ES2697691B2 - Procedure for obtaining a material for bone regeneration and material thus obtained - Google Patents

Description

DESCRIPTION

PROCEDURE FOR OBTAINING A MATERIAL FOR BONE REGENERATION AND

MATERIAL SO OBTAINED

The object of the present invention is a new formulation for obtaining biocompatible and bioactive rough surface porous (non-polished) materials for potential application as substitutes for a bone graft in those clinical situations where biological stimuli are required to achieve regeneration. that is.

The present invention falls within the technical field of manufacturing biocompatible and bioactive materials for bone reconstructive surgery in general, in the medical fields of traumatology and orthopedic surgery, oral surgery, maxillofacial surgery and neurosurgery.

Prior art

To this day, the standard of bone replacement remains the autologous sponge bone graft. The technique of using the autograft consists of taking bone tissue from another area of the skeleton (donor area) of the same patient, either of the same bone or another. The main drawbacks of autografts are those arising from the search and sacrifice of the donor zone that causes a comorbidity of the patient of up to 25% and limited availability when large amounts of bone are needed to graft [1]. Due to all these disadvantages of autografts, over the last decades, different alternatives to the use of these have been studied, such as natural or synthetic «bone substitutes», including calcium phosphate ceramics (mono and biphasic), biovidrios, calcium sulfate (a-SCH) or plaster of Paris, composites, polymers, etc. Of these, the most used are bioceramics and, specifically, the so-called third generation, these are the matrices of biologically active molecules, which in addition to being bioactive and reabsorbable, stimulate the regeneration of living tissues through a specific cellular response. They are preferred to be used as matrices in tissue engineering.

The requirements of an ideal bone substitute are: biocompatibility, biodegradability, mechanical efficiency for the specific application, bioconductivity, bioactivity, ease of sterilization and storage, and reasonable cost-effectiveness. The comparison with the Spongy autograft, shows that, although the conductivity of the structure is common, osteogenic and osteoinductive abilities are absent. Tissue engineering attempts, adding cells and signal proteins to provide these materials with the standard qualities of grafts.

As a substitute for bone tissue, one of the first materials investigated was the plaster of Paris (CaSO4-1 / 2H2O) [a-SCH]. This is obtained by the calcination process of the natural gipsite at a temperature of 110-130 ° C with which 75% of the water is lost. It was first used in 1892, by Dreesman, to fill bone defects created by tuberculous osteomyelitis. Its reabsorption process causes a controlled increase in porosity while forming new bone. Its use is limited given its rapid reabsorption (4-8 weeks) and its poor structural stability that makes it prone to fractures when loading begins early.

The raw material for its manufacture is cheap and abundant. It has been used both for filling bone defects and as a vehicle for controlled drug release and growth factors [2].

Normally, local dissolution of the plaster causes an increase in the release of calcium ions [Ca] and acidification of the medium that can affect the function of bone cells (osteoblasts) and therefore a certain degree of bone demineralization in situ. Although on some occasion a mitogenic effect on undifferentiated cells has also been described. On the contrary, this fact can inhibit osteoclastic activity causing an imbalance in the bone formation / resorption balance that takes place in the process of bone remodeling. With our material, an inverse process is observed, that is, it causes the alkalization of the medium, which contrasts with what happens with the dissolution of the isolated plaster.

Studies referring to preclinical and clinical applications have been focused on animal trials in the field of dentistry to treat periodontal defects. For this purpose, various compositions have been tested:

- Demineralized and lyophilized allogeneic bone (a-SCH) [3]

- Demineralized bone matrix (DBM) (a-SCH) [4,5]

- Autologous bone (a-SCH) [6]

- Chiotosa (a-SCH) BMP [7]

- Silicated Phosphate-Ca (a-SCH) [8]

- Hydroxyapatite (a-SCH) [9]

- PRP (a-SCH) [10]

- Acrylic cements (PMMA) (a-SCH) [11]

- Antineoplastic (metrotexate) (a-SCH) [12].

- Allografts (a-SCH) [13,14]

- Biovidrio (a-SCH) [15]

- Carboxymethyl cellulose (a-SCH) [16]

- Ac Hyaluronic (a-SCH) [17]

- Polylactic Ac (a-SCH) [5]

- Ac. Polyacrylic (a-SCH) [18]

Unfortunately, the results observed with the use of the different materials or compositions described are not comparable due to the heterogeneity of the designs of the studies carried out (type of animal, study duration, cell lines used, implantation site, etc.).

The association of a-SCH with other inorganic compounds in an effort to improve their handling / manipulation and their physical properties, has not yielded all the optimal results expected [19], although it is true in some situations its bioactivity improved. This bioactivity expressed as a reabsorption / deposition combination of a granular neolayer on the surface of the material and of a composition close to the hydroxyapatite is similar to that observed with the biovidrios.

The addition of TCP to treat critical bone defects in dogs improved the repair of the defect by comparing it with the unfilled control [20].

Plaster has many of the desired qualities of an ideal regenerating material, such as: (a) complete resorption at a short period of time (1 mm per week), (b) excellent biocompatibility, (c) abundant supply of Ca ions , (d) relatively cheap, easy to handle and mold before setting, (e) excellent properties as a carrier for different types of molecules (various antibiotics, growth factors, antineoplastic, etc.). Although it also shows some disadvantages: rapid reabsorption and contact with biological fluids slows the setting which could question its permanence at the implantation site.

Although a part of the trials reference the usefulness of gypsum in bone regeneration, there are some negative reports in animal studies [15]. But a problem that makes comparisons difficult lies in the heterogeneous nature of plaster studies. Despite this, a-SCH today is used for filling bone cavities in both oral and other surgical reconstructive surgery (orthopedics and traumatology, maxillofacial surgery, etc.) [21] [22].

Another aspect of interest is the high temperature reached by certain formulations such as polymethylmethacrylate (PMMA) cements during the polymerization process (setting) a few minutes after mixing. This material retains the same temperature at room temperature (22 ° C) during the entire setting process, which is why we rule out the negative effect that exothermic reactions have on cell adhesion and viability and ultimately on adjacent tissues.

In preclinical studies in vivo that are being carried out in an animal model (rabbit NZ), the effects of the new material in terms of its potential osteogenic, osteoinductive capacity, osseointegration, degree of resorption, etc. are evaluated. In this document the cytotoxicity, viability or cell proliferation, protein adsorption, bioactivity, ionic release, setting time and temperature recorded or detached during the setting of the material object of the present invention have been evaluated.

Bibliography

[1] Gazdag A, Lane J, Glaser D, Forters R. J Am Acad Orthop Surg. 1995; 3: 1-8.

[2] Thomas MV, Pileo D. J Biomed Mater Res Part B: appl Biomater 2009, 88B: 597-610.

[3] Kim CK, Kim HY, Chai JK, et al. J Periodontol 1998, 69: 982-988.

[4] Kim CK, Chai JK, Cho KS, et al. J Periodontol 1998, 69: 13171324.

[5] Rosen PS, Reinols MA. J. Periodotol 1999, 70: 554-561.

[6] Macneill SR, Cobb CM, Rapley JW, et al. J Clin Periodontol 1999, 26: 239-245.

[7] Cui X, Zhang B, Wang Y, Gao Y. J Craneofac Surg 2008, 19: 459-465.

[8] Hing KA, Wilson LF, Buckland T. Spine 2007, 7: 475-490.

[9] Frenkel SR, Simon J, Alexander H, et al. J Biomed Mater Res 2002, 63: 706-713.

[10] Intini GE. Tissue Eng 2002, 104: 384-386.

[11] Tuzuner T, Uygur I, Sencan I, et al. J Orthop Sci 2007, 12: 170-177.

[12] Vechasilp j. J Orthop Res 2007.15: 56-61.

[13] Wright HB. University of Kentucky College of Dentistry, 2004, 62p.

[14] Sottonanti Calciun sulphate. Compedium 1992, 13: 226-228

[15] Melo LG, Nagata MJ, Bosco AF, et al. Clin Oral Implants Res 2005, 16: 683-691.

[16] Vance GS, Greewell H, Miller RL et al. J Oral Maxillofac Implants 2004, 19: 491-497.

[17] Lewis KN, Thomas MV, Puleo DA, et al. J Mater Sci Mater Med 2006, 17: 531-537.

[18] Neira-Carrillo, Yazdani-Pedram M, Retuert J, et al. J Colloid Interface Sci 2005; 286: 134-141.

[19] Huan Z, Chang J. Acta Biomater 2007, 3: 952-960.

[20] Urban RM, Turner TM, Hall DJ, et al. Clin Orthop Relat Res 2007, 459: 110-117.

[21] Kelly CM, Wilkins RM. Orthopedics 2004, 27: 131-135

[22] Kelly CM, Wilkins RM, Gitelis S, Harjten C, et al. Clin Orthop Relat Res 2001, 42-50

Explanation of the invention.

The present invention relates to obtaining pieces composed of a-SCH p-TCP and C ² S that act as a scaffold to promote and direct bone growth at the site of implantation, facilitating the process of regeneration of bone tissues and , as a consequence, the repair of bone defects. Similarly, it is intended to improve certain properties such as: bioactivity, setting time, cell adhesion and manipulation, comparing it with the a-SCH that has been used, in the present invention, as a control material because it is a resorbable material. widely used for decades, both experimentally and in clinical practice.

The invention consists in obtaining a material with the characteristics described as a result of the mixture of three components calcium a-sulfate hemihydrate (a-SCH) [CaSO ⁴ -1/2 H ² O], p-tricalcium phosphate (p- TCP) [Ca ³ (PO ⁴ ) ² ] and dicalcium silicate (yC ² S) [2CaO.SiO2] in a given proportion. For in vitro tests, compact pieces 7.5 mm in diameter and 2.65 ± 0.2 mm in height have been obtained.

In preclinical in vitro tests , the material has been shown to be biocompatible, with no signs of cytotoxicity being observed, likewise capable of absorbing proteins on its surface and something inside, and being highly bioactive, as observed 24 hours after Immersion in simulated human serum (SBF). These facts facilitate cell adhesion on the surface of the material by recognizing the integrins of the cell wall the glycine, arginine and aspartic (GRD) sequence of the adhered proteins. The reaction of the two phases Liquid / powder corresponding to the three components does not cause an exothermic reaction that could cause cellular damage or alteration of the protein layer. Finally, the setting or solidification time is not longer than that observed with a -SCH. Therefore, we consider that this new three-phase and bioactive compound can be considered a suitable material for application in the filling of bone defects of any etiology, in those fields of bone reconstructive surgery, such as orthopedic surgery and traumatology, oral surgery and Maxillofacial and neurosurgery.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to restrict the present invention. In addition, the present invention covers all possible combinations of particular and preferred embodiments indicated herein.

Brief description of the drawings

Next, a series of drawings that help to better understand the invention and that expressly relate to an embodiment of said invention that is presented as a non-limiting example thereof is described very briefly.

FIG. 1 shows the ultrastructural aspect of the components of the mixture of the biomaterial object of the invention, as well as their respective analysis by X-ray diffraction (EDX).

FIG. 2 schematically shows the steps taken to obtain the appropriate mixture, and prior to adding liquid phase (A) to obtain a material with characteristics and consistency similar to the “toothpaste”. Appearance of the pieces setting in their mold (B) and finally, the pieces of the disk-shaped material obtained (C).

FIG. 3 schematically shows the setting time and thermal behavior of the mixture of the material object of the invention, including: the mark perceptible on the surface of the material (Fig. 3-A); Fig. 3-B. Comparative images of brands caused by the needle on the surface of the specimens, a-SCH in contact with bovine serum (upper row), a-SCH (central row) and material of the invention (lower row); Fig. 3-C. Temperature variations recorded during the setting process at room temperature

FIG. 4 shows the determination of protein adsorption on the surface of the material by the Coomassie colorimetric test. (a) Blue coloration of the protein impregnated material on its surface (Coomassie test); (b) Quantification data of absorbed proteins.

FIG. 5 graphically shows the results of the ionic release analysis (Ca, P, S, Si) and pH of the material object of the invention submerged in complete culture medium (DMEM) by means of ICP-OES spectrometry ( Inductively coupled plasma-optical emission spectometer).

FIG. 6 shows various aspects of the characterization of the material carried out by: (FIG. 6A) ultra structural aspect of the material by scanning electron microscopy (SEM) at different magnifications and table with results of the microanalysis; (FIG. 6B) X-ray diffraction (EDX); and (FIG. 6C) infrared by Fourier transform-Attenuated Total Reflactance (FTIR).

FIG. 7 In the bioactivity test carried out, it shows by SEM the appearance of the surface of the material, (a) Limit of the layer of neoformed apatite on the surface of the material (arrow), (b) Thickness of the layer in a section perpendicular to the surface, (c) deposited crystallization nuclei, (d) detail of the previous image at higher magnification (3 ^ m bar).

FIG. 8: (a) the results obtained in cytotoxicity tests compared with Paris plaster using SEM and (b) cell proliferation test on the material by reducing the tetrazolium salt (MTT) to 1, 3, 5 , 7 days.

Statement of a detailed embodiment of the invention and example

The present invention relates to a process for obtaining a three-phase biocompatible and bioactive material obtained by mixing a-SCH (main component) pTCP YC ² S in the 4: 1: 1 ratio or ratios (66.6, 16.6 and 16.6 w%, respectively) for its potential use in bone tissue regeneration.

After mixing the solid phase (powders), deionized water was added in the established liquid / powder ratio of 0.33 ml g-1. Then, the material acquires a pasty appearance similar to the "toothpaste" of medium density, and that is when it is poured onto the mold and allowed to set in a stove at 37 ° C for 24 hours. Finally, the samples were removed from the molds and stored in a sealed container in a dry oven environment. The new material obtained shows a discoid shape of 7.5 mm in diameter and 2.65 ± 0.2 mm in height and of average weight 216 ± 5 mg, of compact appearance that avoids the technical complications caused by dispersion of the material during its subsequent handling when carried carry out in vitro studies and in vivo tests (intraosseous implantation). Due to the characteristics of the material, and its in vitro behavior, it is expected that it will be potentially absorbable in the medium term (4-6 months), so it would not require its subsequent removal from the place where it has been implanted.

Figure 1 shows the ultra structural aspect of the components of the mixture (P-tricalcium phosphate (P-TCP) [Ca ³ (PO ⁴ ) ² ], dicalcium silicate ( ^and -C2S) [2CaO.SiO2], and a-SCH ), as well as their respective microanalysis using EDX. Figure 2, on the other hand, schematically shows the steps taken to obtain the appropriate mixture, and prior to adding liquid phase (A) to obtain a material with characteristics and consistency similar to the “toothpaste”. Appearance of the pieces setting in their mold (B) and finally, the pieces of the disk-shaped material obtained (C).

Figure 3 refers to the setting time and thermal behavior. A modified technique of Gillmore [Kokubo T, Shigematsu M, Nagashima Y, Tashiro M, Nakamura T, Yamamuro T, et al. Bull. Inst. Chem. Res. Kyoto Univ., 60, 260, 1982] to obtain qualitative data. The indenter tip is 1 mm in diameter and concentrates a 50 gram load. Defining as setting time, the time elapsed since the Liquid / Powder mixture and the paste obtained is deposited in the molds, until the needle does not leave an indentation or noticeable mark on the surface of the material. (Fig. 3-A) The test was performed on 5 samples (n = 5) of each material. The results have allowed us to affirm that the setting time of the material object of the invention was twelve minutes, similar to that of a-SCH. This time was significantly increased when the deionized water was replaced by fetal bovine serum to simulate a biological environment in contact with organic fluids (Fig. 3B, top row).

A Flash III-Ther-L02 infrared thermometer was used to determine the possible temperature variations during the setting process. The data collection was made to coincide in time with those made to determine the degree of setting of the material (Fig. 3-C).

Figure 4 shows the determination of protein adsorption on the surface of the material by the Coomassie colorimetric test. (a) Level of staining penetration equivalent to protein absorption. (b) Quantitative test by elemental nitrogen analyzer LECO FP-528 and expressed by bar chart. Tests carried out at 1h and 24 hours of immersion of the fetal bovine serum material and kept in an incubator at 37 ° C during that period.

Figure 5 graphically depicts the results of the ionic release analysis (Ca, P, S, Si) and pH of the material object of the invention submerged in complete culture medium (DMEM) by means of ICP-OES spectrometry ( Inductively coupled plasma-optical emission spectometer). Determinations made at 1, 3, 6 hours and 1, 2, 3, 4 and 7 days. The most relevant changes, greater release of ions, are observed at 24h of immersion that cause a slight alkalization of the culture medium (pH 8.16-8.2).

The pH values were measured using an electrode of a digital pH meter. Before taking any measurement, the pH meter was calibrated with a standard pH solution. The samples were placed in 15 ml Falcon tubes containing 10 ml of PBS buffer. The pH measurements of these solutions were made at room temperature at 1 and 24 hours immersion.

Table I collects the data related to the dilution process of the material object of the invention by comparing it with the a-SCH material. The test was carried out on six discs (n = 6) of each material. These were weighed individually before depositing them in their respective wells of a 24-well plate and held in position by grid. They were immersed in MilliQ deionized water and in magnetic stirring continuously for 24 hours. Then, the supernatant was removed, dried in an oven at 60 ° C and finally, weighed again individually.

The results obtained are shown in the table below as the average of the recorded data expressed in grams.

The analysis of these data reveals that, although part of equal weight in the powder phase, and using some type of mold in the paste phase after adding water, the weights after drying, on average, are lower in the material of the invention. In addition, sometimes, there is no direct relationship between the piece that weighs the most with the amount of material lost or diluted, that is, at a higher weight there is no higher dilution rate. Finally, the material object of the invention exhibits a loss of 4.76% higher than a-SCH (0.009 g).

Figure 6 shows various aspects of the characterization of the material carried out by: (FIG. 6A) ultra structural aspect of the material by scanning electron microscopy (SEM) at different magnifications and table with results of the microanalysis; (FIG. 6B) X-ray diffraction (EDX); and (FIG. 6C) infrared by Fourier transform-Attenuated Total Reflactance (FTIR).

The pattern of X-ray diffraction of the a-SCH and of the invention is observed in FIG. 6B. The material object of the invention has, for the most part, corresponding peaks with dehydrated plaster and hemihydrated plaster (ASTM sheets No. 33-0311 and 41-0224 respectively). The rest of the phases are mainly overlapped with the plaster, although the presence of the p-TCP and yC ² S phases (ASTM sheets n ° 55-0898 and 87-1257 respectively) can be seen in the peaks that arise at 27.8 20 and 32.5 - 32.6 20, and in the increase in intensity of the peak located at 29,720, an effect produced by the presence of yC ² S.

FIG. 6C shows the different spectra of the p-TCP, and C ² S materials, plaster and the material of the invention obtained by an IRTF-ATR (Fourier Transformed Infrared - Total Reflectance Attenuated) equipment. As we can see, in the spectrum obtained from the material of the invention you can see the presence of both the plaster and the p-TCP and the yC ² S. The plaster is the major component, as expected, and the p-TCP and the and C ² S appear in the range of 1200 to 800 cm-1.

The ability to form a layer of apatite on the surface of the material when it comes into contact with the SBF is an in vitro procedure always used to determine the Bioactivity of a biomaterial with a view to its application in bone repair. This test was performed by submerging the material for 24 hours with SBF following the protocol of Kokubo et al. [Kokubo T, Shigematsu M, Nagashima Y, Tashiro M, Nakamura T, Yamamuro T, et al. Bull. Inst. Chem. Res. Kyoto Univ., 60, 260, 1982]. FIG. 7 shows the appearance of the material surface, (a) Boundary of the neoformed apatite layer on the surface of the material (arrow), (b) Thickness of the layer in a section perpendicular to the surface, (c) deposited crystallization nuclei and (d) detail of the previous image at higher magnification (Magnification bar 3 ^ m).

Figure 8 shows the results obtained in the cytotoxicity tests compared with plaster of Paris by scanning electron microscopy and cell proliferation test by reducing the tetrazolium salt (MTT) at 1, 3, 5, 7 days. Multipotent undifferentiated cells of the cryopreserved bone marrow were used and donated by the cell therapy unit of the Arrixaca UV Hospital. The test was carried out in accordance with ISO-10993-5, through direct and indirect tests using 24-well inserts and plates that allowed to keep the material without contact with adult stem cells cultured in the wells at a density of 5 x 103 cm-2 cells. Incubation was carried out under standard conditions of temperature, CO ² and relative humidity. (A) Representative images of the adhesion of adult mesenchymal stem cells (MSCs-A) on the surface of the material at 24 hours of incubation. (B) Polygonal morphology with extensive cytoplasmic extensions (phylopodia) of the cells at 7 days in culture. (100 ^ m bar). (C) Results of the cell proliferation assay (MTT) compared with growth on plaster at 1, 3 and 7 days in culture.

Therefore, as indicated, the cytotoxicity tests carried out indicate that the material developed exhibits great potential for application in bone reconstruction, without waiting for an adverse immune response that leads to cellular damage. The material object of the invention shows a degree of bioactivity much higher than a-SCH as can be observed after remaining 24 hours in contact with the SBF. The ionic release by the material of the invention is maximum at 24 hours and does not cause relevant changes, except slight alkalization in the pH of the medium.

The setting time has results be the same for both materials. A prolonged setting time may cause a difficulty in maintaining its structure at the time of its implantation in the bone defect to be repaired, resulting indirectly in a loss of mechanical resistance, at least initially. No temperature changes were detected during the setting process, keeping or decreasing slightly with respect to room temperature. The fact that an exothermic reaction is not triggered is considered favorable for both protein adhesion and cell adhesion. The presence or contact of the materials with biological fluids such as fetal calf serum (FCS) instead of deionized water considerably slows the setting time.

This material was designed, developed and tested with the intention of increasing the degree of bioactivity and cell proliferation, without varying the setting time, maintaining histocompatibility properties, comparing it with a-SCH.

Claims (5)

claims 1. A material for bone regeneration characterized by comprising calcium a-sulfate hemihydrate (a-SCH), tricalcium p-phosphate (P-TCP) and dicalcium silicate (YC ² S) in a proportion or ratio of the mixture of a-SCH, p-TCP and YC ² S of 4: 1: 1. 2. A method of obtaining a bone regeneration material according to claim 1 comprising the steps of: to. Mix in solid phase a-SCH, p-TCP and YC ² S; b. Add deionized water; C. Pour over a mold and set-dry in an oven at 37 ° C for 24 hours. where the solid phase mixture of a-SCH, p-TCP and YC ² S is performed in a 4: 1: 1 ratio. 3. Use of the material defined in claim 1 or obtained according to the method of claim 2 in the preparation of a preparation for bone tissue regeneration. 4. Use of the material defined in claim 1 or obtained according to the method of claim 2 in the preparation of a preparation for bone reconstructive surgery. 5. Use of the material defined in claim 1 or obtained according to the method of claim 2 in the preparation of a preparation as a bone graft substitute.