Dental Ring

Abstract

Disclosed herein is a ring prepared from osteoinductive and/or osteogenic material for a use in a dental and orthognathic surgery in case of bone surface deficiency for the introduction of intraosseous dental implants.

Claims

1. A dental ring, especially for a regeneration of a bone surface with the possibility of a simultaneous introduction of the intraosseus dental implants wherein the ring dimensions are: height (h) from 3 mm to 6 mm, outer diameter (y) from 6.8 to 12 mm, internal diameter (x) from 2.8 to 6.0 mm and the thickness of a wall (z) from 2 mm to 4 mm and it is prepared from the cytocompatible porous material with the open porosity from 70 to 85%, pores size in the range from 200 m to 800 m, preferably from 200 m to 500 m and the size of junctions between pores not smaller than 100 m.

2. A dental ring according to claim 1 wherein its compressive strength is from 0.2 to 0.7 MPa, preferably about 0.7 MPa.

3. A dental ring according to claim 1 wherein it was additionally saturated with a culture medium, particularly a medium for stem cells isolated from tissues of an adult donor.

4. A dental ring according to claim 1 wherein it was additionally seeded with cells, particularly stem cells isolated from tissues of an adult donor, preferably in an autogenic system.

5. A dental ring according to claim 1 wherein it was prepared from a material being a suitable surface for osteogenic cells in a culture.

6. A dental ring according to claim 1 wherein it was prepared from the porous material consisting of 60% of hydroxyapatite and 40% of beta tricalcium phosphate.

7. A dental ring according to claim 1 wherein it was prepared from the porous material consisting of calcium carbonate in the crystal structure of a calcite type.

8. Dental kit wherein it consists of the ring as described in claim 1 and the fitted dental implant.

Description

[0026] For a better understanding of the subject matter the present invention is supplemented with a detailed description of the exemplary embodiments comprising also appended sequencing listing and figures in which:

[0027] FIG. 1. VEGF release by human osteogenic cells in culture (O)values evaluated by means of ELISA assay in days 1 and 4 of a cell culture. For a comparison, analogous values obtained in human endothelium cells culture (S) carried out in identical conditions are shown.

[0028] FIG. 2. VEGF mRNA expression in human osteogenic cells (O) in days 1, 4, and 7 of a cell culture determined by means of a real time PCR technique. GAPDH was used as a reference gene; results were normalized to the value obtained for osteogenic cell culture in day 1. For a comparison, analogous values obtained in human endothelium cells culture (S) carried out in identical conditions are shown.

[0029] FIG. 3. shows the results of a computed tomography performed for a control sample excised from an experimental animal. A sample is a ring prepared from an autogenic bone that was introduced into an acceptor site (in mandible) and stabilized by conical titanium implant. Observations were conducted for 6 weeks.

[0030] FIG. 4. shows the results of a computed tomography performed for a ring prepared according to the invention and excised from an experimental animal after 6 weeks of implantation. A ring prepared according to the description in example 1 was introduced into an acceptor site (in mandible) and stabilized by a conical titanium implant.

[0031] FIG. 5. shows an outline of a ring according to the invention where: xinternal diameter of a ring, youter diameter of a ring, zring wall thickness, hheight of a ring.

[0032] FIG. 6. shows the result of a XTT test performed after 7 days of cells culture carried out on scaffolds prepared from Maxresorb material. The results are expressed as a mean value for 6 measurements, as a percent of a control (value normalized to the result obtained in a control population i.e. cells cultured on a standard culture mediumexpressed as %).

[0033] FIG. 7. shows living cells (stained with green dye fluorescein) seeded and cultured for 7 days in in vitro conditions on a scaffold made of Maxresorb material. Fluorescence corresponds to the presence of living cells on the material.

[0034] FIG. 8. shows a picture of a histological slide taken after the decalcification of the sample and titanium implant removal. Paraffin embedded specimen was prepared by sectioning parallel to the long axis of titanium implant. Hematoxylin/eosin stained specimens depict a whole cross-section of a ceramic scaffold together with recipient's tissue surrounding the ring. The figure was prepared by an automatic assembling of the photographs taken with a 4 lens. It is shown in the picture that the shape of an engrafted scaffold was retained, no signs of resorption are apparent.

[0035] FIG. 9. shows a picture of a histological slide taken after the decalcification of the sample and titanium implant removal. Paraffin embedded specimen was prepared by sectioning parallel to the long axis of titanium implant. Hematoxylin/eosin stained specimen depicts that all pores of the engrafted ring prepared according to the invention are filled with the connective tissue and bone tissue.

[0036] FIG. 10. shows a picture of a histological slide taken after the decalcification of the sample and titanium implant removal. Paraffin embedded specimen was prepared by sectioning vertical to the long axis of titanium implant. Hematoxylin/eosin stained specimen depicts that all pores of the engrafted ring prepared according to the invention are filled with the connective tissue and bone tissue and a very good integration of this ring with the recipient's tissue.

[0037] FIG. 11. shows a picture of a histological slide taken after the decalcification of the sample and titanium implant removal. The picture of a specimen stained with hematoxylin/eosin taken with a large magnification depicts even filling of scaffold pores with a new recipients tissue, penetration of a tissue through individual pores of a scaffold, which confirms the optimal size of the connections between pores.

[0038] FIG. 12. shows a picture of a histological slide taken after the decalcification. A specimen stained with Goldner-Mason trichrome stain. In the picture, vascularization created between fragments of new bone tissue created inside the pores of the material a ring according to the invention is prepared from is shown (example 1).

[0039] FIG. 13. shows results of the XTT test performed in the various time points of a cell culture carried out in calcite medium.

[0040] FIG. 14. shows cellular nuclei stained with Hoechst stain. A picture confirms an even distribution of cells on a porous calcite base.

[0041] FIG. 15. shows cells seeded on a calcite scaffold and cultured for 7 days. Living cells are stained in green (phalloidin staining), dead cells are stained in red (propidium iodide staining).

[0042] FIG. 16. shows a ring formed from CaCO.sub.3 twisted on a dental titanium implant.

[0043] FIG. 17. shows rings made of chitosan (A) the lack of porosity is seen on the picture; (B) a titanium implant screwed into chitosan ring.

[0044] FIG. 18. shows porosity of the rings made of chitosan and modified chitosan rings.

[0045] FIG. 19. shows internal design of chitosan rings (A) and modified rings (B, C).

[0046] FIG. 20. shows rings made of modified PLLA: a) initial sample, b) c) d)rings after culturing in subsequent time points.

[0047] FIG. 21. shows viability of osteogenic cells on the surface of ceramic materials with different phase composition in a culture on flat samples on the 3.sup.rd day of the culture (a) and in three-dimensional porous samples (structure analogous to a ring) in the 3.sup.rd week of the culture (b). The material symbols have the following meaning: Kcontrol i.e. standard culture surface in a form of polystyrene culture plates, 1two-phase ceramic material comprising 60% of hydroxyapatite (phase composition as in Maxresorb material), 2two-phase ceramic material comprising >99% of hydroxyapatite, 3two-phase ceramic material comprising <18% of hydroxyapatite. When cells survival in a flat culture remains on the level that is not lower than in the control, a survival of cells cultured in a three-dimensional structure analogous to the ring is highmaterial 1. When survival in a flat culture remains on the level that is statistically significantly lower than in the control (***-P<0.001), cells survive in a three-dimensional structure analogous to the ring to a very low extent. As an effect, materials 2 and 3 turned out to be a culture surface that was not suitable enough for a culture of osteogenic cells and did not provide suitable survival of those cells in a three-dimensional structure of a ring.

EXAMPLE 1. A RING PREPARED FROM 60% OF HYDROXYAPATITE (HA) AND 40% OF BETA TRICALCIUM PHOSPHATE (-TCP) (DRY RING)

[0048] Commercially available synthetic ceramic material in a form of porous blocks is shaped to a form of a ring of a desired shape:
Sequence of the procedure steps: [0049] 1. from an available block with dimensions of 201010 mm made of Maxresorb material three blocks with dimensions of 10105 mm are formed; from each of them cylinders with a height of 5 mm and diameter of 10 mm are developed using piezosurgery Surgysonic II manufactured by Esacromdevice and equipped with ES 002 diamond drill bit with a diameter of 150 microns and a power of 25 W and vibration amplitude of 160 microns; [0050] 2. in the prepared cylinder an internal aperture with the diameter of 3.2 mm is extracted using piezosurgery Surgysonic II manufactured by Esacromdevice equipped with ES 002 diamond drill bit with a diameter of 150 microns and a power of 20 W and vibration amplitude of 100 microns; [0051] 3. a shaped ring is packed and sterilized by means of radiation of 25 kGy. [0052] 4. a sterile ring is ready for an implantation into acceptor site and fixation with a dental implant.
In order to prepare rings commercial material available under the trademark of Maxresorb was used. It is a synthetic material with a controlled resorption. Material consists of 60% of hydroxyapatite (HA) and 40% of beta tricalcium phosphate (-TCP) (dry ring) It was prepared on the matrix of pores connected to each other that form material with a porosity of about 80% and pores with a size of from 200 to 800 m.

EXAMPLE 2. A RING SATURATED WITH CELL CULTURE MEDIUM (WET RING)

[0053] Commercially available on the market synthetic ceramic material in a form of porous blocks is specifically shaped to the form of a ring with the desired dimensional shape (outer diameter10 mm, internal diameter3.2 mm, height5 mm). The prepared ring is sterilized by means of radiation in a dose of 25 kGyring preparation according to the description in example 1.
The sterile ring is placed in a culture medium and using vacuum pump vented under vacuum. To vent a ring a pressure of about 0.5 to 0.6 Bar was applied.
While the ring is being vented, a standard medium for cell culture is transferred into its pores. Culture medium composition: DMEM medium (Life Technologies) enriched with an inactivated fetal bovine serum (FBS) in a concentration of 10%, supplemented with antibiotic in a form of an Antibiotic-Antimycotic preparation (product of Life Technologies, containing 10000 units of penicillinin a form of sodium salt, i.e. penicillin G, 10,000 g streptomycinin a form of streptomycin sulphate), L-glutamine in a concentration of 2 mM (Life Technologies). After venting, rings are individually transferred into wells of 24-well plate (the diameter of a well is 15 mm) and from 1.5 ml to 2 ml of culture medium of a composition as described above is added. All the procedure steps are carried out in a laminar flow cabinet ensuring aseptic working conditions and samples remain in the culture medium for a period of time from 12 do 24 hours in order to wash out residual material created while material was developed technologically, if needed. After that time, the ring is being washed at least 3 for 5 min in a culture medium without FBS. Such prepared sterile ring is ready for an implantation into an acceptor site and fixation with a dental implant.

EXAMPLE 3. A RING SEEDED WITH CELLS

[0054] Cells are isolated from fragments of recipient's adipose tissue. For cells isolation a tissue fragment of a volume of min 20 ml to 40 ml is required. The tissue is mechanically purified to remove all kinds of tissues other than adipose and grinded to fragments of 2 mm. Tissue fragments are then washed at least 3 in PBS solution (product of Life Technologies) and then enzymatically digested in a collagenase solution (product of Life Technologies) in a concentration of 400 U/ml=0.15%. The volume ratio of the amount of adipose tissue to collagenase should remain 1:1. Such prepared solution should be incubated in 37 C. for 4 h with applied constant shaking of about 200 rpm. During this time adipose tissue is dissolved in collagenase. Having obtained possibly homogenous suspension, it is then centrifuged at 1500 rpm for 10 min then, the resultant supernatant is removed and remaining pellet is washed in a culture medium of a composition described in example II and centrifuged again at 1500 rpm for 5 min. Again, the resultant supernatant is removed and a pellet is suspended in a culture medium. Such an obtained mixture should be filtered through the nylon filter of a density of 100 m. A filtrate comprising isolated cells is mixed with culture medium of the composition described in Example II and seeded into culture flasks (in the volume of about 15 ml) in the number of 1 million cells/1 flask. Sterile culture flasks manufactured by NUNC with the culture surface of 75 cm.sup.2 are used. Cell culture is conducted in an incubator ensuring constant culture conditions, i.e., humidity of above 95%, temperature of 37 C. and presence of 5% carbon dioxide. The culture medium is replaced with a fresh one every 3-4 days. Cell culture is continued until confluencepreferably 70-80% confluence.
Then, cells are detached from the bottom of the flasks by means of trypsinization and subsequently are centrifuged at 1500 rpm for 10 min and counted in hemocytometer and resuspended in the adequate volume of a culture medium.
Ready to use, sterile (sterilized by means of radiation in the dose of 25 kGy) scaffold in the form of rings, prepared according to the description comprised in Example I and vented according to the procedure described in Example II, was seeded with cells isolated from adipose tissue and propagated during in vitro culture. The process of seeding scaffolds with cells is carried out in vacuum to ensure an even distribution of cells throughout the whole available surface of a ring.
Seeding method: vented scaffold in the form of a ceramic ring, prepared according to the procedures described in examples I and II, is transferred to a sterile 10 cm syringe. Cells detached from the culture surface are suspended in the culture mediumDMEM medium (Life Technologies) enriched with inactivated fetal bovine serum (FBS) in the concentration of 10%, supplemented with an antibiotic in the form of Antibiotic-Antimycotic preparation, L-glutamine in the concentration of 2 mM (Life Technologies) and vitamin C in the form of L-phospho-ascorbic acid in the concentration of 100 M (SIGMA) in an amount of 700.000 cells/2 ml medium. Prepared cells solution is withdrawn with a syringe containing the ring inside. The air contained in a syringe is removed, then, after closing a syringe a vacuum is created inside by delicate dragging and loosing a piston. Such an activity is repeated 3 to 5 times. The scaffold seeded in such a manner is placed inside of a well of 24-well plate and covered with a volume of cells suspension that remained in the syringe. Such prepared scaffolds are transferred to an incubator ensuring constant culture conditions i.e. the humidity of above 95%, temperature of 37 C. and the presence of 5% carbon dioxide. After 24 hours, the culture medium is replaced with the fresh portion of a culture medium of the same composition in order to remove cells that do not adhere to scaffold and a scaffold itself is transferred into a new well within the culture plate. The cell culture on such a prepared scaffold is carried out for another 7 days in standard culture conditions, in an incubator with the constant humidity of above 95%, temperature of 37 C. and in the presence of 5% carbon dioxide. After that time, scaffold is washed at least 3 for 5 min in a culture medium without FBS. Such a prepared scaffold/ring is ready for an implantation into an acceptor site and fixation by a dental implant.
To ensure the quality control of the prepared graft, each time 2 additional scaffolds/rings seeded with cells are prepared. These scaffolds are used to assess viability of the cells cultured in applied conditions. Simultaneously, the cytocompatibility of the material used to prepare a ring is assessed. To evaluate cells viability, the XTT test (product of SIGMA)measuring an activity of cellular mitochondrial dehydrogenase and live/dead type fluorescence stainingstaining with fluorescein and propidium iodide reagents (products of Life Technologies) are performed enabling determination and comparison of the quantity of living and dead cells attached to the scaffold at the time of implantation. The biocompatibility of the material/ring was stated if the viability of cells cultured thereon quantified by XTT test was min. 70% when compared to the control cells. Controls are the cells cultured on the bottom of a culture plate in the same conditions as cells cultured on scaffolds.

EXAMPLE 4. PERFORMANCE VERIFICATION FOR THE PRODUCTS DESCRIBED IN EXAMPLES II AND ILL CARRIED OUT BY MEANS OF AN OBSERVATION AFTER IMPLANTATION INTO EXPERIMENTAL ANIMALS TISSUES

[0055] The rings seeded with cells and cultured in in vitro conditions were observed in the tissues of experimental animals (the description for the rings preparation method is comprised in example II and III). The rings were transplanted into the mandible of a small Gttingen minipig. Each implantation was carried out in an autogenic system. Two rings were transplanted into each animal: one seeded with cells according to the description comprised in example III and second non-seeded and prepared according to the description comprised in example II. Individual rings were implanted on the right and left side of a mandible in parallel. The observation in in vivo condition was carried out for 6 weeks. After that period of time, animals were euthanized and implanted rings together with a surrounding new tissue and a portion of mandible were excised.

[0056] Macroscopically no difference between seeded and non-seeded rings was noted. All rings were overgrown and grown through with tissue and stably embedded in a mandible bone (the mean implant stability was about 70 ISQ). After in vivo culture neither connective tissue capsule around rings nor macroscopic inflammation marks were observed. The excised rings were transferred into 10% buffered formalin solution (product of SIGMA). The prepared histological sections of the tested product after in vivo observation reveal the presence of well vasculated and organized connective and bone tissue (HE staining) that fill pores of both type materials (FIGS. 8 to 12). The collagen fibers that are luminescent in a polarized light were visualized with Sirius red. The observations carried out by light microscopy confirm that the rings according to the invention serve their function and can be utilized to reconstruct vertical bone losses.

EXAMPLE 5. A RING PREPARED FROM CALCIUM CARBONATE

[0057] From a ceramic material made of calcium carbonate (CaCO.sub.3) characterized with the calcite type crystal structure scaffolds in a shape of rings with the dimensions of: internal diameter 10 mm, outer diameter 3.2 mm, height 5 mm were prepared. The rings were characterized by the open porosity of 70-80% and the pores size of 200-500 m; the compressive strength of the tested samples is about 0.7 MPa. The shape and technical parameters of the prepared samples suggested that the rings made of calcite might be utilized for prosthetic reconstruction. In further experiments rings were exposed to the culture medium of the following composition: DMEM medium (Life Technologies) enriched with an inactivated fetal bovine serum (FBS) in the concentration of 10%, supplemented with antibiotic in the form of an Antibiotic-Antimycotic preparation (product of Life Technologies, containing 10000 units of penicillinin the form of sodium salt i.e. penicillin G, 10,000 ug streptomycinin the form of streptomycin sulphate), L-glutamine in the concentration of 2 mM (Life Technologies). The rings were transferred into medium and incubated in the standard culture conditions i.e. the humidity of above 95%, temperature of 37 C. and in the presence of 5% carbon dioxide. After incubation lasting up to 35 days none changes in the structure of the material were observedits integrity was sustained, the external and internal dimensions were not altered. In the further experiments human osteogenic cells were seeded on calcite rings (the response of the commercially available cell lineMG-63 (ATTC) was also studied in the separate experiments) to carry out a culture in a contact with ceramic rings. Cultures were maintained in dynamic conditions for the period of 7 to 35 days. After this time, a test evaluating cells viabilityMTT test assessing the activity of cellular mitochondrial dehydrogenase was performed. Tests results indicate high tolerability of cells on the used biomaterial (FIG. 13). Additionally, the possibility for an even distribution of living cells on the entire available culture surface of calcite biomaterial was confirmed (FIGS. 14 and 15).

EXAMPLE 6 COMPARATIVE

[0058] Introduction. Selection of the Material Suitable for a Ring Preparation.
During the conducted research work a number of types of cytocompatible materials were tested against osteogenic cells, such materials potentially could be utilized to prepare a ring designated to regenerate losses within mandible or jaw.
As a result of the conducted in vitro and in vivo experiments, it has been recognized that in order to prepare rings according to the invention only cytocompatible material with the open porosity of 70 to 80%, pore size in the range from 200 m to 800 m, preferably from 200 m to 500 m and the size of junctions between pores not smaller than 100 m can be used.
Also, preferably, its compressive strength is comparable to the compressive strength of a dried human cancellous bone, namely from 0.2 to 0.7 MPa, preferably, it should be about 0.7 MPa.
It is also desired that the material utilized to prepare the ring according to the invention is biodegradable, however, its degradation should not occur too rapidly and the aforementioned mechanical parameters should be sustained for at least the period of 30 days from the day of ring transplantation.
Additionally, it is desired that the material the ring is made of is hydrophilic. This feature ensures better absorption of the active substances from blood, which improves ring healing and bone tissue reconstruction as well as it facilitates cells seeding.
Moreover, it is desired that the ring is characterized by the microporosity and grittiness of a surface. The ring should be characterized by the ease of surgical convenience that should be understood such that despite its fragility typical for ceramic materials, it preserves its shape and does not crush while handling during the stage of the preparation for an implantation and the implantation itself.
In case of the ring designated to be seeded with osteogenic cells (as in Example 3) the material that the ring is made of should be a suitable culture surface for the osteogenic cells in culture. According to the invention, a material meets such criteria if cells survival on the surface made of a material identical in terms of chemical composition, in a form enabling to culture cells in the conditions comparable to a routine cell culture on the standard culture plates (polystyrene) is not statistically significantly lower than in such a routine culture. During work undertaken to obtain the invention, it was surprisingly founded that statistically significantly lower survival in the plate culture conditions is correlated with survival on the three-dimensional ring structure, and disadvantageous influence of a material that is observed in a plate culture is surprisingly potentiated in the three-dimensional structure. This effect is presented in FIG. 21. Selected examples describing unsuccessful embodiments of a ring prepared from materials that did not meet the criteria of the invention are presented below.

Chitosan Rings

[0059] During the performed preliminary studies and an identification of the biomaterial suitable to reconstruct existing bone losses within a jaw or mandible rings made of chitosan were prepared. Chitosan is a polymer of natural origin. An elementary unit of a polymer chain is (1-4) 2-amino-2-deoxy-D-glucose (or D-glucosamin). It is a material belonging to the resorbable polymers group that is being utilized in a production of various medical products. Accordingly, porous chitosan scaffolds of a ring shape and selected internal and external dimensions were prepared. Scaffolds were formed by the extrusion of the previously prepared chitosan granules carried out in an adequate form. The advantage of the obtained scaffolds is their high compressive strength and high biocompatibility. It was suspected that such properties of a material would ensure a suitable supporting construction for cells as well as future bone tissue reconstruction after sample implantation into an acceptor site. Cells were seeded on the prepared scaffolds and subjected to culture in standard experimental conditions. Additional studies on scaffold characteristics were performed in parallel. Based on the obtained results, it was stated that rings prepared by the described method were characterized by a low porosity and a small diameter of junctions between the pores (FIGS. 17, 18 and 19). Results of the cells observations also confirmed that the architecture of the scaffold disables even cells distribution on the sample surface. Accordingly, such scaffold cannot be utilized in clinic because pores size disables blood vessels to invade the scaffold and therefore tissue nutrition.
Within additional experiments attempts for the modification of the chitosan rings preparation were undertaken, however, such attempts did not result in a significant improvement of the scaffolds architecture.
Porous Ring Prepared from Lactic and Glycolic Acid Copolymer
The rings were prepared in a following manner: the mixture of polymers PDLLA-PLGA and PLLA is dissolved in 1,4-dioxan and porogen (NaCl) with the granule size of 250-500 m in the amount of 270 mg per sample is added. Then the mixture is frozen in the liquid nitrogen and freeze-dried for a minimum of 10 days. After freeze-drying, samples are pressed in forms under vacuum. Then NaCl is washed out from samples and the samples of scaffolds are dried in air for 24 hours following by vacuum drying. Due to such a procedure the material of a desired porosity and pore size was obtained.
Following seeding with cells rings are maintained in cell culture conditions i.e. in the culture medium analogous to the medium in an example presented in present application. Cells well tolerate the culture surface material and divide intensively in the culture, which proves the cytocompatibility of such a biomaterial.
In a continuous culture occurs macroscopically and microscopically visible material degradation such that eventually in in vitro condition (still outside the body system, before implantation to tissues) a product with complete scaffold disintegration is obtained. It is shown in FIG. 20.
Such a disintegrated product does not meet the criteria that are stated for rings for augmentation despite the fact that the criteria regarding scaffold porosity and material cytocompatibility were met. Therefore, according to the invention it is required that the material a ring is made of was characterized by the stability in aqueous and biologically active environment lasting at least 30 days.