HK1048076B - Endoprosthesis with long-term stability - Google Patents

Description

Endoprosthesis with long-term stability

The invention relates to the use of active substance complexes having at least one supplementary component, at least one adhesion component, and at least one growth and/or maturation component, which are different from one another and are specifically adapted to the respective biological body part to be formed, namely at least one extracellular substance-based structural component, which is specifically adapted to the cells of the respective biological body part to be formed.

Complexes of active substances with the stated components for forming parts of living organisms, in particular living organs, are known from the prior art. In the known active substance complexes, the structural components can consist, for example, of different collagens, elastins or proteoglycans. As a supplementary constituent of such active substance complexes, mention may in particular be made of chemokines, for example peptides, such as N-F-Met-Leu-Phe-, and/or metabolites of, for example, arachidonic acid, such as leukotrienes. Proteins of the fibronectin or laminin type may play a role as adhesion components, but also cell adhesion molecules, such as L-CAM, N-CAM, and matrix adhesion molecules, such as tenascin, collagen IV, V, VII, synthetic peptides and transmembrane compound proteins, such as integrins. With regard to the active substance complexes discussed here, the first-mentioned examples of adhesion components, namely fibronectin and laminin, fall within the class of matrix adhesion molecules. As a further component, the active substance complex comprises at least one growth and/or maturation component, preferably in the form of one or more cytokines. Examples of such cytokines are colony stimulating factors in blood production; fibroblast growth factor in connective tissue production; epidermal growth factor in skin production; cartilage-inducing factors in chondrogenesis; lymphocyte activating factor and spleen peptide in spleen or lymph node generation; t-cell growth factor and thymosin peptides for use in thymopoiesis; bone growth factors and transforming growth factors for osteogenesis; and angiogenic factors for angiogenesis. The following cytokines were also used: interleukins, insulin-like growth factors, tumor necrosis factors, prostaglandins, leukotrienes, transforming growth factors, platelet-derived growth factors, interferons, and endothelial-derived growth factors.

For more details on such active substance complexes see european patent No.0,500,556, the contents of which are expressly included herein.

The object of the invention is to make the active substance complexes more versatile.

This object is achieved by the use of the active substance complexes for the formation of endoprosthesis implants. This provides long term stability of the endoprosthesis compared with conventional endoprostheses without active substance complexes.

In this use according to the invention, use is made of active substance complexes which are suitable for forming a biological part in the form of bone and which have components which, differing from one another, are specifically adapted to form bone, i.e. at least one structural component based on extracellular substances which is specifically adapted to the cells of the biological part to be formed in each case, at least one supplementary component, at least one adhesion component, and at least one growth and/or maturation component.

The finding of such a use according to the invention is the result of extensive studies aimed at combining the active substance complexes with different support materials, in particular metal support materials. The binding of the support material to the active substance complex is not unproblematic. Based on previous experience with active substance complexes and their complex mode of action, it has at least to be expected that the formation or reformation of the specific part of the organism to be treated, for example bone regeneration, is reduced. The risk of tissue toxicity is also questionable.

The solution of this problem is therefore not obvious, for the reason that, as already explained, it is extremely problematic to combine the active substance complex with a support, which in this case is an endoprosthesis, since the function of the active substance complex in a bone defect can be destroyed or at least complicated by possible immune reactions.

The endoprosthesis to be stabilized has an outer surface which is at least partially coated with an active substance complex and/or which has at least one cavity which is filled with an active substance complex.

The purpose of the coating and/or impregnation with the active substance complexes is to make the endoprosthesis more rapidly and permanently integrated in the organism. The integration of the endoprosthesis at the implantation site is accelerated and improved, leading to long-term stability and a higher and earlier loadable loading of the endoprosthesis.

According to a further embodiment, the endoprosthesis has at least one cavity which is filled with the active substance complex, the active substance complex being additionally applied to a further supporting material. Collagen or a suitable polymer may be used as such a further support material. Types of collagen I, IV, V and VII may be mentioned specifically here. Collagen can be used, for example, in the form of a mesh or gel, which has, in particular, an inherently good immunological compatibility and is easy to process.

Polymeric support materials that can be employed are precisely polymers of natural monomers, such as polymers of polyamino acids (polylysine, polyglutamic acid, etc.) and lactic acid. Copolymers, such as copolymers of polylactic acid and glycolic acid, may also be employed.

Polylactates are polyesters of lactic acid having the following chemical formula:

direct polymerization of the monomers results in polymers having relatively low molecular weights. The upper limit is about 20,000 Da. Higher molecular weights can be obtained by linking the cyclic dimer in the presence of a catalyst at high temperature and low pressure. Lactic acid polymers are biodegradable, biocompatible, water insoluble, and are characterized by high strength.

A further supporting material, for example collagen or the polymer, has the additional effect of reducing the amount of active substance complex required to completely fill the endoprosthesis body cavity without adversely affecting the basic efficiency of the active substance complex. In this way, the use of active substance complexes is economically more advantageous.

The invention also relates to an endoprosthesis which, in one of the embodiments according to the use, is coated with or contains an active substance complex.

The invention is explained in more detail below on the basis of embodiments and with reference to the drawings, in which:

FIG. 1 is a graphical representation of the neo-osteogenesis effect of rabbits using active substance complexes compared to untreated samples;

FIG. 2 is a graphical representation of the osteogenesis of sheep using an active compound complex with tricalcium phosphate as a support material, as compared to pure tricalcium phosphate;

FIG. 3 is a graphical representation of the new osteogenesis of rats using active substance complexes and different collagens as supporting materials, compared to pure collagen;

FIG. 4a is a side view of an endoprosthesis coated with an active substance complex;

figure 4b is a side view of the endoprosthesis of figure 4a rotated 90.

I. Preparation of active substance complexes

The main steps for preparing the active substance complexes are described below: tubular bones of calves, sheep, rabbits or rats are washed, in particular the bone marrow is removed, and the bones are frozen. The frozen bone is ground to a particle size of less than 2 mm. The ground bone fragments were defatted in acetone and decalcified in 0.6N hydrochloric acid. The product was then freeze dried to give demineralized bone matrix, which was extracted in 4 molar guanidine hydrochloride solution. Dialyzing the extractive solution against distilled water, centrifuging, and freeze-drying the precipitate to obtain active substance complex.

This basic preparation is illustrated in the following flow chart.

FIG. 1: flow chart showing the preparation of active substance complexes

Fresh tubular bone shaft after slaughter

↓

Grinding to obtain particles with a diameter of less than 2mm

↓

Defatting in acetone

↓

Decalcification in 0.6N HCl

↓

Washing, and freeze drying

↓

Demineralized bone matrix

↓

Extraction in 4M GuHCl

↓ ↓

Supernatant of the residue

↓

Dialyzing against distilled water

↓

And (3) precipitation: active substance-containing complex

II. Efficacy of active substance complexes without the use of support materials

To illustrate that the active substance complex is effective per se, an experiment was first carried out in which no additional support material was present during implantation of the active substance complex.

1. Animal for test

Female chinchilla with an average body weight of 3089g were used. They ingested rabbit maintenance feed and double ozonated tap water acidified with hydrochloric acid to ph 4.5.

Animals were anesthetized by subcutaneous injection of a mixture of ketamine and xylazine.

2. Preparation of rabbit bone defect

An implant bed of 4mm diameter and about 9mm depth was prepared in the rabbit knee joint (distal femur) using an internal cooling drill. The resulting cavities were then filled in each case with 30 and 90mg of the active substance complexes which had been prepared as described under I. In each case one additional well was left untreated and served as a control for new bone formation.

Fig. 1 shows the new osteogenesis in the untreated cavity and the cavity after implantation of the active substance complex 28 days after surgery, and the density of the surrounding pre-existing sponge substance (n 2/active substance mass).

Analysis of the test results revealed that the sponge material around the holes was 45% higher in density than the untreated holes after implantation of 30mg of the active substance complex and 69% higher in density than the untreated holes after implantation of 90mg of the active substance complex. The amount of the preexisting sponge mass has no influence at all on the regeneration action of the defect, since the new osteogenesis action after insertion of the active substance complex does not start from the periphery of the cavities but is distributed uniformly in the defect site.

III production of sheep mandible using tricalcium phosphate (TCP)

Tricalcium phosphate (TCP) is based on CaO/P₂O₅The calcium phosphate ceramic of the system is prepared by pressing and then sintering raw materials of calcium oxide (CaO) and phosphorus pentoxide (P)₂O₅) And then the preparation. Alternatively, it can be prepared by a hot pressing step.

1. Animal for test

Mature domesticated sheep from Viehzentrale Sudwest AG, Stuttgart were used in the following experiments. They were supplied with hay and water, and three days prior to surgery with Altromin granulation slurry.

Animals were given 1ml xylazine/1 ml ketamine i.m. beforehand. The sheep were then anesthetized with sodium pentobarbital.

2. Preparation of implants

TCP is suspended in 10ml of aqueous solution of 100mg of the active substance complex and deep-frozen with liquid nitrogen under constant stirring. After 24 hours of freeze-drying, the TCP with the active substance complex incorporated therein was implanted in the mandibular defect described below in sheep by gas sterilization (ethylene oxide). In addition, another mandible defect for control purposes was loaded with un-doped TCP that had been sterilized in an autoclave.

3. Preparation of sheep mandible defect

The sheep mandible was suitably prepared, cut with a 5mm diameter trepan using saline as a coolant, and standard cylinders of bone were taken, respectively. One of the formed holes was then filled with TCP, which had been doped with the active substance complex according to test procedure 1, and the second hole was filled with undoped TCP.

For clarity, the results of bone growth in the mandibular defect are graphically shown in fig. 2. The duration of the test was 26 days and 41 days, respectively.

It was found that TCP incorporating the active substance complex promoted bone regeneration of the No.811 and No.86 sheep mandibular bone defects by about 100% in the initial phase. After 41 days, the promotion rate of the bone regeneration effect is still 10%. The bone therefore heals more rapidly, in particular in the initial phase, than the osteogenic effect of an implant without the active substance complex incorporated.

This finding is particularly important for endoprostheses coated with active substance complexes. In the case of, for example, a fracture of the femoral neck, an endoprosthesis coated with an active substance complex correspondingly facilitates a more rapid integration of the prosthesis and thus a more rapid regeneration and recovery of the patient. Thus shortening the hospitalization period.

IV, experiments with collagen as a support material

The known active substance complexes discussed above can be used for the incorporation of endoprostheses. In the preparation of the active substance complexes, the quantitative yields with the desired degree of purity are very low. We therefore examined the presence of a support material capable of binding to the active substance complex in order to reduce the quantity of active substance complex required for a specific purpose, without however reducing its osteogenesis efficiency.

1. Active substance complex

The active substance complexes used for the test purposes described below were prepared completely as described under I and using tubular bones from calves.

2. Animal for test

Male Wistar rats weighing between 350 and 400g were used and housed in an air-conditioned animal house at 23 ℃ and a relative humidity of about 50%. They were given rat and mouse maintenance feed.

Two implants of the same support material, one of which was coated with the active substance complex and the other of which remained uncoated, were implanted into the abdominal musculature of each test animal and served as control implants. The animals were sacrificed after 21 days and the area of action of the implant within the abdominal musculature was removed for histological evaluation.

3. The support material

In these experiments, the collagen materials used were all commercially available. Collagen a is pure, sterile, natural, absorbable collagen of the cowhide skin, without any extraneous additives, such as stabilizers or disinfectants.

Collagen B is purified, freeze-dried, lightly cross-linked, sterile and pyrogen-free bovine skin collagen with weak antigenic properties. The helical structure of the collagen is preserved.

Collagen C comprises pure, natural and absorbable bovine collagen fibers.

All collagen used was reticulated. Sections of the collagen mesh weighing 50mg each were cut and 1ml of the active substance complex solution (3mg/ml) was added. In the control implant, 1ml of distilled water was added instead. The treated collagen mesh sections were frozen at-20 ℃ and freeze-dried to obtain implants about 10mm in diameter and about 5mm thick. Fig. 3 shows the results of osteogenesis after 21 days for collagen implants A, B and C, both coated and uncoated with the active substance complex, in immunosuppressed and non-immunosuppressed animals with cyclosporin a. Here, the evaluation value (BZ) corresponds to the arithmetic mean of the evaluation values of six implants by three persons per group.

Collagen a coated with the active substance complex shows an osteogenic effect on immunosuppressed animals after this period of time, while this effect is not demonstrated in collagen B. However, in phase, collagen C showed a very significant osteogenic effect.

It follows that this depends on the formulation of the particular collagen used and shows suitability as a support material. Immunogenic collagen is not suitable for use as a support material.

IV, biocompatibility of test support Material

In tests involving an increase in the long-term stability of the endoprosthesis, titanium disks, TiAl from Friedrichsfeld with different surface roughness (100, 20 and 0.5 μm) were used₆V₄Alloy (0.5 μm) and Al₂O₃Disks, and hydroxyapatite disks from Feldmuhle AG. The hydroxyapatite is obtained by ceramic firing of calcium pentahydroxide triphosphate powder at 1250 ℃. In addition, hydroxyapatite can also be prepared from natural materials, such as the carbonate skeleton of red algae. After washing and drying operations, the organic constituents are first removed by pyrolysis at a temperature of about 700 ℃. Then, a phosphate solution is added under high pressure and high temperature to convert into hydroxyapatite.

In a further method of preparing hydroxyapatite ceramics, starting from a natural coral skeleton, the calcium carbonate of the coral is hydrothermally converted into hydroxyapatite or a mixture of hydroxyapatite with other mineral structures. In the resulting material, the coral-like structure, i.e., the interconnected pore system of the coral, remains.

The active substance complexes which have been prepared by the general procedure described above are applied by dip coating. Dip coating is understood as a coating process in which the object to be coated, in this case a disk, is dipped into a solution of the coating agent, in this case an active substance complex, having the desired, predetermined concentration. And then freeze-dried. A thin coating or layer is obtained. The biocompatibility of the indicated materials, in particular with respect to surface roughness, was determined (n-20; four discs each).

This biocompatibility test of the materials studied revealed that titanium is very suitable as a support material, with the highest number of living cells and the best ratio of living to dead cells. Hydroxyapatite gives approximately good results, TiAl₆V₄Is quite poor.

As regards the surface roughness, it has generally been found that the smoothest surfaces, i.e. surfaces with a pore size of 0.2-0.5 μm, TiAl, give the best results₆V₄With the exception of this. As the coarseness or pore size increases, the number of live cells and the ratio of live to dead cells decreases. A pore size of about 0.5 μm maximizes the proportion of living (bone) tissue in direct contact with the disc surface.

Table 1:

support material	Per cmNumber of living cells of	Per cmNumber of dead cells
			Hydroxyapatite 0.2-0.5 μm	1792±700	200±37
20μm	7469±2614	2238±715
			50μm	4477±408	1692±427
Osprovit(Feldmuhle)	7930±2007	1638±377
			Titanium 0.5 μm	11377±2538	1054±308
20μm	9600±3038	1754±439
			100μm	2308±669	2085±623
TiAlV0.5μm	7200±1062	2800±954
			AlOUltra pure, polished	11446±1500	2292±600

These test results can now be converted into the coating of the endoprosthesis with an active substance complex. A view of the endoprosthesis used is shown in fig. 4.

Before the use of the endoprosthesis, their outer surface (I) is coated with the active substance complex by a dip coating process, and the active substance complex is additionally introduced into the lumen of the stem (II) of the prosthesis, the lumen having an opening at the surface of the stem. The advantage is that in case of possible future loosening of the endoprosthesis, the active substance complex can subsequently be applied without great effort and causing osteogenesis, thus stabilizing the endoprosthesis. If desired, the region of the screw connection (III) can also be coated with the active substance combination.

Table 2 illustrates the fact that the loading capacity is higher with the active substance complexes than with uncoated surfaces, as exemplified by Hydroxyapatite (HA). Tensile strength values at the interface between different implant materials in N/mm²Expressed as ± standard deviation. The hydroxyapatite prepared by a Hot Isostatic Pressing (HIP) process was compared with hydroxyapatite additionally coated with an active substance complex. The implant material was implanted into the distal femoral region of rabbits and examined 84 days later. Tensile strength values are listed in the table below.

Table 2:

material	SR(μm)	Number of days	n	Tensile strength
					HA HIP	0.5	84	10	1.53±0.24
HA HIP AS	0.5	84	6	2.27±0.31

Number of implants

AS ═ active substance-coated complexes

Hot Isostatic Pressing (HIP)

Surface roughness (SR ═ surface roughness)

Finally, it must again be pointed out that the tests carried out for the purposes of the present invention are carefully designed model tests, since the actual subject (i.e. the endoprosthesis, for example, implanted in the femoral region) cannot be utilized for the tests, since these tests would not be acceptable for the human body.

In addition, the invention can be applied to all conceivable endoprostheses. The description of the example of the endoprosthesis in the femoral neck region is merely illustrative.

Claims (4)

1. Endoprosthesis implant, having an outer surface (I) which is at least partially coated with an active substance complex having a pore size of 0.2 to 0.5 μm, the active substance complex having the following components which are different from one another and which are specifically adapted to the respective biological body part to be formed, namely at least one structural component based on extracellular substances which is specifically adapted to the cells of the respective biological body part to be formed, at least one supplementary component, at least one adhesion component, and at least one growth and/or maturation component.

2. An endoprosthesis implant as claimed in claim 1, characterized in that the endoprosthesis has at least one cavity (II) which is filled with the active substance complex.

3. An endoprosthesis implant as claimed in claim 1 or 2, characterized in that the active substance complex is further combined with a supporting material.

4. An endoprosthesis implant as claimed in claim 3, characterized in that collagen or a polymer other than immunogenic collagen is used as further supporting material.