Medical device based on Aloe schweinfurthii (anteriacae) for the preservation and regeneration of living cells, specifically the cells of the periodontal ligaments of an avulsed immature permanent tooth

Abstract

Medical device based on Aloe schweinfurthii (anteriacae) for the preservation and regeneration of living cells, specifically the cells of the periodontal ligaments of an avulsed immature permanent tooth. A medical device based on Aloe schweinfurthii (anteriacae) for the preservation and regeneration of living cells, specifically periodontal ligament cells, from avulsed permanent teeth. The present invention relates to the technical field of preserving living cells, specifically periodontal ligament cells, using concentrations of 5 to 50% of Aloe schweinfurthii mucilaginous gel in avulsed permanent teeth. The invention provides a low-cost preservation method with maximum safety; in fact, studies on its general oral toxicity in rats have shown no organ damage during the study period. This has enabled the development of a medical device for the safe preservation of living periodontal cells from avulsed teeth.

Description

[0001] Medical device based on Aloe schweinfurthii (anteriace) for the preservation and regeneration of living cells, in particular the cells of the periodontal ligaments of an expelled immature permanent tooth

[0002] A- Description of the invention

[0003] 1- Introduction

[0004] The present invention relates to the technical field of living cell preservation. The object of the invention is to create a new medical device containing a medium for preserving and regenerating living cells, in particular periodontal ligament cells, a medium containing Aloe schweinfurthii gel. The recovery of an expelled tooth that cannot be reimplanted immediately requires a suitable preservation medium.

[0005] The aim of treatment is therefore to maintain cell vitality through a preservation medium and limit pulp contamination by infectious agents. Periodontal ligament (PL) cells, immediately immersed in a suitable medium, can maintain their vitality for a long time. Some preservation media have been studied in the literature, however these media are expensive and not always available.

[0006] A / oe schweinfurthii (A. schurenfurthii) has shown superior efficacy to conventional media in preserving periodontal ligament cell vitality. In the search for cost / dose ratio, accessibility, efficacy and safety, the present work was undertaken to contribute to solving public health problems related to the preservation of ejected immature permanent teeth through the use of A / oe schweinfurthii gel.

[0007] It allowed us to create a medical device that allows the preservation of living cells. For a better justification and knowledge of the elements favoring the preservation of the cellular vitality of A.schurenfurthii gel, we aimed to evaluate the effectiveness of a conservation medium based on A.schurenfurthii gel on the cellular survival of the periodontal ligaments of an expelled immature permanent tooth. This specifically involved:

[0008] - Determine the minimum effective concentration of A. schweinfurthii gel capable of maintaining cell survival of the periodontal ligaments of an ejected immature permanent tooth and compare it with usual media.

[0009] - Identify its composition in secondary metabolites as well as its biological activities (antioxidant, anti-inflammatory and antibacterial).

[0010] - Assess its oral toxicity on Wistar strain rats.

[0011] This study facilitated the process of developing a medical device that can be used for primary health care in general and in odontostomatology in particular at a lower cost. A medium for preserving living cells based on A. schweinfurthii that can be used in the management of expelled teeth. - State of the art known to the inventor

[0012] Reimplantation is an effective treatment option for an expelled tooth. However, the survival of replanted teeth depends on various factors, such as the time interval between expulsion and reimplantation, the type of conservation medium, and the vitality of periodontal tissues. The appropriate use of a conservation medium is an important clinical factor affecting the postoperative prognosis of expelled teeth after reimplantation.

[0013] Hank's balanced salt solution (HESS) and pasteurized milk are considered the clinically recommended storage media for ejected teeth, Osmanovic and Allen 2018 conducted a literature review on periodontal cell viability in different media

[0014] 1

[0015] REPLACEMENT SHEET (RULE 26) of conservation; They showed that HBSS, milk, vispan and propolis preserved more than 80% of living periodontal cells after 2 hours.

[0016] After 24 hours, only Vispan still showed 88% of live periodontal cells. The International Dental Association recommends the use of HBSS, which is marketed under the name "Rescue Box". Live cell preservation media marketed under the name Euro-Collins are also used in oral medicine, however all these media are marketed at a very high cost that is not within the reach of vulnerable populations.

[0017] Moreover, in the case of dental trauma, it is difficult to find this kind of solution at hand. Plants contain secondary metabolites with biological activities and a great diversity of chemical structures. This is the case of Aloe, which has more than 400 varieties, and therefore only half a dozen have medicinal properties. Hu et al in 2003 demonstrated the antioxidant potential of Aloe barbadensis.

[0018] Aloe vera leaf extract showed in vitro antibacterial properties against Gram + and Gram bacteria and also on two multi-resistant strains: Staphylococcus aureus ATCCC 25923 and Escherichia coli ATCCC 25922. Pandey and ail studied the antibacterial activities of crude extract of Aloe bcirbadensis on isolated bacterial pathogens. The fresh gel reduced acute inflammation in rats (carrageenan-induced paw edema).

[0019] A comparative study shows that mice treated topically with Aloe vera gel regain healthy skin while those treated with placebo still show signs of atopic dermatitis. Guo and Ail in 2016 evaluated the toxic effect of Aloe barbadensis. Very few studies have worked on Aloe schweinfurthii in oral medicine and even fewer on media for preserving expelled teeth.

[0020] Unresolved issues

[0021] For cell survival, the International Dental Association has recommended the use of Hans' Balanced Solution (HBSS) as an effective medium for preserving living periodontal cells. However, this product is rare, expensive, and not always available to our populations. Preservation media based on pasteurized milk at 4 degrees has a very short shelf life, limited to 6 hours, and is non-regenerative. Knowing that accidents sometimes occur on weekends, some populations do not have the necessary immediate means for the treatment of the ejected tooth or they live very far from the health center that should accommodate them. Therefore, these studies did not resolve the problem of time required for a large mass of populations who are often far from hospitals or do not have immediate financial means.By seeking the cost / dose ratio, accessibility, efficacy and safety, the present invention was undertaken in order to contribute to the resolution of public health problems related to the preservation of expelled immature permanent teeth through the use of A. schweinfurthii gel. In this context, the present invention aims to provide a medical device containing a living cell preservation medium based on Aloe schweinfurthii.

[0022] It preserves for a time greater than 12 times that described in the literature with milk. Another objective is to provide a low-cost preservation medium because Aloe schweinfurthii is available in many gardens and therefore the marketing of the leaf is accessible to the general public. The preservation device according to the present invention must also present maximum safety in terms of sterility, regeneration and harmlessness.

[0023] It has the advantage of being easy to handle and therefore reproducible from one batch to another. It is sterile due to its antibacterial activities, it is anti-inflammatory. It is

[0024] 2

[0025] REPLACEMENT SHEET (RULE 26) regenerating due to its antioxidant activities. It is accessible due to its presence in gardens and the quantity required to prepare the solution is small. The preservation medium of the invention can be described as a medical device repairing and regenerating living cells.

[0026] Indeed, the preservation medium according to the invention contains anti-inflammatory, antioxidant and antibacterial activities. We used concentrations of 5 to 15% which showed 99% of living cells at the 1st hour and 94 to 99% after 24 hours. The concentration of 50% allowed us to preserve the living cells for 72 hours, which is 12 times more than what is described in the literature. The preservation media in the literature have not evaluated these effects. This is what we did by evaluating in vitro the antioxidant, antibacterial, anti-inflammatory activity as well as the assessment of the toxicity of the gel (T Aloe schweinfurthii . This allowed us to move towards the formulation of a medical device based on Aloe schweinfurthii capable of preserving and regenerating living cells in complete safety. - Brief description of the invention

[0027] The medical device based on Aloe schweinfurthii is a medium for preserving living cells, in particular the periodontal ligament cells of expelled immature permanent teeth. The present invention relates to the technical field of preserving living cells, in particular the periodontal ligament cells, through the use of Aloe schweinfurthii as a medium for preserving living cells. The recovery of an expelled tooth that cannot be reimplanted immediately requires an adequate preservation medium. For cell survival, some preservation media have been studied in Western countries. However, they are expensive, not always available and sometimes non-regenerative. Hence the present invention is seeking an alternative medium based on Aloe schweinfurthii compared to the usual media.

[0028] In the search for cost / dose ratio, accessibility, efficacy and safety, the present work was undertaken in order to obtain a device preserving immature permanent teeth expelled through the use of A. schweinfurthii gel. The study lasted 8 years, the national herbarium identified the plant. The preservation of cell vitality by concentrations of 5 to 50% was studied by aqueous eosin staining and observation under a light microscope.

[0029] Phytochemical characterization was evaluated by colorimetric and complexometric techniques of Harbome, Trease and Evans. Total phenols by Lolin-Ciocalteu test. Evaluation of antioxidant biological activities by 2,2-diphenyl-l-picrylhydrazile radical activity and Ferric Reducing Capacity of Antioxidant. Anti-inflammatory activity by carrageenan-induced rat paw edema inhibition method.

[0030] Antibacterial activity was evaluated by well diffusion and microdilution methods. Acute oral toxicity was assessed on female rats of Wistar strain while subacute toxicity was assessed on male and female rats of the same strains. Liver, kidney and hematological parameters were measured after animal sacrifice as well as organ section observation.

[0031] Results: The pH of the different solutions ranged from 7 to 7.2. All the minimum concentrations used from 5 to 50% of Aloe schweinfurthii maintained 95 to 99% of the cells alive after one hour. The concentration of 15% maintained 99% of the cells alive over 24 hours. The maximum time of preservation of the live CLPs was 72 hours with the 50% concentration. Seven groups of chemical compounds were identified.

[0032] The total flavonoid content was 75.28 ± 0.22pg eqv Querc / mg DM. The gel showed a DPPH radical scavenging power with an effective concentration (Ce) of 748.90 ± 0.17, (IC50)

[0033] 3

[0034] SUBSTITUTE SHEET (RULE 26) was 224.67 ± 0.52 pg / mL, with a free radical scavenging power of 0.13 ± 0.00 ax 102. The FRAP test showed a reducing power of 83.76 ± 1.76 pg eqv Ac Gal / mg dry matter. Its anti-inflammatory activity was superior to that of aspirin between the 5th and 6th hour.

[0035] The diameters of the inhibition zones of Streptoccocus mutans ATCC 49619 were 9.40 ± 0.25mm. The ratio between the minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) was between 1 and 2, so the gel was bactericidal according to the Kamanzi scale in 2002. The A / oe schweinfurthii gel administered at doses of 2000 and 5000mg / kg did not cause any physical disturbance or weight loss in the animals.

[0036] The maximum tolerated dose was found to be greater than 5000 mg / kg. Doses of 2000 and 5000 mg resulted in a significant increase in organ weight at p < 0.01. No damage to the removed organs was observed on organ micrographs. Therefore, the mucilaginous gel of A. oe schweinfurthii can be used in the standardization of available preservation media, regenerating at concentrations of 5 to 50% in complete safety. - Description proper

[0037] The present invention relates to the technical field of the preservation of living cells, in particular periodontal ligament cells, a medium containing A / oe schweinfurthii gel.

[0038] The invention aims to create a new medical device, a medium for preserving living cells, particularly periodontal ligament cells. Indeed, during trauma with dental expulsion, when the tooth cannot be reimplanted at the accident site, a medium for preserving the dental organ is often necessary. The recovery of an expelled tooth that cannot be reimplanted immediately requires an adequate preservation medium.

[0039] The aim of the treatment is therefore to maintain the vitality of the cells by means of an adequate preservation medium capable of maintaining the vitality of the periodontal ligament cells (PLC) for good reimplantation and limiting pulp contamination by infectious agents. The PLCs, immediately immersed in an adequate medium, can maintain their vitality for a long time and promote the success of the reimplantation process of the expelled tooth.

[0040] For cell survival, the use of a preservation medium is an important factor affecting the postoperative prognosis of ejected teeth after replantation. Hank's balanced salt solution (HBSS) and pasteurized milk are considered the recommended preservation media for these teeth. A literature review showed that HBSS, milk, vispan, and propolis preserved more than 80% of live CLPs after 2 hours.

[0041] After 24 hours, only Vispan still showed 88% of living periodontal cells.

[0042] The International Dental Association recommends the use of HBSS marketed under the name "Rescue box." Live cell preservation media marketed under the names ViaSpan and Euro-Collins are also used in oral medicine, however, all of these media are marketed at a price that is not affordable for vulnerable populations.

[0043] Furthermore, in the case of dental trauma, it is difficult to find this type of solution at hand. In this context, the present invention aims to provide a preservation device containing a medium for preserving living cells for a time greater than 12 times the time described in the literature. Another objective of the invention is to provide a preservation medium at low cost.

[0044] The preservation medium according to the present invention must also offer maximum safety in terms of sterility and availability. It has the advantage of being easy to handle and therefore reproducible from one batch to another. It is accessible due to its presence in the Gardens. The preservation medium of the invention can be described as a medical device regenerating CLPs due to its anti-inflammatory, antibacterial and antioxidant activities.

[0045] 4

[0046] SUBSTITUTE SHEET (RULE 26) As Trope et ail says, if the preservation medium has antioxidant ingredients, the effectiveness of the medium will be increased. In the search for the cost / dose ratio, accessibility, effectiveness and safety, this work was undertaken to contribute to solving public health problems related to the preservation of DPIE through the use of D. schweinfiirthïi gel, which allowed us to create a medical device for preserving living cells.

[0047] For a better justification and knowledge of the elements favoring the conservation of the cellular vitality of the gel of A.schurenfurihii. we have:

[0048] To determine the minimum concentration of A. schweinfurthii gel capable of maintaining the survival of CLPs in an expelled PI tooth.

[0049] Identify its composition in secondary metabolites as well as its biological activities.

[0050] Assess the level of oral toxicity.

[0051] This study facilitated the process towards the development of a medical device; preservation medium based on A. schweinfurthii usable in primary health care in general and in odontostomatology in particular at a lower cost. Our research was of an experimental type.

[0052] This study lasted eight years, from April 2015 to January 2021. The collection of plant material was carried out in the central region of Cameroon. The National Herbarium of Cameroon (HNC) was used for the identification of the A. schweinfurthii plant, family Anteriaces.

[0053] At the Yaoundé University Hospital in its Odontostomatology departments, we recruited patients to obtain premolar periodontal ligaments for DPIE.

[0054] The Pathological Anatomy laboratory for microscopic experiments.

[0055] The Biochemical, Bacteriology, Hematological and Pathological Anatomy laboratories for the various blood analyses. The Multidisciplinary Laboratory of the Department of Galenic Pharmacy and Pharmaceutical Legislation (LMSB)Yaoundé I was used for the drying of plant material, phytochemical characterization, investigations for the various antioxidant and anti-inflammatory activities; as well as studies related to the evaluation of the toxicity of A / oe schweinfurthii gel.

[0056] Target population consisted of patients of both sexes aged under 18 years free from general pathologies who came for consultation during the study period and presented with anomalies requiring the extraction of an immature permanent premolar. Male and female albino rats of the Norwegian Rattus species of Wistar strain. Convenience sampling was carried out. Therefore, 5 biological strains were retained, the streptococci of which were taken from samples of the periodontal region before extraction.

[0057] There were 59 albino rats of both sexes of Wistar strain, aged 6 to 10 weeks: 34 female rats and 25 male rats. 9 female rats were used in acute toxicity, 25 female rats and 25 male rats in subacute toxicity. All experiments were conducted in accordance with the rules and regulations adopted by the Institutional Research Ethics Committee of the Faculty of Medicine of Yaoundé I.

[0058] Determine the minimum concentration of AAloe schweinfurthii mucilaginous gel:

[0059] The harvested A. schweinfurthii leaves were cut and placed in trays, then rinsed with tap water before being treated with physiological saline. They were spread out for 30 min to dry in the open air. Each leaf was weighed before being cut longitudinally for gel extraction. A cut A. schweinfurthii leaf weighed on average 80 g.

[0060] Procedure for obtaining A.schweinfurthii gel:

[0061] The artisanal chgel harvest was done by cutting the leaf lengthwise with pruning shears and scraping the central pulp. The pulp obtained per leaf weighed on average 31.5g and was collected in

[0062] 5

[0063] SUBSTITUTE SHEET (RULE 26) a jar. It was poured into the blender to be ground and then filtered. The resulting gel was stored in amber glass bottles in the refrigerator at 5°C for the experiment. The ambient working temperature was 27°C.

[0064] Procedure for obtaining concentrations of A.schweinfurthii gel (planchel):

[0065] The ability of A. schweinfurthii gel to maintain periodontal ligament cell viability was tested using 1% aqueous eosin as a diluent and chemical indicator of cell vitality at a constant volume of 100 ml. 150 milliliter test tubes and 10 cc intramuscular syringes were used. Empty syringes were weighed on a precision balance; the weight of the empty syringe was obtained (p=9.6 g).

[0066] Samples of 5 to 50g of mass of mucilaginous gel were taken using the millimeter syringe, the contents of the syringe + gel were weighed, a large weight P for each sample was obtained. The mass of the gel was obtained by the formula: M=Pp (M: mass of AS, P weight of syringe + AS, p: weight of empty syringe). The concentration was calculated by the ratio of mass of the gel to the volume of 100ml of aqueous eosin.

[0067] Table I: Obtaining gel concentrations of A. schweinfurthii

[0068] Concentration C= mx / v Which gave us the concentrations ranging from 5,10,15,20,25 to 50%. Experimental conditions: The pH set had allowed to evaluate the pH of the solutions of the gel of A.schweinfurthii as illustrated in Table II. The ambient working temperature was 27° Centigrade. The prepared solutions of the gel of A.schweinfurthii were kept in the refrigerator at 5°. For thirty minutes to twenty-four hours while waiting for the dental extractions. 18 healthy immature premolars free of caries and periodontal pathology associated with no systemic pathology were extracted according to the availability of the cases. These teeth were extracted in a traumatic way for orthodontic reasons. They were then rinsed with physiological saline, then introduced into the different concentrations of A.schweinfurthii from 5 to 50%.

[0069] Each medium received an immature permanent tooth. This experiment was carried out 3 times over 8 years.

[0070] Obtaining periodontal ligaments:

[0071] Periodontal ligament cells were obtained by extirpating the ligaments on the roots of extracted premolars using a number 15 scalpel blade (planchel). For collection and

[0072] 6

[0073] REPLACEMENT SHEET (RULE 26) preparation of the alveolo-dental ligaments, the teeth once extracted were held by the crown with sterile precedents.

[0074] They were rinsed three times with physiological saline to remove blood clots. Then they were immersed in the solution of Al.schweirfurthii concentration. The periodontal ligaments were obtained by scraping the middle parts of the dental roots, then were dissected into a thin layer using a number 15 scalpel blade. The ligaments obtained were cut into small particles on the reading slide and returned to the preservation solution, a time T was noted.

[0075] Aqueous Eosin Staining Technique:

[0076] Aqueous eosin measures viable cells. Viability based on the presence of a yellow tetrazolium salt that turns an insoluble formozan crystal purple in color. A 10 cc syringe without a needle was used every 3 hours, for the collection of the gel concentration mixture - 1% aqueous eosin and ligaments; Three drops of the solution were placed on the slide and then covered by the coverslip before reading under a 40 objective light microscope.

[0077] Study of the effect of Concentrations on cell survival:

[0078] This study consisted of interpreting the results of the optical microscope reading by observing or not the cellular vitality of the ligaments. The reading was done according to the storage times in the different concentrations of A.schweinfurthii according to the chronology below: 1, 3, 6, 9, 24, 48 and 72 hours respectively. The percentage of viable cells was noted by counting a reading field then multiplied by 4. Each medium was tested 3 times.

[0079] Cell viability was determined using chemical tests based on aqueous eosin, which is an acid with selective affinity for cell cytoplasm. The existence of viable periodontal ligament cells was identified by red staining of cells that retained their activity in the preservation solution diluted with aqueous eosin. Changes were made exclusively in metabolically active cells by the action of the enzyme dehydrogenase.

[0080] Therefore, the browner the shade, the more indication there was of the presence of viable cells.

[0081] Success status: Viable cells stain red with aqueous eosin.

[0082] Negative control: The percentage of dead cells under optical microscope was determined by the absence of red coloration of the cells or brown coloration and by the decrease in the number of viable cells or the absence of living cells.

[0083] Phytochemical screening, biological activity (antioxidant, anti-inflammatory and antibacterial)

[0084] The aim was to identify the different chemical groups contained in A.schweinfurthii gel by qualitative phytochemical screening, to search for its antioxidant content by quantitative phytochemical screening, and to evaluate its antioxidant, anti-inflammatory and antibacterial activity in vitro.

[0085] The phytochemical profile was determined in order to highlight the different classes of secondary metabolites present in the mucilaginous gel "A.schweinfurthii" which could suggest the mechanism

[0086] 7

[0087] REPLACEMENT SHEET (RULE 26) of their action on the conservation of pulp vitality of expelled immature permanent teeth and justify the desired pharmacological activity. It was highlighted using the colorimetric and complexometric techniques of Harbome, Trease and Evans.

[0088] The different secondary metabolites tested were: Alkaloids, phenols and polyphenols, tannins, saponins, flavonoids, terpenes and steroids, anthocyanins. The dosage of total polyphenols, by colorimetric methods was used to evaluate the quantity of phenolic compounds in the gel. Total antioxidants were evaluated according to the spectrophotometric method using the Lolin-Ciocalteu reagent. The results were expressed in pg gallic acid equivalent / mg of gel tested.

[0089] The evaluation of the antioxidant activity consisted of determining the antioxidant power of the gel by the Benzie and Strain method. The antibacterial activity was carried out according to three successive stages of the culture of the bacterial strains and preparation of the inocula to the determination of the diameters and inhibition parameters. The evaluation of the oral toxicity consisted of highlighting the maximum tolerated concentration of the rats used after oral administration of a single dose for 24 hours.

[0090] Two dose levels were tested: 2000 and 5000 mg / kg body weight. The gel was dissolved in distilled water and the mixture was homogenized by stirring for 2 minutes. The 9 rats used were divided into three groups; then weighed and marked with 1 to 3 identification marks. The control group consumed distilled water, the other two consumed the 2000 mg and 5000 mg gel. They were deprived of food but not water for 12 hours before administration by gavage of 1 ml per 100 g of body weight.

[0091] They were further deprived for 4 hours after administration of the test dose. The weight of each animal was determined shortly before administration of the gel (T Aloe Schweinfurthü Baker, and then daily for 14 days. Weight changes (Vp) were calculated as well as the relative weights of the organs. The subacute oral toxicity assessment of the gel was conducted in accordance with OECD Guideline No. 407, amended on 16 October 2008, for the testing of chemical substances. It consisted of performing a test limited to a sequence of low decreasing doses, the dose 2000 mg / kg and 5000 mg / kg of body weight as the maximum lethal dose in order to highlight any dose-related effects as well as a concentration not observed at the lowest dose. The solution administered to the rats was prepared by solubilizing the gel in distilled water to obtain a stock solution in acute toxicity.1 ml / 100 mg of body weight for 28 days was administered to rats divided into 5 groups. The animals were observed daily for 28 days for signs of subacute poisoning. An individual record was established for each animal or was noted if there was the appearance of particular signs and weighing. The sacrifice of the animals on the 28. e and 42 e days for normal and satellite batches respectively, after injection of ketamine at 1 ml / kg. The collected blood was used for the determination of hematological and biochemical parameters and a histopathological examination of the organs was carried out.

[0092] RESULTS

[0093] To determine the minimum concentration of Aloe schweinfurthü mucilaginous gel effective in the survival of LP cells of a DPIE over time.

[0094] The pH of the solutions and ambient temperature: The pH of the different concentrations used varied from 7 to 7.2. The ambient temperature was constant: 27 in degrees centigrade (Table II).

[0095] 8

[0096] REPLACEMENT SHEET (RULE 26) Table II: pH of the solutions used and ambient temperature

[0097] Maintenance of periodontal ligament cell survival by minimal gel concentrations: The results obtained are illustrated in plate 2: The A.schweinfurthii gel at a concentration of 50% on the vitality of periodontal cells gave us a cell viability of 95% of the periodontal ligament cells at the first hour. The viability at twenty-four hours was 91.5%, after 72 hours there were still 67% of living cells.

[0098] Vitality of periodontal cells using a 25% concentration of A. schweinfurthii: This concentration gave us a 99% cell viability of CLPs in the first hour. 82% of live cells After 24 hours, at 72 hours there are only 35% of live cells.

[0099] Using a 15% concentration of A. schweinfurthii: Gave us a 99% cell viability of the periodontal ligament cells in the first hour, after 24 hours it was still 99%. This cell viability rate decreased significantly because after 72 hours with 41% of live cells.

[0100] Viable periodontal cells by using 10% AA.schweinfurthii: We gave a cell viability of 97.8% of periodontal ligament cells at the 1st hour. A significant decrease in the viability rate of periodontal ligament cells was observed after 24 hours with 89.1% of live cells, after 72 hours there are only 45.6% of live cells.

[0101] Percentage of viable periodontal cells using a 5% concentration of AA.schweinfurthii: We gave a cell viability of 97.8% of periodontal ligament cells at the first hour. We observed a non-significant decrease in the viability rate of periodontal ligament cells after 24 hours with 94.3% of live cells compared to the control of the first hour. The cell viability rate significantly decreased after 72 hours there are only 43.4% of live cells compared to the control of the first hour with a significance rate stopped at P < 0.001.

[0102] Viable periodontal cells using AA.schweinfurthii at different concentrations (plate 4): Cell survival is observed at all concentrations used after 24 hours of tooth expulsion (plate 3).

[0103] Concentrations of 5.15 and 50% maintain 94.3, 99 and 91% of periodontal cells alive after 24 hours, respectively (Figure 8). A periodontal cell vitality rate of 67%

[0104] REPLACEMENT SHEET (RULE 26) is observed after 72 hours with the 50% concentration (Figure 10). A significant decrease in the viability rate of periodontal ligament cells is observed after 72 hours with 67% of live cells compared to the control of the first hour.

[0105] Phytochemical screening, biological activity (antioxidant, antibacterial and anti-inflammatory activity) of A.schweinfurthii gel: Chemical group detection tests revealed that the mucilaginous gel of A.schweinfurthii is an important source of secondary metabolites. We identified 7 groups (plancheô) (Phenol and polyphenols, flavonoids, saponins, alkaloids, sterols, polyterpenes, the absence of anthocyanins and tannins.

[0106] Phytochemical quantification: During the determination of total phenols, the total flavonoid content in the 50% A.schweinfurthi gel was 75.28 ± 0.22 pg eqv Querc / mg DM). Antioxidant activity assessment: The anti-radical activity, the 50% gel showed a DPPH radical scavenging power at 224.67 ± 0.52 (pg / mL) which is a high concentration compared to that of gallic acid. It has a significant antioxidant capacity giving it its capacity for cell repair and regeneration.

[0107] However, it is lower than that of gallic acid. Aloe Schweinfurthii gel has anti-radical activity with an effective concentration EC50 of 224.67 ± 0.52 (pg / mL), an anti-radical power of 0.13 ± 0.00 (ax 10 2) , an inhibitory concentration (IC50) of 224.67 ± 0.52 (pg / mL). This concentration is higher than that of the reference molecule which is gallic acid.

[0108] However, its activities are lower than those of gallic acid. Tests of the reduction capacity of A.schweinfurthii gel 50% of ferric iron into ferrous iron allowed us to observe that the mucilaginous gel of A.schweinfurthii has a metal chelating activity with a reducing power of 83.76 ± 1.76. Its metal chelating activity was lower than that of gallic acid, which was used as a reference molecule with the maximum desired antioxidant activity.

[0109] Antibacterial activity.' Diameters of the inhibition zones of A. schweinfurthii gel; The diffusion of A. schweinfurthii gel in the selected culture media showed the presence of inhibition zones on almost all the bacteria tested after incubation. The Minimum Inhibitory Concentrations of the gel and Ciprofloxacin showed that A. schweinfurthii gel inhibited the growth of all the bacteria tested with S. aureus ATCC 25923 as the most sensitive bacterium which had a MIC of 750 pg / ml.

[0110] The activities of A. schweinfurthii gel are lower than those of Ciprofloxacin with a MIC of 2 pg / ml for the most sensitive bacteria. The minimum bactericidal concentrations of A. schweinfurthii gel and Ciprofloxacin, A. schweinfurthiii gel inhibited the growth of all bacteria that were tested with Staphylococus aureus ATCC 25923 which had a concentration of 1500 pg / ml and Streptoccocus mutans ATCC 49619 that of 1600 pg / ml.

[0111] Report of the CMI and CMB of the freeze 1 A. schweinfurthii: The inhibition parameters made it possible to calculate the MIC / CMB ratio showing a bactericidal effect with a ratio between the CMB and MIC which was understood to be 1 and 2 therefore less than 4. This shows that gel 1 A.schweinfurthii is bactericidal for the strains tested according to Kamanzi's theory in 2002 while ciprofloxacin is bactericidal only on 2 strains out of the 4 tested.

[0112] The evaluation of the anti-inflammatory activity of A.schweinfurthii gel allows us to observe in general that from the first hour of treatment, A.schweinfurthii already exerts a statistically significant anti-inflammatory activity (p<0.05) compared to the negative control batch (distilled water). This activity highlights a percentage of inhibition of edema of 53%, and is maintained throughout the duration of the 6-hour experiment with a rate of 569 compared to

[0113] 10

[0114] SUBSTITUTION SHEET (RULE 26) aspirin which was only at 372.

[0115] A more precise analysis shows that at the third hour, the percentage of inhibition of edema of A.schweinfurthii is similar to that of aspirin respectively 46 and 42%. But from the fifth hour, the anti-inflammatory effect of A.schweinfurthii becomes superior to that of aspirin (p<0.05) i.e. 163 against 13%, suggesting that V A.schweinfurthii would have a more intense anti-inflammatory activity between the fifth and sixth hour of use.

[0116] We can therefore conclude from these results that A.schweinfurthii gel has an anti-inflammatory activity statistically similar to that of aspirin up to the third hour of use, an activity which becomes more intense than that of aspirin from the fifth hour of use as can be seen in the figure above.

[0117] To evaluate the oral toxicity of A. schweinfurthii gel on Wistar strain rats in the survival of LP cells of a DPIE: Evaluation of the oral toxicity of the gel: During the first 30 minutes, slight drowsiness was observed. Observations on the color of the animals' coat, their feces, perception of pain, salivation, motor skills, and appetite showed no particularity.

[0118] These doses did not cause the death of any animal. No visible signs suggesting the toxic nature of A.schweinfurthii gel were observed. Evolution of the weight mass of rodents during acute toxicity: The weight gain of the animals according to the different doses of A.schweinfurthii gavaged compared to the control group having consumed distilled water was observed. The respective initial body weights of the treated rats and the controls showed an increase since the first day.

[0119] This increase becomes more significant for animals having consumed the mucilaginous gel of A. schweinfurthii compared to the control from the 4th day particularly with the dose of 2000mg with a significant difference (P < 0.001). From the 7 ème day the rats of the group having consumed 5000mg undergo a weight loss paraport to the control group with a significance (P < 0.05) and to the group having consumed 2000mg with a significant difference (P < 0.01).

[0120] In general, the weight of all batches increased with a marked increase in the batch of rats having consumed 2000mg of A.schweinfurthii gel compared to the control batch and that of 5000mg from the 10th day with significance (P < 0.05). An identical mass is observed at the last weighing. Effect of A' Aloes schweinfurthii gel on relative organ weight during acute toxicity: The relative organ weight of rats in acute toxicity is marked by a significant decrease in the lung weight of rats having consumed A.schweinfurthii gel at both doses of 2000 and 5000mg compared to the control batch with a significance threshold set at p < 0.001. The relative weight of the brain and lungs increased in rats consuming A. schweinfurthii gel at both doses of 2000 and 5000 mg compared to the control group with a significance threshold set at p < 0.01.The relative weight of the spleen increased in rats that consumed the 5000mg gel compared to the control group with p<0.05.

[0121] Evaluation of the subacute oral toxicity of Aloe schweinfurthii gel: General signs, In the present repeated dose toxicity study for 28 days with the dose 5000 mg / kg, 2 deaths were observed on day 19 following incorrect gastric gavage. In fact, the animals would have received the gel on the wrong route during which the gavage product would have entered the lungs. No visible signs suggesting the toxic nature of the gel were observed during the experiment on the

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[0123] REPLACEMENT SHEET (RULE 26) rats. In terms of general behavior, the rats did not show any particular signs suggesting the toxic nature of the gel.

[0124] Effects of A.schweinfurthii gel on weight parameters in acute toxicity: Monitoring of the variation in animal growth during subacute toxicity for 28 days by gel in males highlighted the weight gain of animals according to the different doses of A.schweinfurthii force-fed compared to the control group having consumed distilled water.

[0125] The respective initial weights of treated and control rats showed an adaptation period of about three days, especially for animals that consumed 5000 mg of A. schweinfurthii gel. In general, the weight of all groups of normal rats that consumed A. schweinfurthii gel increased compared to the control group with a significance threshold of P < 0.05. Rats in the satellite group that consumed 5000 mg increased in weight compared to the control group with P < 0.01.

[0126] The group that consumed 5000mg lost weight in the last days compared to the control with a significant difference stopped at P < 0.01. This increase was greater for the satellite animals that consumed the A.schweinfurthii mucilaginous gel compared to the normal group with a significant difference stopped at P < 0.001.

[0127] Effect of A. schweinfurthii gel on ALT levels in rats. The gel decreased ALT levels in animals consuming the normal 2000mg and 5000mg doses in males compared to the control with a significance level stopped at P < 0.05. A. schweinfurthii gel caused an increase in ALT levels in animals of both sexes at the satellite doses, which is a sign of hepatotoxicity of the gel at this dose with a significance level stopped at p 0.01%.

[0128] Effect of A. schweinfurthii gel on ASAT levels: There was an increase in ASAT levels in all animals at doses of 5000 satellite. This demonstrates that the gel causes hepatomegaly at satellite doses of 5000 mg in both sexes. Indeed, the dose of 50% satellite corresponds to prolonged exposure of the animals to the gel and to a high concentration, thus increasing the toxic risk. A significance level stopped at P < 0.001. The ASAT level for animals having consumed the dosage of 2000 mg decreased significantly with a difference in significance stopped at P < 0.001 in males compared to the control.

[0129] Aloe schweinfurthii gel induced a decrease in ASAT levels in animals of both sexes at doses of 5000 mg normal with a significance level stopped at P < 0.05.

[0130] Effects of A.schweinfurthii gel on uric acid levels: The gel induced a significant increase in uric acid levels in male rats at the satellite doses of 5000 mg at a significance level stopped at P < 0.001, which means that the gel caused nephrotoxicity at the satellite doses in both sexes. The uric acid level for animals that consumed the dosage of 2000 mg decreased significantly in female rats with a difference of significance stopped at P < 0.01 compared to the control.

[0131] Effects of A. schweinfurthii gel on serum creatinine levels: An increase in serum creatinine levels was observed in both female and male animals consuming 5000 mg of the gel compared to control rats with a significance level of P < 0.05. SA gel increased serum creatinine levels in rats consuming 5000 mg of the gel at both doses in female rats. This increase is related to the increase in kidney weight observed; this indicates probable nephrotoxicity at these doses.

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[0133] REPLACEMENT SHEET (RULE 26) Effects of A. schweinfurthii gel on total serum protein levels: A slightly significant decrease in total serum protein levels was observed in male animals at all concentrations having consumed A. schweinfurthii gel compared to the control with a significance level stopped at P < 0.05. The very small variations in total protein do not allow us to conclude that there is liver damage at these doses.

[0134] Effects of A. schweinfurthii on hematological parameters in male and female animals: Administration of the gel for 28 days caused a moderately significant increase in the white blood cell count in animals of the normal groups at doses of 2000 P<0.01. The mucilaginous gel of A. schweinfurthii caused a highly significant increase in the lymphocyte count in normal animals that consumed 5000mg and the monocyte count in those that consumed 2000mg compared to the control with a significance level of P<0.001. The red blood cell count increased in animals that consumed A. schweinfurthii gel 5000mg / kg normal and satellite compared to the control with a significance level of P<0.05.

[0135] Aloe schweinfurthii mucilaginous gel increased the white blood cell count, particularly lymphocytes and monocytes, in the normal groups at both doses. This indicates protection of the cellular environment and therefore contributes to the survival of periodontal cells. This means that the gel contains bioactive substances that have an amplifying and stimulatory effect.

[0136] Effect of A. schweinfurthii gel on histological sections of the removed organs: The administration of Aloe schweinfurthii gel did not cause any cellular disorganization, nor damage to the portal vein and bile canaliculi. No abnormalities were observed in the liver and kidney tissues. Indeed, microphotographs of sections of the liver, kidney, and heart, obtained from control and treated rats (males and females) in the different groups did not show any histopathological signs. This demonstrates the absence of toxicity of the gel in the doses used.

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[0138] SUBSTITUTION SHEET (RULE 26)

Claims

B- The demands 1. Medium for the preservation and regeneration of living cells characterized in that it is composed of a concentration of Aloe schweinfurthii mucilaginous gel of between 5 and 50%.

2. Medium for preserving and regenerating living cells according to claims 1 and 2, characterized in that it contains a pH of 7 to 7.2%.

3. Medium for preserving and regenerating living cells according to the preceding claims, characterized in that it contains secondary metabolites consisting of phenols, flavonoids (total flavonoid content of 75.28 ± 0.22 pg eqv Querc / mg DM), terpenes and steroids giving it an antioxidant activity responsible for its capacities for preserving, repairing and regenerating cells.

4. Medium for the preservation and regeneration of living cells according to the preceding claims, characterized in that it is used for the preservation and regeneration of the periodontal ligaments of expelled teeth.

5. Medium for preserving and regenerating living cells according to claims 2 and 5, characterized in that the concentrations of Aloe schweinfurthii of 5%, 10%, 15%, 25% and 50% allow preservation and regeneration of the periodontal ligaments of the expelled teeth for 24 to 72 hours.

6. Medium for preserving and regenerating living cells according to claim 6, characterized in that the concentrations of Aloe schweinfurthii gel of 15% allow preservation and regeneration of the periodontal ligaments of the expelled teeth with a vitality rate of 99% after 24 hours.

7. Medium for the preservation and regeneration of living cells according to claim 6, characterized in that the concentrations of Aloe schweinfurthii of 50% allow preservation and regeneration of the periodontal ligaments with a vitality rate of 67% after 72 hours.

8. Medium for preserving and regenerating living cells according to claims 1 to 5, characterized in that it is anti-inflammatory with an anti-inflammatory activity greater than that of aspirin from the 5th hour, antibacterial and having no toxic activity.

9. Method for obtaining the medium for preserving and regenerating living cells according to claims 1 to 4, characterized in that it contains the following steps: Determination of the minimum concentration of Aloe schweinfurthii mucilaginous gel by dilution to 1 acquese eosin. Determination of pH of solutions and ambient temperature; Aqueous eosin staining and light microscope observation of cell vitality of the 5-50% concentrations studied; Phytochemical screening (qualification and phytochemical quantification) Biological activity (antioxidant, antibacterial and anti-inflammatory activity) of A. schweinfurthii gel Evaluation of acute and subacute oral toxicity of A. schweinfurthii gel on Wistar strain rats.

10. Use according to any one of claims 1 to 10, for the preservation, repair and regeneration of living cells in the manufacture of a device from concentrations of 5 to 50% of Aloe schweinfurthii gel. 14 SUBSTITUTION SHEET (RULE 26)

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