USE OF 5-AMINOLEVULINIC ACID FOR BONE REGROWTH

FIELD OF THE INVENTION

The invention relates to 5-aminolevulinic acid and/or the salts thereof for use in favouring the proliferation of osteoblasts and bone mineralisation, and hence bone regrowth, by means of photodynamic therapy.

BACKGROUND OF THE INVENTION

Photodynamic therapy is used to treat various pathologies. For example, in the field of oncology, photodynamic therapy (abbreviated as PDT) is based on the absorption and/or preferential retention of photosensitive substances by tumour cells. The photosensitive substances are generally inert but are capable of stimulating the production of toxic substances that can cause cell damage or death and thus ultimately kill or inactivate tumour cells and pathogenic organisms when they are exposed to radiation at a given wavelength.

In the field of dermatology, photodynamic therapy consists in the application of a preparation containing b-aminolevulinic acid or 5-aminolevulinic acid (abbreviated as ALA) within a carrier that enables the topical release thereof in the skin area to be treated. The desired biological effects of the therapy derive from the interaction between light radiation and protoporphyrin-9 (PpIX), which is the product of the metabolic conversion of ALA; this molecule is a potent photosensitizer, whilst ALA in itself does not interact with light radiation. The conversion of ALA into PpIX takes place selectively in cells in which the metabolic rate is accelerated by pathological conditions (as in tumours or dysplasia), the presence of inflammations (as in the case of acne or due to mechanical stimulations: micro-punctures) or in organisms, such as bacteria, in which the metabolic rate is structurally higher; this is the basis of the treatment’s selectivity.

The conversion of ALA into PpIX takes place proportionally to the contact (incubation) time, which normally ranges from 45 to 240 minutes.

PpIX converts light radiation into chemical energy by means of a photochemical process; the biological effects derive from the production of reactive oxygen species (abbreviated as ROS) within a cell (or bacterium) in which a sufficient concentration of PpIX has arisen; if the ROS production rate exceeds the tolerable value, phenomena of apoptosis or necrosis will occur in the target cell, so the desired therapeutic action will be achieved.

The effectiveness of the method has recently also been verified for antimicrobial treatments against a broad spectrum of pathogens, in wound care applications (photodynamic therapy of skin ulcers) and in dental applications (photodynamic therapy for periodontitis and periimplantitis and other gum and oral cavity infections).

For these applications, it is of particular importance to use a carrier that allows a topical release of the active substance directly to the site to be treated and to maintain the required concentration of the active substance for an extended period.

In this regard, the use carriers based on mucoadhesive polymers capable of adhering to the epithelial surface or mucosa and allowing a sustained release of active ingredients (e.g. a drug) directly to the body site of application is well known. One category of carriers of this type that is particularly advantageous for topical applications uses triblock copolymers of polyethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) known as poloxamers, which, in addition to having mucoadhesion properties, are also thermoreversible (or thermosensitive), i.e. they have a solution-to-gel (sol-gel) transition temperature that can be suitably regulated according to the preparation, thanks to the fact that the PEO block has hydrophilic properties whilst the PPO block has hydrophobic properties.

Patent application EP3127557 describes a preparation containing ALA and poloxamer P407 which favours the absorption of the active ingredient for topical use in a photodynamic therapy.

Patent application WO2019123332 describes a pharmaceutical preparation comprising 5-aminolevulinic acid and a thermosensitive carrier with good characteristics of adhesion to the skin and/or mucosa, such as to remain on the application site long enough to release the active substance, while at the same time having adequate characteristics in terms of compatibility with the skin and/or mucosa, sol-gel transition temperature and active ingredient release. This patent application relates to the use of such a composition in the treatment of a pathology or lesion of the skin or mucosa by photodynamic therapy.

In the pharmaceutical sector there remains a need to provide new uses of known compounds/formulations.

SUMMARY OF THE INVENTION

The present invention relates to the use of 5-aminolevulinic acid (ALA) and/or the salts thereof to stimulate bone regrowth or regeneration, in particular mandibular bone regrowth or regeneration. In particular, the bone regrowth is stimulated by 5-aminolevulinic acid in combination with the application of photodynamic activation, preferably by subjecting the area in which the preparation containing ALA has been applied, after the incubation period, to light radiation at a wavelength ranging from 405 to 650 nm.

In particular, protoporphyrin-9, resulting from the metabolic conversion of ALA, is the substance sensitive to light radiation. Therefore, the photodynamic activation is preferably applied after an incubation time ranging from 45 to 240 minutes .

Preferably, the 5-aminolevulinic acid is comprised in a pharmaceutical formulation in combination with a thermosensitive carrier comprising poloxamers.

Poloxamers are triblock copolymers of polyethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO), wherein the PEO block has hydrophilic properties, whereas the PPO block has hydrophobic properties. Poloxamers have characteristics of mucoadhesion and are also thermoreversible (or thermosensitive), i.e. they have a solution-to-gel (sol-gel) transition temperature that can be suitably regulated according to their preparation.

The use to stimulate bone regrowth in fact entails subjecting the formulation, after having applied it on bone tissue, and after an incubation time ranging from 45 to 240 minutes has elapsed, to photodynamic activation, in particular by means of light radiation with a wavelength ranging from 405 to 650 nm. The time of light application is preferably between 5 and 30 minutes.

After application of the 5-aminolevulinic acid, preferably in a formulation with a poloxamer-based carrier, incubation and subsequent activation by light radiation, a considerable increase in osteoblast proliferation and a notable improvement in the mineralisation of the extracellular matrix were observed. Both parameters are indicative of a stimulation of osteoblast growth, and hence of bone tissue regeneration.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the PpIX content determined by measuring cell fluorescence at Oh, 48h and 72h after incubation of osteoblasts with different concentrations of the formulation of the invention containing 5% ALA (called ALAD) and subjected to photodynamic therapy. An increase in the PpIX fluorescence was observed only when the osteoblasts were incubated with 100% ALAD at 0 hours. At 48h and 72h no increase in the PpIX fluorescence was observed. The data are presented as the mean±SD of three independent experiments in triplicate and are expressed in comparison to untreated, unexposed cells (CTRL). (** p < 0.01 ) (*** p < 0.001 ).

Figure 2 shows the effect of the photodynamic therapy applied to the formulation containing 5% ALA (ALAD) on the cell culture. The sensitivity of the osteoblasts at different concentrations of ALAD (%) in combination with LED irradiation was determined with the MTS assay at 48h (A) and 72h (B). The data are presented as the mean±SD of three experiments independent experiments in triplicate and are expressed in comparison to untreated, unexposed cells (CTRL). (* p < 0.05), (** p < 0.01 ).

Figure 3 shows the alkaline phosphatase (ALP) activity of human osteoblasts cultured after 3 days of treatment with ALAD-PDT. The ALP activity of the cells was increased by the ALAD gel, used at concentrations of 50% and 100% in combination with red light, irradiated for 7 minutes, +12.91 %±0.154 and +14.01 %±0.146%, respectively. The data are presented as the mean±SD of three independent experiments in triplicate and are expressed in comparison to untreated, unexposed cells (CTRL). (** p < 0.01 ).

Figure 4 shows the effects of ALAD-PDT on the osteoblast mineralisation capacity at the 14th day of culture. (A) The Ca deposits of the extracellular matrix were stained with Alizarin red S to highlight the mineralised nodules. (B) A quantitative measurement was performed with cetylpyridinium chloride. An increase in the mineral deposit was detected in osteoblasts treated with 100- ALAD-PDT compared to untreated, unexposed cells (CTRL). A significant decrease compared to the control was observed in osteoblasts exposed only to the LED, as was the case in osteoblasts treated with 100-ALAD alone. (* p <0.05), (*** p <0.001 ).

Figure 5 shows the results of the toxicity assay carried out on osteoblasts (HOBs): the cells were subjected to an ALAD-PDT treatment at different concentrations and the release of LDH was measured at 24 hours. The data are expressed as percentages compared to the control (CTRL).

Figure 6 shows the cell morphology after treatment of the HOBs with the ALA- PDT protocol and staining after 24 h with toluidine blue: Fig. 6A) control (CTRL); Fig. 6B) 0-ALAD-PDT; Fig. 6C) 100-ALAD; Fig. 6D) 100-ALAD-PDT. Figure 7 shows the X-ray images of a molar furcation subjected to hygiene treatment and the application of 5% ALA (ALAD) thermosensitive gel followed by incubation for 45 minutes and then irradiation with red light for 7 minutes, before (Figure 7A) and after, at the 90-day follow-up examination (Figure 7B).

DETAILED DESCRIPTION OF THE INVENTION The terms “bone regrowth” and “bone regeneration” are understood as synonymous.

The term “gel” means an elastic two-phase colloidal material, consisting of a liquid dispersed and incorporated in a solid phase. The liquid fills the structure consisting of the solid, which in turn exploits the surface tension of the liquid to avoid collapsing.

The term “sol-gel system”, means a colloidal suspension capable of solidifying to form a gel.

The term “carrier” means any inert substance or mixture of substances, i.e. one devoid of pharmacological activity which can be mixed with an active substance in order that the latter can be used in the desired form, by facilitating, for example, its administration and/or release, in particular topical administration and/or topical release.

The term “thermosensitive carrier” means a carrier characterised by a sol-gel system in which the transition between states takes place above a certain transition temperature (gelling temperature); below that temperature the system is liquid, above it is solid and the transition is reversible.

The term “poloxamer” means a triblock copolymer consisting of a central (hydrophobic) block of polypropylene oxide)(PPO) with two (hydrophilic) blocks of polyethylene oxide)(PEO) connected at either end and which falls into the following general formula:

Given that it is possible to modify the length of the polymer blocks, there exist different poloxamers with different physicochemical properties, identified by the letter "P" followed by three digits; the first two digits multiplied by 100 indicate the mass of the PPO component, whilst the last digit multiplied by 10 indicates the percentage of PEO. Preferably, the pharmaceutical preparation according to the invention comprises a mixture of poloxamers P407 and P188, i.e. of a poloxamer with a molecular mass of PPO of about 4000 g/mol and 70% PEO content (P407) and a poloxamer with a molecular mass of PPO of about 1880 g/mol and 80% PEO content (P188). In relation to the general formula above, the poloxamer P407 has a value of x and z equal to 101 and a value of y equal to 56, corresponding approximately to 70% PEO units and 30% PPO units, whereas the poloxamer P188 has a value of x and z equal to 80 and a value of y equal to 27, corresponding approximately to 80% PEO units and 20% PPO units. The poloxamers are also known by their trade names Synperonics®, Pluronics®, and Kolliphor®.

The term “photodynamic activation” means the combined use of a formulation containing an active ingredient and/or a photosensitive metabolite thereof, i.e. a metabolite capable of interacting with light radiation, and light radiation applied to the formulation at an appropriate wavelength.

In the present description and in the appended claims, unless indicated otherwise, the percentages are by weight relative to the total weight of the preparation of the invention.

As previously noted, the 5-aminolevulinic acid and/or the salts thereof is used for bone regeneration or regrowth preferably in association with a thermosensitive carrier, preferably a poloxamer-based carrier, and in combination with a photodynamic therapy, preferably by subjecting the ALA- based formulation to light radiation at a wavelength ranging from 405 to 650 nm, preferably from 600 to 650 nm, after an incubation time ranging from 45 to 240 minutes has elapsed.

The poloxamers used as carriers are as defined above. In particular, the pharmaceutical preparation according to the invention contains a thermosensitive carrier comprising from 19% to 24% by weight of poloxamer 407 and from 4% to 8% by weight of poloxamer 188.

Preferably, the poloxamer 407 content ranges from 21 % to 23% and the poloxamer 188 content ranges from 5% to 7%.

In this manner, advantageously, the preparation according to the invention has a sol-gel transition temperature lower than body temperature (i.e. lower than about 37°C), thus enabling the application thereof in the liquid state on a bone surface to be treated, such as, for example, the mandibular bone surface.

At the same time, once in contact with the surface to be treated, the preparation according to the invention gels rapidly in situ (in-situ thermosetting), adhering to that surface and permitting the release, for example an extended release, of the ALA or the salts thereof on the surface to be treated in order to perform the desired therapeutic treatment, in particular by photodynamic therapy.

Furthermore, at the end of the therapeutic treatment, any excess product can be easily removed by washing with cold water, that is, water at a temperature below the sol-gel transition temperature, thus avoiding any pain caused by rubbing of the treated surface.

Furthermore, it has been found that, with the mixture of the aforesaid poloxamers, preferably used in the specified ranges of ratios, the preparation according to the invention shows improved adhesion properties.

In this manner, one also obtains a more effective release of ALA or the salts thereof and it is also possible to reduce the content of the active substance in the composition while obtaining the same the desired therapeutic effect.

The content of ALA or the salts thereof in the composition according to the invention can vary according to the specific bone regrowth needs.

For example, the content of ALA or the salts thereof ranges from 1 % to 30%, preferably from 1 % to 10%, more preferably from 4% to 6%.

The salts of ALA are preferably acid addition salts, selected from salts with a pharmaceutically acceptable acid having a pka lower than 5, preferably a pka lower than 4, and even more preferably a pka lower than 3. Examples of pharmaceutically acceptable acids are inorganic acids such as hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid and particularly nitric acid, and organic acids, preferably sulfonic acid, derivatives of sulfonic acid, aryl, aralkyl or naphthyl acids.

In addition to water, the pharmaceutical formulation according to the invention can further comprise pharmaceutically acceptable excipients and/or additives to the extent that they are compatible with the active substance, i.e. to the extent that they do not negatively impact the characteristics of stability and absorption or release of the active substance or the rheological mechanical characteristics or adhesion of the preparation.

Suitable excipients can include, for example, inert substances such as lactose, starch, magnesium stearate, calcium phosphate, mannitol, colloidal silica, and microcrystalline cellulose. Suitable additives can include diluent agents, emulsifying agents, humectant agents, and preservative agents. Preferably, the content of each excipient or additive ranges from 0.1 % to 1 %, preferably from 0.1 % to 0.5%.

Preferably, the formulation according to the invention comprises at least one preservative in a content ranging from 0.1 % and 0.5%.

According to a preferred embodiment, the formulation according to the invention comprises potassium sorbate and sodium benzoate as preservatives, each of them being present in a content ranging from 0.1 % to 0.5%.

If appropriate, the formulation according to the invention can further comprise buffering agents such as, for example, carbonates, phosphates, or acetates to adjust the pH to a value that is optimal for the use of the therapeutic treatment on the skin or mucosa.

The ALA or the salts thereof or the pharmaceutical formulation comprising said active ingredient and a poloxamer-based carrier, are particularly suitable for use in stimulating the proliferation of osteoblasts.

The ALA or the salts thereof or the pharmaceutical formulation comprising said active ingredient and a poloxamer-based carrier, are particularly suitable for use in stimulating the mineralisation of the extracellular matrix of osteoblasts.

The ALA or the salts thereof or the pharmaceutical formulation are thus particularly suitable for use in stimulating bone regrowth or regeneration, in particular for mandibular bone regrowth or regeneration.

The ALA or the salts thereof or the pharmaceutical formulation exert their activity of proliferation, mineralisation and hence bone regrowth or regeneration through the application of a photodynamic therapy, preferably wherein the ALA or the salts thereof or the pharmaceutical formulation are subjected to light radiation at a wavelength ranging from 450 and 650 nm, preferably from 600 to 650 nm, preferably after an incubation time ranging from 45 to 240 minutes has elapsed.

The time of application of light ranges from 5 and 30 minutes, preferably from 5 to 20 minutes, more preferably from 5 to 10 minutes.

The invention also relates to a treatment method for stimulate bone regrowth or regeneration, in particular mandibular bone regrowth or regeneration, which comprises applying ALA or the salts thereof, or a pharmaceutical formulation comprising ALA or the salts thereof and a poloxamer-based carrier, in an effective amount, to a bone surface of a human or animal patient, followed by the application of light radiation at a wavelength ranging from 405 to 650 nm, preferably from 600 to 650 nm, preferably after an incubation time ranging from 45 to 240 minutes has elapsed.

The pharmaceutical formulation according to the invention can be prepared by means of a process that comprises the steps of:

- dissolving ALA and/or a salt thereof and optionally at least one excipient and/or at least one additive, in a solvent selected from water and an aqueous buffer previously cooled to a temperature of 3-6°C, and kept under stirring, thereby obtaining a solution,

- adding poloxamer 407 and poloxamer 188 in predetermined amounts, individually or in a mixture, to said solution kept under stirring at said temperature,

- continuing to stir the solution until the components are completely dissolved. The at least one additive preferably consists of potassium sorbate and sodium benzoate. EXAMPLES

Preparation of compositions according to the invention

A pharmaceutical composition containing the ingredients specified in the following Table 1 was prepared. Table 1

The composition was obtained by cooling a predetermined amount of purified water at 4°C, then dissolving potassium sorbate and sodium benzoate in the water, in the respective predetermined amounts, under stirring, thereby obtaining a solution. 5-aminolevulinic acid, poloxamer 407 and poloxamer 188 were then added under stirring to that solution, maintained at a temperature of 4°C, and the stirring continued until the components were completely dissolved.

The amounts of poloxamers used were 22% P407 and 5% P188. The solution is liquid at temperatures lower than 28 ^S C and becomes a gel at higher temperatures. The gel obtained is indicated as ALAD.

Light source and irradiation parameters

The light source used is an AIGaAs power LED TL-01 device (ALPHA Strumenti, Italy). This LED device is capable of emitting red light at a wavelength of 630 nm ± 10 nm FHWM, visible to the naked eye. During the experiments, the LED was positioned perpendicularly to the wells at a distance of 0.5 mm (contact mode), in order to have an output irradiation surface of 380 mW/cm ² (spot area) and a specific total dose of 23 J/cm2 per minute of irradiation. The irradiation was performed in the dark under a laminar flow hood in all the experiments.

Experimental design

For the experiments, the cells were seeded and after 24 hours they were incubated with increasing concentrations (0%, 10%, 50%, 100%) of ALAD gel in a serum-free medium at 37°C for 45 minutes, as suggested by the manufacturers. The ALAD gel was added in order to reach these final concentrations: 10% v/v (10-ALAD-PDT), 50% v/v (50-ALAD-PDT), and 100% v/v (100-ALAD-PDT). The cultures were then exposed to irradiation with the LED device for 7 minutes. In the absence of ALAD gel the cells were exposed solely to the LED light (0-ALAD-PDT). In order to explore the effectiveness of the combination of the gel studied with the LED light, the cells were also treated with the ALAD gel at the 100% concentration without irradiation (100-ALAD). The untreated, unexposed cells were considered as a control (CTRL). The time-dependent concentration of PpIX was determined fluorometrically. The cells were subsequently washed three times with PBS and cultured in a medium containing 10% fetal bovine serum (FBS) and the effects of ALA-PDT were evaluated up to 24h for LDH release, up to 48h and 72h after treatment for proliferation, up to 3 days for ALP activity and up to 14 days for mineralisation. The experiments were conducted in triplicate using different cultures each time.

Cell cultures

Human osteoblasts (hOBs) were isolated from fragments of human mandibular bone obtained from 12 patients undergoing treatment at the G. D'Annunzio University dental clinic. All the procedures were in accordance with the ethics standards of the Institutional Committee for human experimentation (reference number: BONEISTO N. 22 10.07.2021 ) and with the principles of the Declaration of Helsinki. After the protocol was approved by the Institutional Review Board, signed informed consent was obtained from every participating subject.

The bone fragments were subjected to three enzymatic digestions at 37°C for 20'; 30' and 60' using a solution composed of collagenase type 1A (Sigma- Aldrich, St. Louis, Missouri, USA) and trypsin-EDTA 0.25% (Sigma-Aldrich) dissolved in Dulbecco's Modified Eagle's medium (DMEM, Corning, New York, USA) with 10% fetal bovine serum (FBS, Gibco-Life Technologies, Monza, Italy). The solution obtained from enzymatic digestion was centrifuged at 1200 rpm for 10'. The pellet was resuspended and placed in a T25 culture flask with DMEM with a low glucose content (1 g/L) supplemented with 10% FBS, 1 % antibiotics (100 pg/ml-1 streptomycin and 100 IU/ml-1 penicillin), and 1% L- glutamine in order to favour a final spontaneous migration of cells. The isolated hOBs were cultured in a controlled atmosphere (5% CO2 and 37°C) after reaching confluence and were used for all the experiments between the 3rd and 5th steps after characterisation by means of cytometric analysis. After 10 days of culture, the bone fragments were removed.

Cytotoxicity assay

Protocol

Cytotoxicity was quantified by measuring the activity of LDH (lactate dehydrogenase) released in the supernatants of the cell cultures of 1 X10 ³ cells/well after the treatments with ALAD-PDT at 24h. The release of LDH was determined using an LDH cytotoxicity detection kit (Roche, Basel, Switzerland) in accordance with the method of cytotoxicity analysis according to the manufacturer’s protocol. Absorbance was read at 490 nm with a microplate reader (Synergy H1 Hybrid BioTek Instruments, Winooski, VT, USA). LDH release was calculated as a percentage compared to the control (CTRL).

For staining with toluidine blue, an amount of 2X10 ⁴ osteoblasts per well was seeded and subjected to the ALAD-PDT protocol. After 24h, the adherent cells were fixed with 70% cold ethanol and stained with 1 % toluidine blue and 1 % borax (Sigma Aldrich, St. Louis, MO, USA). The cells were then observed by means of a microscope connected to a 40X camera (Leica, Wild Heerbrugg, Wetzlar, Germany).

Experimental design

A determination was made of the cytotoxicity of different concentrations of ALAD in combination with light at 24h (10-ALAD-PDT, 50-ALAD-PDT, 100- ALAD-PDT), the cytotoxicity of ALAD alone in a concentration of 100% (100- ALAD), and the cytotoxicity of light without ALAD (0-ALAD-PDT: exposure of cells to 7 minutes of LED irradiation only). As indicated in figure 5, ALAD-PDT was not cytotoxic for the cells in any of the concentrations considered: the cells released a small amount of LDH after incubation with ALAD at the different concentrations and after irradiation. Furthermore, neither irradiation in the absence of a load of ALAD, nor incubation with ALAD alone in the absence of irradiation showed cytotoxicity.

For this reason, the cell morphology at 24h was studied solely for 100-ALAD with and without irradiation. Staining with toluidine blue confirmed the data of the LDH assay, since the osteoblasts were spindle-shaped in appearance and well distributed after the treatment with ALAD-PDT (see Figure 6).

Determination of PpIX

In order to determine the time-dependent intracellular content of PpIX, 6x10 ³ cells/well of human osteoblasts were seeded in 96-well plates and subjected to (%)-ALAD-PDT photodynamic therapy according to the experimental design. After Oh, 48h and 72h the PpIX was extracted with 0.5M perchloric acid (HCIO4) in 50% methanol and the fluorescence was measured at 608 nm after excitation at 405 nm, using a spectrofluorometer for microplates (Synergy H1 Hybrid BioTek Instruments).

Cell proliferation

The effects of ALAD-PDT on the proliferation of hOBs were evaluated with a CellTiter 96 assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) after 48 and 72 hours of treatment, according to the manufacturer’s instructions (MTS, Promega, Madison, Wisconsin, USA). Briefly, 6x10 ³ cells/well were cultured in 96-well plates and treated with ALAD-PDT (10, 50 and 100% v/v) according to the experimental design. At the end of every incubation period (48h and 72h), 10 pl of MTS solution were added to every well and the cells were then incubated for 2h at 37°C and 5% CO2. The spectrophotometric absorbance (optical density, OD) at 490 nm was measured with a spectrophotometer for microplates (Synergy H1 Hybrid BioTek Instruments). The number of cells was calculated in relation to the OD values. The cell proliferation rate was calculated as a percentage compared to the control.

Activity of alkaline phosphatase (ALP)

In order to evaluate osteoblast function after (%)-ALAD-PDT, the activity of alkaline phosphatase (ALP) was determined according to the protocol (Abeam Inc, Cambridge, UK) based on the splitting of p-nitrophenyl phosphate. Briefly, the osteoblasts, 5x10 ⁴ cells/well in 24-well culture plates, were subjected to the (%)-ALAD-PDT treatment. After 3 days, the cells were washed three times with cold PBS and resuspended in the test buffer. The cell suspension was then homogenised by means of a TissueRupture device (QIAGEN, Hilden, Germany). After centrifugation at 10,000 g for 15 minutes, the relative ALP activity of the supernatant was measured using p-nitrophenyl phosphate (pNPP) as a substrate for 1 hour. After incubation the reaction was stopped and the product of the enzymatic reaction, p-nitrophenol, was determined by measuring absorbance at 405 nm.

Alizarin (ARS) staining

The effect of various concentrations of ALAD gel in combination with LED irradiation on the mineralisation capacity of human osteoblasts was analysed by Alizarin red (AR) staining. The untreated, unexposed cells were considered as a control for comparative evaluation. The cells were seeded at the density of 5x10 ⁴ cells/well on the surface of the coverslips (Thermo Fisher, Waltham, MA, USA) in a 24-well culture plate. After 14 days of culture, the samples were rinsed three times with PBS and fixed with a glutaraldehyde solution (2.5%) for 2 hours. After fixing, 1 ml of AR staining solution (Sigma-Aldrich) was added; then followed a 1 -hour incubation at room temperature. The excess stain was washed off using deionised water, and the presence of mineral deposits was qualitatively evaluated by observing the intensity of the red stain on the surface of the coverslips.

Quantification of calcium

The calcium from mineralisation was quantitatively measured with the addition of cetylpyridinium chloride (CPC) to the coverslips after the qualitative measurement of the AR staining. The slides, washed with deionised distilled water, were treated with 1 mL of 10% CPC solution (Sigma-Aldrich) for 1 hour to chelate the calcium ions. After the incubation time, the absorbance was read at 540 nm in a microplate reader (OD540) (Synergy H1 Hybrid BioTek Instruments) and normalised with the number of cells.

Statistical analysis

All the experiments were carried out in triplicate, and the data are reported as the mean ± SD. A t-test was used to compare the two groups. The statistical analyses were performed using GraphPad Prism8.

Results

Accumulation of PpIX

The time-dependent level of PpIX was studied at Oh, 48h and 72h after the osteoblasts had been subjected to different concentrations of ALAD gel for 45 minutes (Figure 1 ). An increase in the intracellular content of PpIX, measured after extraction with perchloric acid, was observed immediately (Oh) after treatment only in the osteoblasts incubated with ALAD gel at a concentration of 100% (100-ALAD), with an accumulation of the highest levels of PpIX when this concentration was combined with red LED light (100-ALAD-PDT). Neither irradiation on its own nor treatment with 10%-ALAD-PDT and 50%-ALAD-PDT showed an increase in the cellular accumulation of PpIX at this time point (Oh), or at the other time points (48h), (72h). The PpIX content did not show an increase in any of the cases. At 48h and 72h the fluorometric measurements of PpIX in the treated and/or exposed cells did not show any differences compared to the untreated/unexposed cells used as a control.

Figure 1 shows the time-dependent PpIX content. The cell fluorescence of PpIX was measured at Oh, 48h and 72h after incubation with different concentrations of ALAD (%). An increase in PpIX fluorescence was observed only when the osteoblasts were incubated with 100% ALAD at 0 hours. At 48h and 72h no increase in PpIX fluorescence was observed. The data are presented as the mean±SD of three independent experiments in triplicate and are expressed in comparison to untreated, unexposed cells (CTRL). (** p < 0.01 ) (*** p < 0.001 )

ALAD-PDT promoted the proliferation of human osteoblasts

The effect of ALAD-PDT on the metabolic activity of osteoblasts was evaluated at 48h and 72h by means of the MTS assay. The results in Figure 2(A, B) show a similar trend at both time points. The cells subjected solely to exposure to the LED (0-ALAD-PDT), like the cells treated with gel without irradiation (100- ALAD), showed the same proliferation rate as the control. The lower concentrations of ALAD gel (10-ALAD-PDT, 50-ALAD-PDT), by contrast, had no appreciable effect on osteoblast proliferation. A significant increase in cell proliferation was observed only in osteoblasts subjected to 100-ALAD-PDT compared to the untreated, unexposed cell cultures. In particular, 100-ALAD- PDT increased cell proliferation by +46.83%±0.230 and +127.75%±0.154 at 48 and 72 hours, respectively. These results suggest that the ALAD gel used as indicated by the manufacturer in combination with LED irradiation could promote osteoblast proliferation.

Figure 2 shows the effect of ALAD-PDT on the cell culture. The sensitivity of the osteoblasts to different concentrations of ALAD (%) in combination with LED irradiation was determined with the MTS assay at 48h (A) and 72h (B). The data are presented as the mean±SD of three independent experiments in triplicate and are expressed in comparison to untreated, unexposed cells (CTRL). (* p < 0.05), (** p < 0.01 )

ALAD-PDT favoured the enzymatic activity of ALP

ALP activity is a fundamental in vitro biomarker of osteoblast functions and was determined, in terms of rate of hydrolysis of p-NPP, 3 days after the cells had been treated and exposed. The graph in figure 4 shows that though irradiation with red light did not exert any negative effect in the ALP activity of osteoblasts exposed to the LED alone (0-ALAD-PDT) compared to the control cells, a marked increase was shown. Similarly, the cells treated with ALAD gel without exposure to the LED showed the same ALP activity as observed in the control. Whereas the lowest concentration of ALAD (10-ALAD-PDT) added to the culture media showed a significant decrease in ALP activity compared to the control. Among the concentrations used to treat the cells in combination with exposure to the LED, both 50 and 100-ALAD-PDT showed stimulatory effects on osteoblast ALP activity. Though a stimulation was observed when the osteoblasts were subjected to 50% and 100% ALAD gel compared to the untreated, unexposed cells (CTRL), no significant increase in ALP activity was measured.

Figure 3 shows the ALP activity of human osteoblasts cultured after 3 days of treatment with ALAD-PDT. Cellular ALP activity was increased by the ALAD gel, used at concentrations of 50% and 100% in combination with red light, irradiated per 7 minutes, +12.91 %±0.154 and +14.01 %±0.146%, respectively. The data are presented as the mean±SD of three independent experiments in triplicate and are expressed in comparison to untreated, unexposed cells (CTRL). (** p < 0.01 )

Deposition of minerals by alizarin staining and treatment with cetylpyridinium chloride

In addition to osteoblast proliferation and ALP activity, the mineralisation of the extracellular matrix is also an important part of bone formation. Therefore, the treated and exposed osteoblasts were examined after 14 days of culture in order to evaluate whether the ALAD-PDT treatments stimulated the mineralisation of the matrix, which is normally correlated to a greater anabolic activity in bone metabolism. The calcified nodules, which appeared as a bright red colour after Alizarin red S staining, took on a more intense colour in the culture treated with 100% ALAD gel following LED irradiation, whereas the intensity of the colour decreased when the osteoblasts had only been exposed to the LED or treated only with 100-ALAD (Fig. 4A). The quantitative results showed that mineralisation occurred in the presence of 100% ALAD gel and increased significantly compared to the control. More conclusively, an increase of +72.03%±0.320 was measured in the mineral deposit for 100-ALAD-PDT. In the untreated osteoblasts (0-ALAD-PDT), by contrast, the deposition of calcium showed a significant decrease compared to the untreated, unexposed cells (CTRL), indicating that exposure to the LED on its own, without the ALAD gel, was not able exert a positive influence on cell mineralisation. Similarly, when the osteoblasts were treated with 100-ALAD on its own, a significant decrease in calcium deposition was observed (Fig. 4B).

Figure 4 shows the effects of ALAD-PDT on the osteoblast mineralisation capacity at the 14th day of culture. (A) The Ca deposits of the extracellular matrix were stained with Alizarin red S to highlight the mineralised nodules. (B) A quantitative measurement was performed with cetylpyridinium chloride. An increase in the mineral deposit was detected in osteoblasts treated with 100- ALAD-PDT compared to the untreated, unexposed cells (CTRL). A significant decrease was observed in osteoblasts exposed only to the LED compared to the control, as was the case in osteoblasts treated with 100-ALAD on its own. (* p <0.05), (*** p <0.001 ).

Evidence of clinical effectiveness

Furcation of a molar subjected to hygiene treatment and the application of 5% ALA (ALAD) thermosensitive gel followed by incubation for 45 minutes and then irradiation with red light for 7 minutes, before (Figure 7A) and after, at the 90-day follow-up examination (Figure 7B).

The 5% ALA thermosensitive gel was applied after the mechanical procedure for removing tartar (scaling); then, about 1 hour after the initial application of the gel, red light was irradiated for 7 minutes.

The molar had an infection in the area where the roots of the tooth branch off (furcation). No remineralisation of this area has ever been clinically observed, but 90 days after treatment with 5% ALA (ALAD) thermosensitive gel a complete resolution of the infection and remineralisation of the bone was observed.