CA1310918C - Gamma irradiation of collagen/mineral mixtures - Google Patents

Abstract

GAMMA IRRADIATION OF COLLAGEN/MINERAL MIXTURES

Abstract A process for sterilization of collagen/mineral compositions using .gamma. radiation is conducted under conditions which produce a product of desired handling and biocompatibility properties.

Description

1310ql8 GAMMA IRRADIATION OF COLLAGEN/MI~ERAL MIXTURES

Technical Field The invention relates to preparation of implants and prostheses for hard tissue repair composed of collagen and a mineral. In particular, mixtures of ateloeeptide fibrillar reconstituted collagen are mixed with a calcium phosphate mineral and the mixtures are treated with ~ irradiation to improve both biological and handling properties.

Backaround Art A wide range of material6 has been proposed for use in cepairing hard tissues. For weight-bearing areas, prostheses which are capable of withstanding stress have ranged from metal rods to reconGtituted animal bone. Various packing materials have also been used for augmentation of bony structures, such as the use of cross-linked collagen for alveolar ridge augmentation. It is desirable to have available a variety of materials suitable for the various types of skeletal repair, as each application has its unique set of parameters to determine the optimum implant. In addition, the physical handling properties of the, material as it is manipulated by the medical practitioner is significant in permitting the practitioner to achieve a successful result, in part because the ease of manipulation determines the ability to succeed.
Attempts have been made to compose suitable materials of the chief organic and inorganic components of bone, namely, collagen and calcium phosphate mineral. Reports of attempts to use the collagen/mineral combination are numerous. For example, l~Uql~

Lemons, J., et al., reported at the Second World Congres~ of Biomaterials in Washington, D.C., 27 April-l May 1984, on attempts to utilize collagen along with commercial hydroxyapatite and calcium phosphate to repair artificially created lesions in rabbits. The use of these mixtures did not result in reunion of the lesions. A control experiment using fresh autogenous bone, however, was successful in producing a union. -~
Similarly, Levy, P., et al, J Periodontal (1981), 50:303-306, were unsuccessful in their attempts to utilize collagen/mineral gel implants to repair intra-bony defects in root canals of canine or monkey teeth. Gross, B.C., et al., Oral Surq (1980), 49:21-26, reported limited success in using mixtuces of reconstituted lyophilized calfskin collagen in admixture with a hydroxyapatite preparation to induce bone growth through subperiosteal implants in monkeys. Various others have reported use of forms of collagen which clearly contain telopeptides, a major source of immunogenicity of collagen, in combinaticn with minerals in bone repair. See, for example, Hayashi, K. et al., Arch OrthoP Traumat Surq (1982) 99:265-269: Battista, U.S. Patent 4,349,490 (using a hydrated gelatin); Cruz, Jr., U.S. Patent 3,767,437 (using a calcium-precipitated form of collagen); and Battista, et al., U.S. Patent 3,443,261 (utilizing, in addition to calcium phosphate, a "new form" of collagen which contains microcrystals of aggregated tropocollagen units.
Miyata, et al., U.S. Patent 4,314,380, utilized a mineral backbone prepared directly by treatment of animal bone to remove all organic materials, which was then coated with an atelopeptide collagen. Jaeanese Application J5B~058041, published 6 April 1983, discloses a spongy porous calcium phosphate material 13~0918 having pores treated with atelopeptide collagen. The collagen derives from collagen-in-solution having a concentration of not more that 2~ by weight. The Japanese application reports the advance of osteoblasts into the pores of the material and new bone growth. European patent application, Publication No. 030583, published 24 June 1981, discloses use of Collagenfleece* in admixture with hydroxyapatite in bone repair. This collagen material is a commercial product, is obtained from animal hide by proteolytic digestion, and is lyophilized and sterilized by gamma irradiation. This collagen preparation forms a soft membrane-like material but does contain telopeptides and is partially degraded by the processing.
EPO application Publication No. 164,483, published 18 December 1985, discloses a process which is asserted to provide biocompatibility of a mineral/collagen mixture. In this mixture, solubilized collagen is cross linked either in the presence of, or before the addition of, a calcium phosphate mineral component just to the point wherein it retains its resorbability and absorptive capacity with respect to body fluids, rather than permitting the cross-linking to proceed to completion. U.S. 4,516,276 to Mittelmeier discloses the combination of a nonfibrillar, nonreconstituted collagen along with hydroxyapatite.
U.S. patent No. 4,795,467 issued 11 January 1989, and Canadian patent No. 1,260,391 issued 26 September 1989, both assigned to the same assignee as the application herein, disclose novel compositions containing reconstituted fibrillar atelopeptide collagen in admixture with a calcium phosphate mineral. Various methods are also disclosed for strengthening the (*) Trademark composition, which methods include incubation of the mixture at specified temperatures and times, and the treatment of the dried mixture with heat. The preparation of the referenced application6, in order to S be non-infective to treated subjects, must be prepared under aseptic conditions, as there i8 no provision in the disclosed procedure6 for dicect sterilization.
Typically, asceptic processing resul~s in products with sterility assurance levels (i.e., probability of a non-sterile product unit) between 10 and lO
The material which results after the various curing treatments disclosed in the above-referenced applications has a compressibility above 6 Newtons per square centimeter (N/cm ). Both this strength and further improvement in the compressibility indices are achievable by the curing processes disclosed therein.
The art offers no suitable composition for bone defect repair which is readily and efficiently sterilizable while retaining the efficient handling properties desired to eermit effective insertion of the implant. The material should be resistant to compression, and yet sufficiently resilient to permit shaping into place, or, alternatively, if to be used in a weight-bearing area, should be suitably rigid. The procesg and resulting product of the present invention remedies this omission in the art.
The invention takes advantage of an irradiation erocess which has previously been disclosed with regard to its impact on physical properties only in regard to preparations containing collagen alone. A summary of the effect of y-ray irradiation on collagen sutures, for example, is found in Artandi, Technical Report #149, Intl Atomic Energy Agency, Vienna, Manual on Radiation Sterilization of Medical & Bioloaical Material~ (1973) chap. 15, and a review of the effect of radiation on collagen as a tissue component is published by Bailey, A.J., in Internat Rev Connect Tis (1968), pp.233-281.
In addition, PCT application W081/00963 discloses that collagen materials can be increased in physical strength by heat treatment and by subjecting them to treatment with gaseous hydrogen halide. However, Applicant i~
aware of no disclosure in the art which shows the effect of y-ray ieradiation on the physical properties and handling properties of collagen/mineral mixtures, although y-ray irradiation has been used to Eterilize the lyophilized preparations disclosed in EP0 publication No. 164,483 (Eupra) without further comment concerning either properties or further use.
Disclosure of the Invention The invention provides a process whereby collagen/mineral preparations can be efficiently sterilized and simultaneously have conferred upon them eroperties which are especially favorable for handling of the material in defect repair, and for their behavior as implants. The heart of the process is irradiation of the preparation with sufficient total energy to effect sterilization to the required level, wherein the , collagen/mineral preparation is furnished in such form that the irradiation also provides a satisfactory compressibility modulus as well as the resilience and rigidity combination desired. A range of desired properties iB available, depending on the adjustment of the condition oc status with regard to celevant parameters of the collagen/mineral sample during the irradiation period.
Accordingly, in one aspect, the invention relates to a method for conferring desired physical properties and stsrility level6 on a collagen/min2ral mixture, which proce~6 compri~es irradia~ing the mixture with a sterilizing amount of y radiation, typically between 0.5 and 4 Mrad, wherein the mixture compri6e6 about 60-90% of a calcium phospbate mineral and 2-40% of an atelopeptide fibrillar reconstituted collagen exclusive of moisture. During the irradiation, it is important that the collagen portion of the preparation undergo or have undergone sufficient cros6-linking to stabilize the physical properties. Thi6 can be achieved in a variety of way6, for example, by preheating the sample to effect partial cros6-linking or by adjusting the humidity under which irradiation occur6 so that the radiation itself effect6 the desired level of cro66-linking. Thus, under these conditions, not only does 6terilization to a 6terility assurance level of at lea~t as low as 10 take place, but al60 adjustment of the phy6ical properties occurs by achieving a balance between cross-linking and degradation due to the radiation Another aspect of the invention is to provide a biocompatible collagen/mineral composition which comprises a mixture containing 2-40% reconstituted fibrillar atelopeptide collagen and 60-90% calcium phosphate mineral by weight exclusive of mois-ture, which composition has a compressive modulus of at least 10 N/cm and a sterility assurance factor of at least as low as 10 6, .~

Brief DescriPtion of the Drawina Pigure 1 ~how~ a diagram of alternative method~
for carrying out the invention.
Figure 2a shows the effect of moisture content of collagen/mineral mixtures on compres6ible modulus at various levels of irradiation Figure 2b shows this effect on trypsin sensitivity.
Figures 3a and 3b show the results of independent determinations similar to those of Figures 2a and 2b.
V

t 3 1 09 1 8 Modes of carlYinq Out the Invention The method of the invention is applicable to collagen/mineral mixtures of defined composition. There follows first a discussion of the nature of the individual components and the manner in which they are formed into mixtures.

The Mineral Component ~~
The composition6 of the invention can use a variety of calcium phosphate mineral component materials. As used herein, ~calcium phosphate mineral~
materials refers to those materials compo~ed of Ca and phosphate ions, regardles6 of the microstructure, protonation status of the phosphate. or extent of hydration. Calcium phosphate mineral materials include a variety of forms, such as the commercially available forms of tricalcium phosphate, for example, Synthograft~ tricalcium phosphate, or of hydroxyapatite such as Periograf~, Alveograf~, Interpore~, OrthoMatrix~ HA-1000~, or OrthoMatrix~ HA-500~ hydroxyapatite particulate preparations. The hydroxyapatite or tricalcium phosphate may also be prepared by known methods, such as those disclosed by Termine, et al., Arch Biochem BiophYs (1970) 140:307-325, or by Hayashi, K. et al., Arch Orthop Trauma Surq (1982, supra). In any event, the mineral i6 generally and preferably of nonbiological origin and is supplied as a powder of appropriate mesh.
Preferred particle sizes are in the range of 100-2000 ~. While the mineral content of bone could be harvested and purified for this purpose, more economically prepared and controlled compositions are preferable, both as a matter of cost and of quality. If -B-solid blocks are desired, these are prepared from the particulate form as described below.

The Collaqen The collagen component of the composition is critical to it~ efficiency. The collagen suitable for use in the invention i8 a purified atelopeptide fibrillar reconstituted collagen: it is typically prepared from skin.
Numerous forms of collagen have been prepared and they differ in their physical properties as well as in their biocompatibility. Where it is not intended to seecify the particle size within the range of diameters over which a mixture will be a solution, colloid, or suspension, a single generic term, "collagen dispersion"
is used. This term refers to any collagen preparation in aqueous medium where the collagen particle size is not specified -- i.e., the preparation may be a solution, suspension, or gel.
Native collagen consists mainly of a triple helical structure containing repeating triplet sequences composed of glycine linked to two additional amino acids, commonly proline and hydroxyproline. Native collagen contains regions at each end which do not have the triplet glycine sequence, and thus do not form helices. These regions are thought to be responsible for the immunogenicity associated with most collagen preparations, and the immunogenicity can be mitigated by the removal of these regions to produce "atelopeptide~' collagen. This can be accomplished by digestion with proteolytic enzymes, such as trypsin and pepsin. The nonhelical telopeptide regions are also responsible for natively occurring cross-linking, and atelopeptide collagen must be cro6s-linked artificially if cross-linking is desired.
Naturally occurring collagens have been subcla66ified into about ten types, depending on the amino acid sequence in the individual chains, the carbohydrate content, and the presence or absence of disulfide cro6s-links. The most common subtypes are Type I, which is present in skin, tendon, and bone, and -~~
which is made by fibroblasts; and Type III, which is found primarily in skin. Other types reside in specialized membranes or cartilage, or at cell surfaces. Types I and III contain similar numbers of amino acids in their helices and have a high degree of homology; however, Type III, but not Type I, contains two adjacent cysteine6 at the C-terminal ends of the triple helix, which are capable of forming inter-chain cross-link6.
Therefore, collagen preparations may differ from each other by virtue of their initial compositions, which is a function of their origin, or by virtue of their modes of preparation. Collagen derived from bone, for example, contains exclusively Type I collagen; while collagen derived from skin also contains Type III.
Also, the process of preparation may or may not remove the telopeptides. Thus both unaltered and "atelopeptidell collagen are po6sible. Cros6-linking may be effected deliberately or accidentally. Sterilization by ~-ray irradiation or by high heat may result in cross-linking without control of extent or nature and result6 in partial degradation of the triple helix;
deliberate cros6-linking may be carried out by a variety of means, including treatment with glutaraldehyde.
Differences arising from perhap6 more 6ubtle cause6 are perhap6 the result of variations in the details of the preparation procedure. For example, the collagen may be solubilized and reprecipitated, or may simply be finely divided and kept in 6uspension. When the solubilized material is reaggregated, the aggregation may be done in S ways BO as to form nonspecifically bonded solid6, or the collagen may be reconstituted into fibers which simulate the native form. Also, of course, the degree of purity may vary.
As used herein, "free from impurities" or "purified~ as regards collagen preparations refers to those impurities which are normally associated with collagen in its native state. Thus, collagen prepared from calfskin is free from impurities when other components of calfskin have been removed; that from bone when other components of bone are eliminated.
"Reconstituted" collagen refers to collagen which has been disassembled into individual triple helical molecules, with or without their telopeptide extensions, brought into solution and then regrouped into "fibrillar" forms. In this form, the fibrils consist of long, thin collagen molecules staggered relative to one another by multiples of about one-fourth their length. Thus results in a banded structure which can be further aggregated into fibers.
Collagen which is "substantially free from cross-linking" refers to collagen which has had the atelopeptides removed, and thus lacks the native capàcity for cross-link formation. These preparations remain substantially cross-link free if not deliberately cross-linked by, for example, being treated with glutaraldehyde or subjected to treatment which itself results in cross-linking--for example, treatments often used for sterilizing purposes, such as high temperature -11- 131091~

and the y-radiation described herein when conducted under appropriate conditions.
One collagen preparation which i6 suitable for the mixtures of the invention is an atelopeptide collagen which is reconstituted into fibrillar form and supplied as a dispersion of 5-100 mg/ml, preferably around 50-70 mg/ml. Such dispersions as Zyderm~
Collagen Implant (ZCI), which is commercially available -~~
in preparations containing 35 mg/ml collagen or 65 mg/ml collagen in saline, manufactured by Collagen Corporation, Palo Alto, California, are appropriate.
For use in the compositions of the inventions, the ZCI
or other collagen dispersions are used without lidocaine or other sedative drugs. As used herein, "ZCI" refers to the aqueous collagen dispersion, rather than to the ; collagen component per se.

The Collaqen/Mineral Mixtures The compositions of the invention which are eventually subjected to irradiation are generally initially prepared by mixing 50-85% by weight of calcium phosphate mineral component, preferably 65-75% mineral component, with the balance as a collagen dispersion in aqueous medium, such as ZCI. In terms of the mineral/collagen ratio (excluding the water content of the collagen dispersion), the mixtures are 60-98%
mineral, preferably 75-98% mineral and the rest collagen. The composition may be prepared simply by thoroughly mixing the two components into a cohesive mass. The mixture can also be cast into a desired shape (e.g., blocks, square6, sheets). Cross-linking can be superimposed using, for example, glutaraldehyde to a level of 0.001-0.1% for either a dry or wet product, as further described below.

The mixtures are then dried to less ~han 1%
moisture content and either rehydrated or heat treated before subjecting them to the sterilizing radiation procedures of the invention described below. The percentage compositions of the collagen/minera-l and moisture content are calculated as follows: percentages of collagen and mineral are given as dry weights relative to the total weight of these two components alone, not including water. Percent moisture i5 the weight water divided by the total weight (water +
collagen + mineral) times 100.
The sterilized material resulting from the radiation process may be used as mineral/collagen per se or may be mixed with additional components, which are also stecilized, as aepropriate, for administration to the subject. The preparations, while being described in terms of collagen and mineral, are always supplied to the subject in a wetted condition and contain either the inherent moisture of the original mixture or are rewetted with sterile water or saline before administration. In addition, components designed to increase the efficacy of the compound may be added, such as blood or bone marrow. As stated above, the percentages of collagen and mineral reflect their, relative amounts, and the collagen/mineral mixture can focm as little as 10% of the total preparation applied in some instances. Any additives must them6elves also be sterilized, or be derived from such source that sterilization is irrelevant, as is the case for blood, for example.

Desired ProPerties of the Mixture The collagen/mineral mixture itself, depending on its application, needs to exhibit certain physical properties. Specifically, it needs to be resilient enough to permit some shaping, but at the same time must be sufficiently rigid to resist total disorganization when stres6ed. Resistance to compression can be measured as the compressive modulus, using commercially available equipment, such as Instron Universal Testing Instrument Model 4202, and according to guidelines for measurement of compres6ive modulus as published by the American Society for Testing Materials (ASTM).
To conduct this measurement, the mixtures are first soaked for 5-24 hours in physiological saline.
This gives more relevant data, as the material will be wetted when implanted. The soaking is done for a sufficient time to insure complete wetting; the mixture is then placed in the test apparatus. If the material is resilient, it will compres6 easily until a point is reached wherein, in order further to compres6 the material, it is necessary to disrupt the inherent structure at the microscopic level. If the material is rigid, this point will be reached with les6 deformation than for resilient material. For collagen/mineral mixtures, the microscopic organization is maintained first by the triple helix per se, but also by interaction between the collagen triple helical po,rtions of the individual components of the fibrils as well as binding of the fibrils to each other. Compression disrupting any of these levels of organization will be more difficult than general compression which decreases the volume of empty space. Of course, the more highly organized and cross-linked the collagen chains in the composition, the more difficult this microscopic compression is.
Thus, a high compressive modulus (measured in Ntcm ) indicates a high level of organization at the 13~0918 microscopic level, specifically, a high level of cross-linking. A low compressive modulus indicates that cross-linking i5 low. For appropriate physical handling properties and for maintenance of integrity a~ an implant, it is important that the compressive modulu~ be reasonably high, at least about lO N/cm or more, and may be as high as 35-45 N/cm . The upper levels of compressive modulus are imposed by the nature of the materials, and it is believed that mixtures of this type cannot, in fact, attain modulus values of much greater than 100 N/cm under any degree of cross-linking. In any event, it is significant in maintaining suitable physical properties for the compositions of the invention that the compressive modulus be above 10 N/cm , and a preferred range is 10-60 N/cm , most preferably 25-45 N/cm . The resultant composition after the treatment according to the process of the present invention is assessed by this measure in order to verify that the appropriate compressive resistance strength is attained.
While the mixture needs to maintain integrity at a microscopic level, it must also be sufficiently porous and vulnerable to have biological properties which permit ingrowth of surrounding hard tis6ue,,and in some cases should exhibit resorbability when placed in a subject. However, this is a property that needs ~o be optimized rather than maximized. It is reflected as a modest degree of degradation of the collagen fibrils, which makes them susceptible to biological processes when placed in the 6ubject.
One in vitro mea6urement of this chaIacteristic is su6ceptibility to hydrolysi6 by tryp6in or "trypsin 6ensitivity". To effect this measurement, the samples are treated with the protease trypsin, which is capable -15- 1310~18 of attacking only fragmented portions of the collagen protein. The extent of hydrolysis is measured by fluorescamine assay for solubilized peptides, and the results are expres6ed as percentage nonhelical collagen. For example, and for comparison, gelatin preparations of collagen are 100% non-helical, collagen in solution is about 10% non-helical, and ZCl is 10%
non-helical. Desirable ranges depend on the use intended.
An alternative measure of fragmentation at a microscopic level i8 the transition temperature as measured by differential scanning calorimetry (DSC).
lowering of the transition temperature indicates an increase in fragmentation at a microscopic level in a manner similar to that measured by tcypsin sensitivity.
The process of the invention permits adjustment of the foregoing parameters to achieve optimum physical and biological compatibility properties. The process also results in efficient sterilization of the material, assuring sterilization levels at least as low as 10 Method of the Invention Sterilization and optimization of physical properties are achieved by subjecting the compositions to irradiation using a y radiation source in the range of 0.5-4 Mrad, preferably 1-3 Mrad, and most preferably 2.5-3 Mrad. These dosages are known to effect sterilization of pceparations containing only collagen (see Artandi, (suPra)). The irradiation process itself is carried out uging standard procedures known per se in the art for sterilization of foodstuffs, cosmetics, and the like. The irradiation is conducted using a y-emitting source, such as 131I 137C

commonly, Co. These materials are supplied in .,..:.. -1310ql8 standard forms and applied to samples using standard equipment by AEC licensees according to establi6hed guidelines. Reference is m2de to Proces6 Control Guidelines for Gamma Radiation Sterilization of Medical Devices published by Assoc. for Advancement of Medical Instrumentation (1984) as ~AMI Recommended Practice.
Reference is made also to Technical Reports Series 149;
"Manual on Radiation Sterilization of Medical Biological Materials", Intl Atomic Energy Commission, Vienna 1973.
The significant factors in the effect of the radiation on the sample are the total dosage (Mrad) and the state of the sample while being irradiated. Other factors, such as the rate at which the energy is supplied, total radiation time, distance of the sample from the source, and so forth, are generally irrelevant except as to their combined effect on total dosage.
The condition of the sample subjected to the radiation is of the utmost importance, and forms the basis for the invention herein. The sample must either be provided with a desired level of cross-linking before being subjected to the radiation, or must be placed in a condition ducing the radiation so as to permit the radiation itself to effect this cross-linking, or,a combination of these factor6 must be used.
In one preferred method of carrying out the invention, the mixture is assured to contain a moisture content of 1-6%, preferably 1-2%, during the application of the y-radiation. This is most conveniently achieved by first drying the mixture to a moisture content of less than 1% by dry heat at 35-45C, preferably 35-37C, and then rehydrating the mixture by treating it for 6-24 hours at 35-45C at 50-95% relative humidity (RH), preferably 35-37C at 50-80% RH, to achieve the desired equilibrium moisture content. The moisture content can be measured by standard techniques such as that described by Fischer, K., Anqew. Chem.
(1935) 48:394 to assure that the desired range i6 achieved. Other protocols to achieve the desired level of moisture can also be used, and the water content verified as described. When the mixture has the desired level of moisture, it is subjected to the radiation dosage described. Cross-linking to the desired level then occurs during the irradiation.
In an alternative embodiment, cross-linking is induced by heating prior to irradiation. In one preferred protocol, the sample is first dried, to a moisture content of less than 1%, or preferably 0.5-1%
as above, and then heated for 4-24 hours at about 60-90C, preferably 70-80C at 20-80% relative humidity, preferably 50-60% relative humidity to effect a desired level of cross-linking, as measured by the compressive modulus. Suitable values for the compressive modulus are 10-45 N/cm . Alternative means to achieve this level of cross-linking are also available, including treatment with cross-linking agents, such as glutaraldehyde or formaldehyde. In any case, the sample is subjected to these cross-linking treatments un,til a suitable measure of cross-linking by compressive modulus is attained. The sample is then subjected to the radiation.
Thus, in the first embodiment above, cross-linking is believed to occur during the radiation process due to the presence of moisture in the sample;
in the second approach, the cross-linking is effected prior to the radiation treatment and is not greatly increased during sterilization. However, it is clear that a combination of the two foregoing treatments can be employed by reducing the degree of cross-linking in the preradiation treatment and adjusting the moisture content of the sample during radiation so as to complete the desired process. The general aspects of the foregoing preferred procedures are set forth in Figure 1.
For the irradiation step, the compositions, suitably prepared for radiation treatment as above, are packaged in materials compatible with y radiation to preserve the sterilization of the samples contained, and are then subjected to 0.5-4 Mrad of radiation, according to standard procedures. The samples as then packaged are in a form suitable for reconstitution under sterile conditions and application to ths subject. For such use, the sample is removed from the package under sterile conditions and soaked in sterile saline or mixed with blood or bone marrow, as desired, and used for its desired purpose.

Use of the Composition The resulting composition is used to augment bone and fill bony defects, for example, periodontal bony pockets, tooth extraction sockets, and jaw cysts.
An important example of onlay procedures includes alveolar ridge augmentation. The procedures for tpe surgical implantation are known in the art. For alveolar ridge augmentation, the composition is inserted under the periosteum in places where augmentation is desired. In orthopedic and reconstructive applications, mineral in the form of porous blocks may also be indicated, particularly where the graft must bear stress. Implantation of the collagen-impregnated blocks is also effected by standard surgical techniques.

Examples The following example~ are meant to illustrate the invention, but not intended to limit its scope.

Example 1 Preparation of a Basic ComPosition A mineral/collagen preparation was obtained by mixing 65 parts by weight of OLthoMa~rix~ HA-1000 hydroxyapatite with 35 parts by weight of Zyderm~
Collagen Implant (65 mg/ml) without lidocaine. tSince ZCI is a 6.5% collagen-in-saline preparation, the final composition is 65 parts HA, Z.3 parts collagen (0.065 x 35) and 32.7 parts (35-2.3) saline, all by weight).
Th~ mixture was thoroughly mixed, and portions measuring 0;.55 ml were extruded into blocks and dried under a laminar flow hood for about 48 hr at 36-37C.
The resulting preparation had a moisture content of 0.87%, as measured by the method of Fischer, K., Anqew.
Chem. (1935) 48:394. The composition is thus 0.87%
20 water, 3.37% collagen, and 95.76% mineral, all by weight as defined above.

Example 2 Effect of Moisture Content The blocks prepared to according to Example 1 were set into vials for rehumidification. Twenty vials were incubated at 75% relative humidity, 35C for about 24 hr to obtain blocks with a moisture content measuring 1.86%. Ten of these were further subjected to 95%
30 relative humidity at 36-43C for 15-1/2 hr to obtain a moisture content of 5.9%.
The dry and rehumidified samples were subjected to varying levels of total radiation ranging from 0.5 to 3 Mrad. The results of the radiation on the compression 13~0918 modulus are shown in Figure 2a, and the effect on trypsin sensitivity is shown in Figure Zb. These results show that samples containing 1.86% moisture content were strengthened by the radiation procedure in terms of compression modulus, while their trypsin sensitivity was not markedly increased. In contrast, samples not rehumidified showed considerable fragmentation during irradiation, and the compressive strength was not measurably improved. (All samples showed a modest decrease in the transition temperature when measured by DSC.) The foregoing procedure was repeated, this time rehumidifying the samples to 1.28% and 1.62% moisture content, and gave comparable results, as shown in Figures 3a and 3b, respectively. Again, the samples containing a higher moisture content exhibited less fragmentation during irradiation, according to the trypsin sensitivity assay (Figure 3b), but markedly increased in compression modulus during radiation, as shown in Figure 3a.

ExamPle 3 Effect of Pretreatment with Heat The samples prepared as in Example 1 werç
placed in vials and 16 vials stoppered and treated at 80OC at 50-70~ RH for 48 hrs. The effects of radiation on these heat-treated samples was compared to samples not heat treated, but containing the original 0.87%
moisture content. The trypsin sensitivity of the heat-treated samples increased from a value showing 10%
non-helical collagen for unirradiated samples to 60%
nonhelical content for samples irradiated with 3 Mrad, in contrast to a relatively low fragmentation increase of 3% nonhelical character to about 25% at 3 Mrad for the samples not heat-treated. The compres6ive 6trength of the 6ample was measurably increased by the heat treatment, measuring about 35 N/cm before radiation and maintaining this level throughout the dosage range.
In a separate experiment, samples containing 0.87~ moisture heated for only 6-1/Z hrs at 80C and 50-70% RH also showed a compressive modulus of 35 Thus, it appears that heat-treated materials maintain their capacity to resist compression after radiation, while having increased trypsin sensitivity.

ExamPle 4 Effect of Heat Curina Alone Samples were prepared as in Example 1, except that the extruded mixture was incubated for 72 hr at 26-34C at 90-95% relative humidity before drying, as described above, to obtain a moisture content of 0.48-0.49~. When this preincubated mixture was treated for varying lengths of time at 80C at 50-70% RH it showed a consistent increase in compressive modulus, from 15 N/cm with no heat treatment, to 25 N/cm after 4 hours at 80C, 30 N/cm after 8 hours, and 40 N/cm after 12 hours. Therefore, heat treatment is - 25 effective in increasing the compressibility of dried samples as is the application of radiation; however, sterilization doefi not necessarily result.

Claims (3)

1. A biocompatible collagen/mineral composition which comprises a mixture containing 2-40% reconstituted fibrillar atelopeptide collagen and 60-90% calcium phosphate mineral by weight exclusive of moisture, which composition has a compressive modulus of at least 10 N/cm2 and a sterility assurance factor of at least as low as 10-6.

2. The composition of claim 1 which has a moisture content of 0.5-6% and a compressive modulus of 25-45 N/cm2.

3. A method of obtaining a collagen/mineral composition suitable for irradiation at 0.5-4 Mrad and having a moisture content of 1-6% which comprises drying said mixture to a moisture content of less than 1%, and rehydrating said dried mixture by incubating it at 35-45°C at a relative humidity of 50-80%.