Bone tissue regenerating composition

[0002] The present application is the national stage under 35 U.S.C. 371 PCT/ES00/00029, filed Jan. 25, 2000.

[0003] 1. Field of the Invention

[0004] The present invention relates to the regeneration of bone tissue within surgical practice in general, and especially in oral surgical practice, by filling a bone cavity with a graft material which stimulates and accelerates the regeneration.

[0005] 2. Description of related Art

[0006] Until the present moment, various graft materials have been used, and the nature of the material used determines how bone tissue is regenerated.

[0007] In this respect, several authors have published studies and reports about the use of different materials and/or compounds for this purpose.

[0008] Yarnazaki Y, Oida S, Akimoto Y, Shiosa S in Clin Orthop Related Res (1988: 234; 240-9) refer to the use of a bone-derived morphogenetic protein bound with plaster of Paris (calcium sulphate) for this type of regeneration.

[0009] Traditionally, both autologous bone and demineralised bone (DFDBA), either alone or combined with other elements, have been used to constitute graft material for bone cavities within this technique.

[0010] Moreover, this type of graft material has also been protected with different barrier materials in order to prevent adjacent tissues encroaching on the graft material.

[0011] Various materials have been used as barriers for the aforementioned graft material, the most popular being a poly-tetrafluoroethylene membrane, despite creating problems due to the fact that it is not biodegradable and can cause infections in certain cases. In addition to this, since it is generally used in a solid form, it must be trimmed during surgery to conform to the graft recipient site and then sutured in place.

[0012] The U.S. Pat. No. 5,366,507 specifies a composition which combines a graft material based on a mixture of dermineralised bone and calcium sulphate, and a barrier material consisting of calcium sulphate. This composition does not greatly improve upon the previous techniques.

[0013] Various studies have shown that the growth factors found in the blood encourage bone formation, especially the growth factors P.D.G.F. and T.G.F.B.

[0014] However, the practice of extracting and concentrating such growth factors in an outpatient setting is unknown, as is the clinically observable and measurable effect of these growth factors.

[0015] In a particular embodiment of the present invention, a gel of plasma rich in growth factors (P.R.G.F.) as a graft material for the filling and regeneration of bone in cavities and bone defects, or for regeneration.

[0016] The present invention provides a method to obtain and prepare the factor rich plasma for out patient use.

[0017] The present invention further provides a method and a composition for the regeneration of bone tissue which represents an improved rate in the regeneration and which may be administered rapidly to the patient.

[0018] In yet another embodiment of the present invention, there is provided a kit to be used for the preparation of the P.R.G.F. gel.

[0019] Advantageously, the use of P.R.G.F. gel for the regeneration of bone tissue brings acceleration and benefits to the bone regeneration which are greater than those of techniques currently in use, as well as quicker and more predictable healing of the soft tissues, as will become apparent below from the results obtained.

[0020] The invention provides a method of preparing the aforementioned gel which begins with the extraction of blood from the patient undergoing treatment, minutes before surgery begins and prior to the administration of the anaesthetic. Between 10 and 40 ml was taken from each patient, using 5-ml tubes in which 10% trisodic citrate had been placed to act as an anticoagulant.

[0021] The tubes were centrifuged at speeds between 160 and 800 G (according to

[0022] different procedures which can be applied), for 6 minutes or less (depending on the speed) and at room temperature. The blood separates into the following three basic components:

[0023] Red blood cells at the bottom of the tube.

[0024] Plasma rich in growth factors (P.R.G.F.) in the middle of the tube, above the red layer.

[0025] Plasma poor in growth factors (P.P.G.F.) in the upper part of the tube.

[0026] A measure, 1 ml, of the upper P.P.G.F. layer of each 5-ml tube was discarded. The platelets present in this P.P.G.F. were less than 15%, as measured with count n=10.

[0027] The factor-rich plasma from the central layer of the tubes was transferred to Eppendorf tubes and to each tube containing 1.2 ml of the P.R.G.F. was added 50 μl of 10% calcium chloride, such that, with the plasma left in this state, the P.P.G.F. gel formed after a time period of 15 to 25 minutes. The plasma can also be mixed with human thrombin (500 units) and calcium chloride in order to obtain the gel instantly (between 3 and 10 sec) and to allow the possibility of applying it with a syringe.

[0028] The gel was used alone to fill bone cavities in need of regeneration, giving optimal results, or with the aid of a conventional barrier material to provide external protection, as will be explained below.

[0029] The gel can be combined with other components, such as calcium sulphate, autologous bone, reabsorbable hydroxiapatite tricalcium phosphate, calcium carbonate or other regenerative materials, osteoconductive or osteoinductive.

[0030] For the production of the P.R.G.F., the preparation kit will contain a centrifuge, pipettes for the separation of the layers of plasma and a system for blood extraction.

[0031] The selection of patients was based on the absence of systematic or localised illnesses which could create counter-indications with the treatment. Consent was obtained from all patients, who were informed of the study. The 20 patients presented a vertical fracture or an advanced periodontal illness which required an extraction, and were chosen because the area of implant would allow a subsequent biopsy without additional discomfort. The patients were randomly divided into a P.R.G.F. treatment group and a control group. The mean age of the P.R.G.F. group was 41 (range: 35-55), 4 patients were men and 6 women. The mean age of the control group was 42 (range: 38-54); 4 were men, 6 women.

[0032] Three additional patients (2 men and a woman) required multiple extractions in different mouth locations. In each patient, P.R.G.F. was used for one site but not the other, the type of treatment being assigned to the sites at random. This made it possible to include the best control group as both types of treatment were carried out in the same patient, with the same surgical procedure, identical microbiological conditions and the same surgeon.

[0033] All patients received antibiotic treatment; amoxicillin (1.5 g/day) was used. Skin flaps were elevated to allow adequate visibility and to make it possible to close up at the first attempt.

[0034] Each site was carefully scraped after extraction. In the 10 patients in the experimental treatment group, the defect sites were filled with P.R.G.F. gel. In 5 of the cases, the plasma was mixed with autologous bone to prevent the skin flap from collapsing. For the control group, the procedure was identical but without using the P.R.G.F. gel. Membranes were not used in either of the groups to exclude the possibility of barrier effects interfering with the possible benefits of P.R.G.F.

[0035] Biopsy Technique:

[0036] The graft recipient site was biopsied between weeks 10 and 15, depending on the availability of the patient. All biopsies were carried out by an examiner who did not know which treatment had been administered to each defect site. The biopsies were taken with hollow trephine dental drills in the centre of the recipient graft site and to a depth of 3 mm. The bone biopsies were fixed for 48 hours using 10% formalin in 5% formic acid on swabs, and mounted in paraffin wax. From each biopsy 5 mm-thick sections were made and these were stained with haematoxylin and eosin. The stained sections were photographed under bright light. All the biopsies were sent to a laboratory for analysis, without specifying which belonged to the control group and which were from the working group.

[0037] Results

[0038] The formation of epithelium tissue in all 10 patients treated with P.R.G.F. was evaluated as very good or excellent (much better than what is normally found, and comparatively better than the control group). The regeneration of the treated areas was practically complete in 8 of the 10 cases. The degree of regeneration was evaluated with a periodontal probe and by comparison with the previous defects, which had been photographed. The biopsies of these areas showed mature compact bone with well-organised and morphologically normal trabeculae.

[0039] The other two cases treated with P.R.G.F. displayed partial regeneration, presenting connective tissue with badly organised trabeculae in the biopsies. Both patients, a man and a woman, were smokers and presented major defects in three alveolar walls.

[0040] There were significant differences in the degree of organisation of the trabeculae between the biopsies taken in week 10 and those taken in week 16, and also depending on the size and shape of the defect site. In patients with major defects treated with P.R.G.F. combined with autologous grafts to avoid the collapse of the skin flaps, greater vestibule-lingual widths were obtained.

[0041] In the control group, a homogeneous situation was found in the patients' biopsies: connective tissue filling the greater part of the defect site, in clear contrast with the cases treated with P.R.G.F. All the control group biopsies showed connective tissue and connective tissue containing bone trabeculae.

[0042] Mature bone was not found in any of the cases. The status of epithelium formation was rated as normal, showing a significant difference to the cases treated with P.R.G.F.

[0043] In the patients with more than one recipient graft site, one treated with P.R.G.F. and the other using the conventional method, the epithelisation of the areas treated with P.R.G.F. was much more rapid. The biopsies of the P.R.G.F. treated areas revealed more mature bone with better-organised trabeculae and greater bone regeneration.

[0044] The use of P.R.G.F. provides conditions for more rapid and more effective bone regeneration. This P.R.G.F. gel is easy to handle but must be used without any delay in order to conserve the effectiveness of the growth factors.

[0045] Although the optimal dosages have yet to be determined, the use of this technique introduces no risk for the patient, whose blood is used in a short period of time after extraction (30 minutes to 8 hours) and is not mixed with any other component of animal or human origin. At present around 250 patients have been treated with good clinical results.

[0046] It has been observed through practice that calcium sulphate is occasionally lacking in consistency when it is prepared for application with sterile biocompatible liquids such as water, saline solutions and local anaesthetic solutions, and this can lead to manipulation difficulties during its application, given that it tends to disintegrate and is very soluble.

[0047] Additionally, it is quickly diluted in the blood, thus its efficiency in the regeneration of bone tissue could be improved upon.

[0048] Results from laboratory experiments have shown that the use [of tricalcium phosphate] offers significant advantages.

[0049] Firstly, by its very nature tricalcium phosphate has a higher calcium content.

[0050] Secondly, the behaviour of tricalcium phosphate (putty, supersaturated solution), autologous bone mixed with P.R.G.F. (plasma rich in growth factors), P.R.P. (plasma rich in platelets), P.R.G.F. autologous bone plus tricalcium phosphate, during the treatment in its diluted state is more homogeneous as it provides enough plasticity to make it easier to handle.

[0051] Thirdly, the dilution of tricalcium phosphate in blood is more limited, lower than that of calcium sulphate, rendering it more effective as a barrier material.

[0052] As was pointed out above, the filler material for the bone cavity can essentially be tricalcium phosphate mixed with gel of plasma rich in growth factors, or other combinations with the condition that the material used as a barrier is tricalcium phosphate.

[0053] In particular embodiments of the present invention, other combinations for the filler material have been tested, such as the following (all in combination with tricalcium phosphate):

[0054] plasma rich in platelets

[0055] autologous bone

[0056] freeze-dried bone

[0057] mixtures of the above

[0058] the combination proposed by the present invention makes the tasks in operations for bone tissue regeneration more manageable and easier to complete, and, more importantly, the process of regeneration is quicker than with currently known methods and preparations.