4.8 and 18
X-ray as well as histological monitoring was performed after al'fJ.

Two weeks later, new bone cell (osteoblast) islands and matrix were histologically observed within the collagen gel. By 4 weeks, in most cases, new bone had spread across the defect, and a fusion between the cut edges of the defect and this new bone had begun to form. 8
During ~16 weeks.

The junction between old and new bone is now almost indistinguishable. The new bone also reorganized into a layer of mine bone with a distinct marrow cavity typical of the bone originally present in the defect. X-rays of skulls removed from rats confirmed the presence of large amounts of radiopaque bone in the defect after 16 weeks. No significant fusion was observed during the 16-week observation period in rats with symmetrical craniolateral wall canal defects that were not transplanted. In the latter case, osteogenic activity was limited to a small portion of the cut edge of the defect, and the remainder of the defect was filled with loose connective tissue.

Thus, reconstituted collagen fibers provide a new and useful method for providing a supporting matrix for conductive bone growth.

C16LCG F Calcium Calcium -' A rat approximately 8 weeks old was anesthetized, the scalp was inverted, and 11 parts of the skull were removed. A 3 x 7 mm penetrating defect was made symmetrically in the bone of the cranial lateral wall using a dental drill. C,
Freeze-dried Kolaken produced according to 2B was cut into strips slightly larger than the defect so that the graft would be tightly encapsulated in the defect. Some of the strips were placed dry into the defect and hydrated therein, while some were hydrated prior to implantation. After transplantation, the scalp was replaced and sutured. The rate of healing within the defect was assessed histologically at 2 and 4 weeks after implantation.

After two weeks, there was sufficient infiltration of connective tissue and blood vessels into the graft, but new bone formation was very limited. Non-implanted defects of the type described in the previous section did not heal and remained filled with soft tissue, whereas repaired defects showed new bone formation throughout the graft by 28 days. In addition, signs of fusion with existing bone were already observed at the interface between the cut end of the defect and the graft. By 56 days, the presence of mature bone with a distinct marrow cavity was observed throughout the defect.

LOG can be easily cut to form inserts capable of supporting conductive bone growth. The resulting mat of freeze-dried reconstituted collagen fibers is a suitable material for this purpose.

C, 76, ゛ BCP 1゜ A 6-week old rat was anesthetized, and a 3 x 7 mm defect was created in the left and right canals of the cranium. A through hole is made in the skull with a dental drill, and the BCP prepared as described above is added to 1f of sterile food.
i The defect was filled with water after moist temperature. Control defects were left unfilled. BCP samples 2B, 3Z, 3A in Table 1 above
, 3B, and 3C were tested. Samples were collected 2 and 4 weeks after transplantation and examined histologically.

Samples 3Z (trypsin digestion not performed) and 3A (trypsin digestion 1 day) showed inflammation with evidence of antigenic components. Samples 2B, 3B, and 3C showed multinucleated cell reaction, digestion of BCP with osteoblast reaction, and new marginal bone deposition at 2 weeks. At 4 weeks, a large amount of bone had formed throughout the graft and there were signs of fusion of old and new bone. The control defect showed only slight bone growth at the cut edge.

Circulating antibodies against BCP were present in the serum of rats implanted with BCP sample 3z. Other BCP samples tested did not elicit antibody responses.

These results are summarized in Table 2.

Table 2 32 + +++ −+73A
＋ ＋
10-3B ++ + --
3G ++ + −−2B +
+ + --BCP sample 2Z, 2A
, 2B, and 3B.

7ml of saline (PBS) buffered with 30mg of phosphoric acid salt
It was suspended and injected into the abdominal cavity of the runt.

1.3. +4. and 28 days later, the peritoneal cavity was treated with PBS 100
The cells were perfused with m-cells, and the cells were selectively counted and counted as a constant and π-dimensional measure of inflammation. BCP grafts were also examined histologically and serum was measured as a measure of circulating antibodies.

A transient neutrophil response was observed in all samples after 1 day, which was thought to be related to the specific properties of this material. 3,
14. At the time point of 28 days, macrophages became predominant as responding cells, and histological examination of the grafts clearly showed that a large amount of BCP had been digested after 28 days.

Very small amounts of immune cells (lymphocytes) were always present. Serological tests revealed that rats implanted with 2Z had significant amounts of antibodies against BCP.

No antibody response developed in rats implanted with 2A, 2B, 2C, or 3B.

ECP is a novel bone-based matrix useful for conductive bone repair. Bone collagen powder (B CP) is a stress-resistant replacement for lost bone that can be shaped into bone defects to support new bone growth.

Twenty-four rats, 6 to 8 weeks old, were anesthetized, and 3 x 71 defects were made in the left and right lateral canals of one skull. A through hole was made in the skull using a dental drill, and the two types of B CP/Z CI mixtures prepared as described above were filled into the hole. Control defects were filled with BCP or ZCI alone, or left unfilled. Samples were collected 2 and 4 weeks after transplantation and examined histologically.

Defects filled with BCP/ZCIg compound experienced faster bone growth than normal 2 weeks after implantation, but BCP/Z
Because CIg compounds are not necessarily homogeneous, the predominant species (i.e., B
Bone formation patterns were altered by CP or zcB.

By the fourth week, a large amount of new bone was formed in the ZC component of the BCP/Z, CI mixture.

BCP particles were being incorporated into the center of new osteogenic activity. This, combined with early reconstruction, resulted in a more uniform appearance of the in situ bone extending into the defect than at 14 days. Control defects implanted (i.e., ECP alone and Z
CI alone) exhibited the characteristic type of bone filling activity that was observed in the above-mentioned studies. Control defects without implantation showed only slight bone growth at the cut edges of the defect.

By varying the relative amounts of purified atelopeptide-reconstituted microfibrillar collagen, which forms an elastic gel-like suspension, and bone collagen powder, which imparts compressive strength, we obtain a matrix suitable for conduction-mediated bone repair. A wide variety of useful implant materials can be produced.

Claims (1)

[Claims] 1.) (a) exposing fresh bone to the defect, (b) (i) bone collagen powder, (ii) purified atelopeptide reconstituted fine fibrous skin collagen, (iii) purified A lyophilized gel of atelopeptide reconstituted microfibrous dermal collagen, and (iv) one collagen formulation selected from the group consisting of a mixture of (i) and (ii) is filled into the defect to remove the fresh bone. A method for conducting conduction repair of a bone defect in a mammal, the method comprising contacting with a bone defect in a mammal. 2.) The method according to claim 1, wherein the collagen preparations (ii) and (iii) are produced from calf skin. 3.) The method according to claim 1, wherein the collagen preparation of (iii) is extruded into a mat shape. 4.) The method according to claim 1, wherein the collagen preparations of (ii) and (iii) are substantially not crosslinked. 5.) A lyophilized mat of reconstituted collagen fibers consisting essentially of a purified atelopeptide reconstituted microfibrous dermal collagen preparation lyophilized from a suspension. 6.) The freeze-dried mat according to claim 5, wherein the collagen is not substantially cross-linked. 7.) Suitable for use as a bone graft, comprising the steps of (a) casting a suspension of purified atelopeptide-reconstituted microfibrous dermal collagen into a plate shape, and (b) freeze-drying the cast product. A method for producing a freeze-dried collagen mat. 8.) A purified collagen preparation, characterized in that it consists essentially of bone collagen powder. 9.) A method for producing bone collagen powder suitable for use in repairing bone defects, which comprises treating bone powder from which inorganic components have been removed with at least one type of proteolytic enzyme selected from trypsin and pepsin.