CA2072244A1

CA2072244A1 - Bone replacement material with fgf

Info

Publication number: CA2072244A1
Application number: CA002072244A
Authority: CA
Inventors: Berthold Nies; Elvira Dingeldein; Helmut Wahlig
Original assignee: Berthold Nies; Elvira Dingeldein; Helmut Wahlig; Merck Patent Gesellschaft Mit Beschraenkter Haftung
Current assignee: Merck Patent GmbH
Priority date: 1991-06-26
Filing date: 1992-06-24
Publication date: 1992-12-27
Also published as: JPH07171211A; AU1854692A; ES2128330T3; NO922511D0; US6118043A; CZ194692A3; MX9203253A; EP0520237A3; ZA924780B; DE59209624D1; DE4121043A1; ATE176161T1; KR930000129A; JP2003093495A; CZ281711B6; AU652839B2; NO922511L; TW293776B; EP0520237A2; HUT65499A

Abstract

Abstract The invention relates to a bone replacement material which comprises one or more polypeptides having the biological action of fibroblast growth factors in a porous matrix. The healing-in properties correspond to those of autologous bone transplantation.

Description

20722~4 Merck Patent Gesell~chaft mit beschrankter Haftung 6100 D a r m s t a d t Bone replacement material with FGF
The invention relates to bone replacement materials which comprise one or more polypeptides having the biological action of fibroblast growth factors in a porous matrix.
Bone replacement materials are to be understood as material~ which can be used as implants for replacing or reconstituting bone ~tructures because of defects following disease- or accident-related surgical interven-tion. Examples which may be mentioned are shaped implant articles, such as bone prosthe~es of the most diverse type, bone-joining elements, for example in the form of medullary space nail~, bone screws and osteosynthesis plates and implant material~ for filling spongiosa bone defects or tooth extraction cavities and for plastic ~urgery of contour defects in the jaw/face region.
Tho~e implant material~ which have a high bio-activity, that is to ~ay to the extent that they are accepted in the organism and integrated into it, are regarded as particularly favourable for the healing-in process. In the ca~e of bone replacement material, this mean~ that it should soon fuse firmly and permanently with endogenou~ tis~ue, in particular with the bone.
It is known that the most favourable healing-in result~ have hitherto been achieved in practice only with endogenou~ material~, that is to say with bone transplants. The availability of bone tran~plant~ i~ of cour~e limited. Autologous tran~plant~, that is to say tran~plants from the same individual, can be removed, if they are present at all in a ~uitable shape and quantity, only by at least one additional ~ur~ical intervention, which in turn neces~itates an additional healing proces~
at the removal 3ite. The same also applies in principle to homologous transplants, that is to say tran~plants from donor individual~ of the same species. These ~re 2~7224~

also accompanied by problems of compatibility, and also the ri~k of infection with viruses, such as, in parti-cular, hepatitis and HIV viruses, which still cannot be excluded completely at present. The storage of donor material in bone banks is furthermore expen~ive and in the end of only limited duration.
Implant materials for bone replacement from synthetic materials not related to the body or from materials related to the body can display bioinert to bioactive properties, depending on their nature and state. However, the healing-in re3ults of endogenous bone transplants have not yet been achieved by any synthetic implant material.
The invention was therefore based on the problem of providing a bone replacement material, the biological activity of which comes as close as possible to that of endogenou~ bone tran~plantation.
It has now been found that this i8 achieved by a bone replacement material which compri~e~ one or more polypeptides having the biological action of fibroblast growth factor~ in a porous matrix.
The invention therefore relate~ to a bone replacement material which comprises one or more poly-peptides having the biological action of fibroblast growth factors in a porous matrix.
The invention particularly relate~ to such a bone replacement material in which the porous matrix i8 a mineral matrix, preferably based on calcium minerals.
Fibroblaqt growth factors (FGF), which belong to the clas~ of endogenous peptide growth factors, were originally detected as substances in the brain and hypophysis and isolated therefrom and displayed an activity which promotes the growth of fibroblasts. FGFs are known as active angiogenic factors which are respons-ible, inter alia, for neovascularisation during woundhealing. Further detailson FGF~, including their modifi-cation products, on their isolation or preparation, their ~tructure, their biological activities and mechanisms 2~7224~

thereof and on corresponding medical uses can meanwhile be found in extensive technical literature. A comprehen-sive review is offered, for example, by A. Baird and P. Bohlen, Fibroblast Growth Factors, in: Peptide Growth Factors and their Receptors I (editors: M.9. Sporn and A.~. Roberts) Springer Verlag Berlin, Heidelberg, New York 1990.
In addition to an abundance of positive actions of FGF~ in widely varying fields of indication, influen-ces of- FGFs in osteogenesis have also recently been reported in individual cases, for example in Biomaterials 11, 3~-40 (1990). It i5 reported in Acta Orthop. Scand.
60, (4) 473-476 (19a9) than an increased content of minerali~ed tissue was found in implants of demineralised bone matrix (DBM) which had been charged with recombinant human basic FGF and implanted intramuscularly into rats.
DBM is known per se as a bone growth-promoting ~ubstance, ~ince it contain~ itself ~till intact endogenous factors of the mo~t diver~e type having a bone growth-promoting activity. However, the biological activity of DBM varies according to its origin and pretreatment and can in no way be standardised to a reproducible level. DBM is moreover unsuitable in practice as an implant material for bone replacement becau~e of a lack of mechanical strength. From the findings published, it was in no way to be deduced that a material which combines the mechani-cal properties of ~ynthetic implant materials with the biological activity which only bone transplants have could be provided by the bone replacement material according to the invention.
The bone replacement materials according to the invention are characterised by the common feature that they comprise one or more polypeptides having the bio-logical action of FGF in a porous matrix. Not only the "clas~ical" FGFs, such as acidic fibroblast growth factor (aFGF) and ba3ic fibroblast growth factor (bFGF), but al~o all peptidic growth factor~ which display the biological action of FGF sre thu~ to be regarded as 2a722~4 growth factors which are suitable according to the invention.
The narrower cector of FGFs include~ natural FGFs, in particular of bovine and human origin, a~ well as FGF~ prepared by recombinant methods. Human aFGF and bFGF prepared by recombinant methods are particularly preferred. Further details on bovine and human aFGFs and bFGF~ prepared by recombinant methods can be found, for example, in the following patent documents: EP 228 449, EP 248 819, EP 259 953 and EP 275 204. The wider sector of FGFs also includes muteins, which differ from aFGF and bFGF to a certain extent in the number and/or sequence of - the amino acids, without thi~ being associated with a substantial change in action. The wider sector of FGF~
finally al~o includes related peptides with sometimes significantly different amino acid sequences and with the action of FGF a~ well a~ wit~l an activity which intensi-fie~ the action of F5F. The following patent document~
may be mentioned a~ examples of literature re~erence~:
~P 148 922, EP 226 181, EP 281 822, EP 288 ~07, EP 319 052, EP 326 907 and WO 89-12645.
FGF~ in the context of the invention furthermore include derivatives of the~e peptides which are obtained with stabili~ing and/or activity-increa~ing agent~. The~e are, in particular, foxms of aFGF and bFGF which are stabili~ed toward~ acid and contain a~ stabili~ing agents, for example, glyco~amine-glycan~, such as heparin, heparin fragments, heparan ~ulphate and dermatan sulphate, or gIucan sulphates, such as dextran ~ulphate and cyclod~xtrin ~ulphate. FGF derivative~ of this type are described, for example, in EP 251 806, EP 267 015, EP 312 208, EP 345 660, EP 406 856, EP 408 146, WO
89-12464, WO 90-01941 and WO 90-03797.
Forms of human bFGF prepared by recombinant methods, such as are described in EP 248 819, are parti-cularly preferred for u~e in the bone replacement mate-rial~ according to the invention.

2~722~4 The FGFs can be present in the bone replacement materials according to the invention in a concentration of 1 ng/cm3 - 1 mg/cm3. The choice o~ concentration within the range mentioned can depend on the nature and form and the activity of the FGF to be employed in the individual case, and on the nature of the implant material propo~ed in the individual case and its possibly inherently present bioactivity. The concentration of FGF i~ prefer-ably in the range between l~g/cm3 to 100pg/cm3.
All the known and customary implant materials can in principle be present in the bone replacement material~
according to the invention if they are or have a porous matrix for accommodation of FGF. Implant materials can be classified into the classes of mineral, in particular ceramic, materials, physiologically acceptable metallic materials, physiologically acceptable polymer material~
and composite materials of two or more material3 of the type mentioned, ~hese materials can form a porous matrix as an entlrety, for example in the form of porou~ ~haped implant articles, or it i~ po~sible for only certain components of the material to be in the form of porous material or for certain regions of a shaped implant material to be a porous matrix. The last two case~ can be realised, for example, in the form in which a composite material or a bone cement contains a porous component, or an implant is provided with a porous surface coating or an appropriately roughened surface.
On the materials side, preferred materials for the bone replacement materials according to the invention are those which are mineral and in particular ceramic in nature. ~ne advantageou~ aspect of the invention is that materials which are bioinert per se, such a~, for example, oxido-ceramic materials, can be activated biologically by being charged with FGF and in thi~ way exhibit ~ignificantly better growing-in and healing-in propertie~.
Nevertheles~, preferred mineral materials are those which are bioactive per se. ~his chiefly applie~ to 2~722~

materials which are based on calcium-containing mate~
rials, such as, in particular, calcium carbonate, calcium phosphates and ~ystems derived from these compounds. From the group of calcium phosphates, hydroxyapatite, tri-calcium phosphate and tetracalcium phosphate are to bementioned as preferred.
However, mineral-based implant materials usually guarantee a high mechanical stability only if they are employed as ceramics, that is to say in the form of materials or workpieces sintered at sufficiently high temperatures.
Bone replacement materials ba~ed on calcium phosphate ceramics, because these are related chemically to the mineral phase of natural bone, are bioactive.
Natural bone chiefly consists in its mineral pha3e of hydroxyapatite, a calcium phosphate having the empirical formula CaS ( P04 ~ 30H.
Hydroxyapatite of synthetic or organic origin, for example from natural bone material, i~ therefore a frequently used raw material for the production of implants for bone replacement. Hydroxyapatite ceramic is largely non-absorbable in the organism. Thi9 means that exogenous material is retained practically unchanged for a long period and integration into the organism takes place essentially by fusion with exi~ting and regenerat-ing bone and by growing into the surrounding tissue.
Under certain circumstances, tricalcium phosphate is absorbable in the organism. Tetracalcium phosphate is e~sentially non-bioab~orbabl~.
Porous calcium phosphate ceramics exhibit parti-cularly favouxable growing-in properties. Particularly preferred materials here are those based on natural bone, which i8 mineralised by various treatments and converted into a ceramic system, in which the structure o~ the bone should be retained as ~ar as possible. The processes have the common feature of the removal of ~he organic bone constituent~ and sub~equent compaction to a ceramic by sintering at appropriate temperatuxe~. Organic content~

20722~
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are removed by chemical solution processe~ or by pyro-lytic processes.
Further details on bone ceramics and particularly favourable processes for their preparation can be found, for example, in the patent documents DE 37 27 606, DE ~9 03 695, DE 41 00 897 and DE 40 28 683.
Because of their excellent agreement with the pore system of natural bone, bone ceramic implant3 ~how considerable biological advantages in growing-in proper-ties and healing in the organism. Spongiosa bone ceramic is particularly preferred because of its high-porosity, three-dimen~ionally open-pored network structure.
Shaped articles of ceramic material, in parti-cular of the abovementioned type, are employed primarily for replacing load~bearing bone structures which must with~tand high mechanical stre~ses. Examples of these are bone prosthe~es and bone-joining elements, such as, for example, medullary space nails, bone screws and osteo-synthesis plates.
More precise clinical studies have shown that exposed mineral contact surfaces in implants of calcium pho~phate ceramic preferentially stimulate regeneration of mineral bone matrix, resulting in a firm fusion of the implant. This is promoted still further in the ca~e of porous implants, where a particularly intensively inter-linked and therefore mechanically stable fu~ion develops because of the higher surface area and by new bone tis~ue form~ng shoots into the implant. In the case of implant materials of mainly polymeric materials or of bioinert materials, connective tissue is initially preferentially formed instead, leading to only a moderately firm fusion.
It has now been found that, due to being charged with ~GF, the bone replacement materials according to the invention, largely independently of the nature of the ~5 material, stimulate con~iderable regeneration of mineral bone matrix in the contact region and, depending on whether bone can grow through them because of porosity and/or absorption, also inside the matrix after 20722~

implantation. This stimulation is in all case~
significantly higher than in the case of corresponding non-charged implants. A pronounced synergistic effect was to be observed here in the~ case of porous implants charged with FGF and based on calcium minerals, in particular calcium phosphate ceramics. In preclinical model experiments on bone ceramic implants charged with FGF, complete incorporation into the bone by regenerated, chiefly mineralised bone matrix growing in and through was found six weeks after implantation. A comparable result was achieved only by autologous bone transplant~, while, for example in the case of uncharged bone ceramic, DBM and DBM-impregnated bone ceramic, fusion by regeneration of bone matrix was to be found only in the contact regions with the existing bone. It is assumed that the bone growth-promoting action of FGF and the bioactivity of calcium-containing implant materials, such as, in particular, bone ceramic, mutually intensify each other and in thls way lead to an accelerated healing-in Z0 and incorporatlon of the implant.
The positive influence of FGF on the healing-in properties of implants for bone replacement can be applied, as already mentioned, to practically all type~
of bone replacement materials and implant materials if these are of a type and shape such that they have a porous matrix for accommodation of FGF and re-release to the organi~m, expediently at least chiefly in the contact region with the body tissue. These requirements are also met, for example, by implant~ of metallic materials which are in themselves porous or have a porous surface coating, preferably of bioactive hydroxyapatite, or which have a surface which has a porous structure or is at least roughened. The same applies to implants of polymeric materials, other ceramic materials or composite material~.
The bone replacement material~ according to the invention can in principle be present not only as shaped implant article~, but also in powder or granule form, 20722~
g depending on what is required by the site of use and the intended use.
Preferred possible composite materials are tho~e in which at least one component is present as a porous matrix for accommodation of FGF. Corresponding bone replacement materials ba~ed on composite material~ in which a porou-~ mineral matrix is present in powder or granule form and forms a shaped article in a~sociation with a physiologically acceptable polymeric active compound are expedient. Compo~ite material~ of this type are to be found in the relevant technical literature, for example Patent Documents WO 90-01342 and WO 90-01955, in which implant materials ba~ed on calcium phosphate particles or bone ceramic particles and bioabsorbable polymer are described.
The bioactivity of bone cements can also be increased in an analogous manner. Bone cements consist mainly of acrylate systems comprising mineral fillers, usually based on calcium compounds. According to the invention, for example, FGF-charged porous hydroxyapatite powder or granules can be employed as a filler component in bone cement.
The preparation o~ the bone replacement materials according to the invention by charging the particular porous matrix with polypeptides having the action of FGF
pre~ents no problem~ in itself. A procedure is expediently followed in which a suitable liquid or semi-liquid preparation of FGF, for example in the form of a buffered aqueou~ solution, a suspension or a gel, i9 u9ed as the s~arting ~ubstance and i~ allowed to soak completely, in the proposed dosage, into the porous matrix of the bone replacement material. The bone replacement material is ~hen, or after any drying which may be necessary, already usable or can be stored in accordance with the safety precautions required for such materials for medical use. Porous shaped implant articles, preferably of bone ceramic, implants provided with a porou~ surface and porous particulate component~

~722~ -for composite ma~erials and bone cements can be charged with FGF in this manner.
In a preferred embodiment, the bone replacement material accordin~ to the invention i8 in the form of a ready-to-use implantation set of two or more ~eparate components, in which one component comprises the porous matrix and another component comprises a solution of the polypeptide having the action of FGF. Such an embodiment is particularly appropriate in order effectively to counteract possible ~tability problems which could arise during long-term storage of already made-up bone replace-ment materials according to the invention. Thus, for example, it i5 reported in the technical literature that calcium ions, which are indeed pre~ent in the materials preferred here, can have a des~abilising influence on FGF. The bone replacement materials according to the invention are used in the ~orm of an implantation ~et of this typ~ such that the porous matrix of the particular implant material is charged with the FGF-containing solution in the manner described above shortly before or during the surgical intsrvention for the implantation.
Such an embodiment is particularly expedient in the case wheré the porous matrix is formed by a shaped implant article itself, mineral, preferably ceramic, materials and in particular sintered bone ceramic being primarily ~uitable as the material.
Depending on the embodiment, the bone replacement material according to the invention is thus an at least equivalent substitute for autologou~ and homologous bone transplant~, or i8 a considerable improvement to other forms of bone replacement in respect of healing-in properties.
Example 1 Production of shaped implant articles Cylindrical shaped article~ lOoO0 mm in height and 9.55 mm in diameter are produced with a diamond milling cutter from sponglo~a hydroxyapatite bone ceramic blank~ prepared according to D~ 40 28 683.

... ._., .. . _ .

' ' , .

Some of these shaped articles are impresnated with in each ca~e 100 ~1 of a solution comprising 50 ~g of human bFGF prepared by a recombinant method, dried and stored at 4-6C until the time of implantation.
The other shaped articles are used for comparison purposes.
Example 2 Comparative animal experiment study Animal species: Mini-pig, adult, female, 6 groups, 8 implants per group Implants: a~ spongiosa hydroxyapatite ceramic with FGF
(according to Example 1) b) spongiosa hydroxyapatite ceramic c) DBM
lS d) spongiosa hydroxyapatite ceramic, impreg-nated with DBM
e) autologous spongiosa transplant, removed with accurate dimension~ u~ing a twin milling cutter.
f) homologous ~pongio~a transplant, removed with accurate dimensions using a twin milling cutter, storage at -30C until the time of implantation Site of implantation: Into the patellar sliding bed of the femur condylus, on the left and right After 6 weeks, the bones were removed by surgery and the bone regeneration and mineralisation were deter-mined by histological examination.
Result:
a) 5pongiosa hydroxyapatite ceramic with FGF
Bone regeneration from the bone bed up to the centre of the implant; complete 1ncorporation b) Spongiosa hydroxyapatite ceramic Marginal osseous contact with the implant; growing-in only round the edge of the implant ~72~4~

c ) DBM
Marginal osseous contact with the implant; growing-in only round the edge of the implant d) Spongiosa hydroxyapatite ceramic, impregnated with DBM
Bone regeneration in the contact region of the bone bed and implant; amorphous DBM still present.
e) Autologous spongiosa transplant Bone regeneration from the bone bed into the centre of the implant; complete incorporation f) ~omoloqous spongio~a transplant sone regeneration from the bone bed, affecting about 1/3 to 1/2 of the implant; partial incorporation.
Example 3 Implantation ~et Porous ~pongiosa hydroxyapatite bone ceramic shaped articles (according to Example l; non-charged) are placed in deep-drawn packaging mouldings of appropriate ~hape, the chamber~ of which correspond exactly to the dimensions (only slight re~idual volume) of the shaped article~. The deep-drawn components are sealed and sterilised, and enclosed in a wrapping.
/~ bFGF solution i~ freeze-dried in citrate buffer (10 mmol; pH 5.0) after addition of sucrose solution 25 ~ ( ,~,r9%) ~ and introduced into ampoules. The ampoule filling and ampoule volume are coordinated 80 that the later charging of the ceramic shaped articles corresponds to 50 ~g of bFGF/cm3 block volume.
Shaped implant article packs and bFGF ampoules form pack units as an implantation set.
Conditioning on the operating table The bFGF solution is reconstituted in citrate buffer (pH 5.0) and then drawn up into a sterile ~yringe.
After the wrapping has been opened, the bFGF
~olution is injected through the sterile internal packaging into the deep-drawn container of the ceramic shaped article. The injection volume is mea~ured ~o that the shaped article is immersed completely in the bFGF

207224~

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solution. After about 1 minute, excess bFGF solution is sucked back into the syringe. The ceramic shaped article retains a~out as much solution as corresponds to its pore volume.
The charged shaped article can be implanted after the primary packaging ha~ been opened.

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Claims

1. Bone replacement material, characterised in that it comprises one or more polypeptides having the bio-logical action of fibroblast growth factors in a porous matrix.

2. Bone replacement material according to Claim 1, characterised in that it comprises basic fibroblast growth factor.

3. Bone replacement material according to Claim 1, characterised in that it comprises acid fibroblast growth factor.

4. Bone replacement material according to Claim 2 or 3, characterised in that it comprises fibroblast growth factor prepared by a recombinant method.

5. Bone replacement material according to one of Claims 2 to 4, characterised in that it comprises muteins of fibroblast growth factors.

6. Bone replacement material according to one of Claims l to 5, characterised in that it comprises acid-stabilised forms of the polypeptides.

7. Bone replacement material according to Claim 1, characterised in that it comprises 1 ng/cm3 to 1 mg/cm3, preferably 1 to 100 µg/cm3, of polypeptide.

8. Bone replacement material according to one of Claims l to 7, characterised in that the porous matrix is formed by a mineral, preferably bioactive material.

9. Bone replacement material according to Claim 8, characterised in that the porous mineral matrix essen-tially consists of calcium minerals.

10. Bone replacement material according to Claim 9, characterised in that the porous mineral matrix essen-tially consists of calcium phosphate.

11. Bone replacement material according to Claim 10, characterised in that the porous mineral matrix consists of one or more compounds from the group comprising hydroxyapatite, tricalcium phosphate and tetracalcium phosphate.

12. Bone replacement material according to one of Claims 9 to 11, characterised in that the calcium minerals are obtained from natural bone.

13. Bone replacement material according to one of Claims 9 to 12, characterised in that the porous mineral matrix is sintered calcium phosphats ceramic.

14. Bone replacement material according to Claim 12, characterised in that the porous mineral matrix consists of sintered spongiosa bone ceramic.

15. Bone replacement material according to one of Claims 1 to 7, characterised in that the porous matrix is formed by a physiologically acceptable metallic material.

16. Bone replacement material according to Claims 1 to 7, characterised in that the porous matrix is formed by a physiologically acceptable polymer material.

17. Bone replacement material according to one of Claims 1 to 16, characterised in that the porous matrix is present as a shaped implant article.

18. Bone replacement material according to one of Claims 1 to 16, characterised in that the porous matrix forms the surface or a surface coating of a shaped implant article.

19. Bone replacement material according to one of Claims 1 to 16, characterised in that the porous matrix is present in powder or granule form.

20. Bone replacement material according to Claim 19, characterised in that a porous mineral matrix is present in powder or granule form and forms a shaped article in association with a physiologically acceptable polymeric material.

21. Bone replacement material according to Claim 19, characterised in that a porous mineral matrix is present in powder or granule form and forms a component of a bone cement.

22. Bone replacement material according to Claims 1 to 21, characterised in that it is present in the form of a ready-to-use implantation set of two or more separate components, one component of which comprises the porous matrix and another component of which comprises a solu-tion or a suspension of the polypeptide.

23. Bone replacement material according to Claim 22, characterised in that the component which comprises the porous matrix is a shaped implant article.

24. Bone replacement material according to Claim 23, characterised in that the shaped implant article consists of a mineral, preferably ceramic, material.

25. Bone replacement material according to Claim 24, characterised in that the shaped implant article consists of sintered bone ceramic.