risedronate-loaded macroporous gel foam enriched with ... risedronate-loaded macroporous gel foam...

Research Article

Risedronate-Loaded Macroporous Gel Foam Enrichedwith Nanohydroxyapatite: Preparation, Characterization, and OsteogenicActivity Evaluation Using Saos-2 Cells

Nadia M. Morsi,1 Rehab Nabil Shamma,1,3 Nouran Osama Eladawy,1 and Abdelfattah A. Abdelkhalek2

Received 24 October 2018; accepted 26 December 2018

Abstract. The application of minimally invasive surgical techniques in the field oforthopedic surgery has created a growing need for new injectable synthetic materials that canbe used for bone grafting. In this work, novel injectable thermosensitive foam was developedby mixing nHAP powder with a thermosensitive polymer with foaming power (PluronicF-127) and loaded with a water-soluble bisphosphonate drug (risedronate) to promoteosteogenesis. The foam was able to retain the porous structure after injection and set throughtemperature change of PF-127 solution to form gel inside the body. The effect of differentformulation parameters on the gelation time, porosity, foamability, injectability, and in vitrodegradation in addition to drug release from the prepared foams were analyzed using a fullfactorial design. The addition of a co-polymer like methylcellulose or sodium alginate intothe foam was also studied. Results showed that the prepared optimized thermosensitive foamwas able to gel within 1 min at 37°C, and sustain the release of drug for 72 h. The optimizedformulation was further tested for any interactions using DSC and IR, and revealed nointeractions between the drug and the used excipients in the prepared foam. Furthermore,the ability of the pre-set foam to support osteoblastic-like Saos-2-cell proliferation anddifferentiation was assessed, and revealed superior function on promoting cellularproliferation as confirmed by fluorescence microscope compared to the plain drug solution.The activity of the foam treated cells was also assessed by measuring the alkalinephosphatase activity and calcium deposition, and confirmed that the cellular activity wasgreatly enhanced in foam treated cells compared to those treated with the plain drug solutiononly. The obtained results show that the prepared risedronate-loaded thermosensitive foamwould represent a step forward in the design of new materials for minimally invasive boneregeneration.

KEY WORDS: risedronate; nanohydroxyapatite; pluronic F-127; gel foam; osteogenic activity.

INTRODUCTION

The development of in situ bone grafts, which allow boneregeneration, has gained a great advance in orthopedicsurgery (1). At present, the application of minimally invasivesurgical techniques (2) shows a major achievement in manyfields like surgery of intervertebral disc and osteoporoticfractures treatment (3). In this field, calcium phosphatecements (CPC) like nanohydroxyapatite (nHAP) have shownseveral advantages (4), such as potential injectability (5), easy

adaption to geometrically complex shapes, similarity to thebone mineral phase (6) in addition to their possibility ofincorporating biologically active molecules or drugs (7).

A major limiting for using calcium phosphate cementsalone was the lack of macroporosity (8). Recently, variousapproaches have been tried to achieve macroporous struc-tures in calcium phosphate cements, like the addition ofsoluble porogenic agent that dissolves after implantation ofthe bone cement, creating the macroporous structure. An-other method of creating macroporosity was foaming a CPCby incorporating a protein-based foaming agent, namelyalbumen, in the liquid phase of the cement (9). However,several drawbacks were detected, like delayed bone forma-tion (10), owing to the immunogenic effect of heterogenicproteins (11). In this regard, the evolution of new injectablefoams using biocompatible synthetic surfactants instead ofusing protein-based foaming agents from protein base presenta new approach that could overcome this serious difficulty.

1 Department of Pharmaceutics and Industrial Pharmacy, Faculty ofPharmacy, Cairo University, Cairo, Egypt.

2 Department of Microbiology of Supplementary General Science,Faculty of Oral and Dental Medicine, Future University in Egypt,Cairo, Egypt.

3 To whom correspondence should be addressed. (e–mail:[email protected])

AAPS PharmSciTech (2019) 20:104 DOI: 10.1208/s12249-019-1292-4

1530-9932/19/0000-0001/0 # 2019 American Association of Pharmaceutical Scientists

http://orcid.org/0000-0002-7635-6842

http://crossmark.crossref.org/dialog/?doi=10.1208/s12249-019-1292-4&domain=pdf

Pluronics are triblock copolymers consisting of poly(oxyethylene) (POE) and poly(oxypropylene) (PO) unitsthat undergo changes in solubility with change in environ-ment temperature (12). Pluronic F-127 (PF-127) is one ofthe very few synthetic polymeric materials approved by theFDA for use in clinical applications. The main drawbacksof PF-127 are rapid erosion and weak mechanical strength.PF-127 was used as an in situ gel forming polymer togetherwith mucoadhesive polymers such as Carbopol 934 andhydroxylpropylmethylcellulose (HPMC) to ensure longresidence time at the application site (13). PF-127 can bemixed with alginate or chemically grafted onto alginate inorder to improve its gelling properties (14). These modi-fications with alginate can improve the physical andmechanical properties of the thermo-reversible hydrogels.Many reports have shown that thermoreversible alginatehydrogels that reversibly form a gel in response to thesimultaneous variation of at least two physical parameters(e.g., pH, temperature, or ionic strength) can be blended totarget their physical and mechanical properties (15,16).The potential application of a thermo-responsive alginatehydrogel as a functional injectable cell scaffold in tissueengineering was studied by the encapsulation behavior ofhuman stem cells, e.g., mesenchymal stem cells andadipose-derived stem cells (14).

Methylcellulose (MC) is a polymer derived from cellu-lose that is widely used as a viscosity enhancing polymer andas a drug excipient. Tang et al. (17) reported that a blend ofMC and chitosan in the presence of salts useful forapplications in tissue engineering.

In this study, we describe an alternative in situthermosensitive foam system prepared by the combinationof PF-127 and a co-polymer like sodium alginate (Na-Alg)or methylcellulose (MC) to prolong its residence time inorder to deliver a bone-building drug in a controlledrelease manner and allow osteogenic cells proliferation.The developed RIS-loaded thermosensitive foam wasfurther reinforced with nHAP, then porosity, foamability,injectability, and in vitro degradation in addition to drugrelease from the prepared foams were analyzed using afull factorial design. The optimized formulation wasfurther tested for any interactions using DSC and IR,and revealed no interactions between the drug and theused excipients in the prepared foam. Furthermore, theability of the pre-set foam to support osteoblastic-likeSaos-2-cell proliferation and differentiation was assessedusing Saos-2-cells and compared with the plain drugsolution.

MATERIALS

Risedronate sodium (RIS) was kindly supplied by AlHikma pharmaceut i ca l company, Egypt . Nano-hydroxyapatite (nHAP) (< 200 nm particle size), Dialysistubing cellulose membrane average flat width 33 mm (1.3 in.),Pluronic F-127, sodium alginate, and methylcellulose(Methocel® A15 LV) were purchased from Sigma AldrichCo., USA. Sodium Chloride (NaCl), Potassium Chloride(KCl), Disodium hydrogen Phoshate (Na2HPO4), PotassiumDihydrogen Phosphate (KH2PO4), (Adwic, El- Nasr pharma-ceutical company, Egypt).

METHODS

Preparation of RIS-Loaded Thermosensitive Foam

To prepare RIS-loaded thermosensitive foam, accuratelyweighted amounts of PF-127 and either MC or Na-Alg weredissolved in distilled water by stirring on a magnetic stirrer(Stuart SB161, UK) in an ice bath till a clear solution wasobtained, then left overnight in refrigerator to remove the airbubbles. After that, accurately weighted amounts of nHAPand RIS (10 mg) were added to the polymer solution andmixed on a magnetic stirrer till complete dispersion.

Characterization of RIS-Loaded Foam Preparations

Gelation Time

The gelation times of different foam combinationsprepared were determined using the phase inversion method(18). The prepared combinations were incubated in athermostatically controlled water bath (Memmert, Germany)at 37°C and the flowability of the solution was examinedevery 5 s by inverting the test tube. The time required to forma gel that did not flow within 5 s after the test tube inversionwas considered as the gelation time.

Evaluation of Foamability

The foamability of different liquid foams prepared wascharacterized by measuring the increase in volume of theliquid during the foaming process. This was done by putting1 mL of the prepared liquid inside a 2-mL graduatedEppendorf tube, then inserting the homogenizer probe (Crestultrasonic corp., Trenton, USA) inside the liquid for 1 min at11,000 rpm (19). The % foamability of the prepared RIS-loaded foams was calculated according to the followingequation:

F ¼ Vf−ViVi

� 100

Where F is the % foamability, Vf is the volume afterfoaming, and Vi is the initial volume of the solution.

Evaluation of Porosity

RIS-loaded foams were lyophilized by placing 2 mL ofeach foam in a pocket of a PVC blister with an internaldiameter 2 cm and height 0.2 cm. The PVC blisters werestored in a freezer at − 18°C for 24 h, and then lyophilized ina freeze dryer (Novalyphe- NL 500; Savant Instruments Inc.,USA) under a temperature of − 45°C and vacuum of 7×10−2 mbar for 24 h. The lyophilized samples were stored in adesiccator until use.

The porosity of different lyophilized thermosensitivefoams was determined by the liquid displacement method(20). Briefly, the lyophilized samples generated in a cylindri-cal mold were weighted and the original weight (W0), as wellas volume (V0), was recorded. The lyophilized samples werethen immersed in dehydrated isopropanol for 24 h forsaturation and the samples were re-weighted after placing

104 of 16 Morsi et al. (2019) 20:104

on a filter paper for 1 min for removing excess liquid (W24).The % porosity was calculated using the following equation:

%Porosity ¼ W24−W0PV0

� 100

Where p is the density of dehydrated alcohol(isopropanol) (p = 0.786 g/mL).

In vitro Degradation Test

The in vitro degradation of the foams was carried out bythe gravimetric method (21). First, the initial weight of a2-mL Eppendorf tube was recorded (Wv). One milliliter ofthe foam was placed in the Eppendorf tube and kept in anincubator for 30 min at 37°C. Samples were then weighedaccurately and the initial weight of the Eppendorf tubecontaining the gel (Wi) was recorded. Subsequently, 1 mL ofphosphate buffer saline (PBS) pH 7.4 was added into theEppendorf tube and incubated at 37°C. The buffer solutionswere removed using a graduated syringe at regular intervals(1, 3, 5, 7 days) and the Eppendorf tubes containing theremaining thermosensitive foams were weighed (Wt). Allexperiments were performed in triplicate. The percentageweight of the remaining foam was calculated using thefollowing equation:

%Weight remaining ¼ Wt−WvWi−Wv

� 100

Injectability Evaluation

The injectability of the foam formulations was assessed,using home-based equipment designed for this purpose. Thedesigned equipment was similar to that previously describedby Leroux et al. (22) with some modifications (23). Acommercial syringe with a volume capacity of 3 mL, 14 mminternal diameter, and an aperture of 2 mm was filled with1 mL of the tested foam. The filled syringe was then fixed to arubbery tube ending with an air pump. To measure thesolution injectability, air was forced from the air pump on thesolution surface. The applied pressure on the solution surfacewas measured in mm Hg units and maintained constant at40 mmHg using a sphygmometer. The time required torelease the sample was recorded. The values of flow-rate(mL/s) were reported to give an indication of the foaminjectability (24).

Evaluation of the In vitro Drug Release of the RIS-LoadedFoams

Drug release from different RIS-loaded foams wasconducted using the dialysis bag diffusion technique (25) ina thermostatically controlled water bath shaker maintainedat 37 ± 0.5°C. Before the experiment, the cellulose dialysismembrane was soaked overnight in the release medium(PBS, pH = 7.4). Two milliliters of each RIS-loaded foam(containing 20 mg RIS) was placed in the dialysis bag thentied at both ends. The dialysis tube was immersed in a

bottle containing 20 mL of PBS (pH = 7.4) and placed in athermostatically controlled shaker (Memmert, Germany)adjusted at 100 strokes per minute. At predetermined timeintervals, the release medium was replaced with an equalvolume of fresh medium, in order to maintain sinkconditions, and assayed spectrophotometrically ((UV-1800PC), Shimadzu, Kyoto, Japan.) at a predetermined λmax

(262 nm) in order to determine the concentration of drugreleased.

Optimization of RIS-Loaded Foam Using Factorial Design

Different formulations of RIS-loaded foams were pre-pared according to 24 factorial experimental design in orderto investigate the influence of formulation independentvariables on gelation time, % foamability, % porosity,in vitro degradation, injectability, and % drug released after2 h (Q2h), and 24 h (Q24h).

Four variables were evaluated, each at 2 levels, namely:concentration of PF-127, type of co-polymer, concentration ofco-polymer, and concentration of nHAP are studied each attwo levels (Table I). The design produced 16 formulations.The composition of different PF-127/Na-Alg or PF-127/MCcombinations prepared is presented in Table I. Finally,Design expert® 7 was used to choose the optimizedformulation.

Differential Scanning Calorimetry

The thermal analysis of RIS, PF-127, Na-Alg, nHAP, andselected lyophilized foam were performed using (Shimadzudifferential scanning calorimetry, DSC- 60). Samples wereplaced in a flat-bottomed aluminum pan and heated at aconstant rate, using dry atmosphere of nitrogen as carrier gas,in a temperature range of 20–400°C. The instrument temper-ature and energy scales were calibrated using purified indium(99.9%) as the standard reference material.

Fourier Transform Infrared Spectroscopy

FT-IR spectra between 4000 and 400 cm−1 (IRAffinity- 1;Shimadzu, Kyoto, Japan) of RIS, PF-127, Na-Alg, nHAP, andselected lyophilized foam were determined using potassiumbromide (KBr) disc technique. Samples were ground, mixedwith potassium bromide for 3–5 min in a mortar and thencompressed into disc by applying a pressure of 5 tons for5 min in hydraulic press. The concentration of sample inpotassium bromide should be in the range of 0.2% to 1%.The pellets were placed in light path and spectrum wasobtained and reviewed for evidence of any chemical interac-tions (26).

Saos-2 Cell Line Cell Viability and Osteogenic MarkersEvaluation

Sterilization by Gamma Radiation

The formulated RIS-loaded foams were sterilizedusing gamma radiation (60Co irradiator, (National Centerfor Radiation Research and Technology, Cairo, Egypt) ata dose of 10 kGy (1.19 kGy/h). The selected foam

104 of 16Risedronate-Loaded Macroporous Gel Foam (2019) 20:104

formulation, and drug solution in PBS were kept inside apolyurethane vessel surrounded by dry ice in order toavoid accumulation or melting during sterilization thatmay happen due to increasing the temperature, by γ-irradiation.

Saos-2 Cells Culture

Human bone osteosarcoma cells (Saos-2) were allowedto grow in McCoy’s 5a Medium supplied with 100 units/mLstreptomycin sulphate, 2 mM L-glutamine, 250 ng/mLamphotericin B, 10% FBS, and penicillin G sodium(100 units/mL).

Effect of Hydrogels on Saos-2 Cell Viability

Determination of the metabolic cell viability was done bythe 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) assay (27). Exactly 200 μg/mL of eachtreatment together with Saos-2 cells (1 × 104 cells/well) wasco-cultured in McCoy’s 5a Medium provided with 15% FBSfor 1 week. The viable cells percentage relative to controlwhich is untreated cells was recorded.

Pattern of Cell Growth and Interaction with FoamFormulation

After 1 week incubation of the Saos -2 cells with theoptimized foam formulation in cell culture of 8-chamberslides (5 × 104 cells/ chamber, CELLTREAT Scientific, USA),cells were dyed with a nucleic acid-binding dye which wasacridine orange (AO), 100 μg/mL in PBS, then cells wereinvestigated by fluorescence microscopy (28)(BX63 Olympuslife science, Japan). By means of digital camera (OlympusDP80) Photos were captured.

Determination of Calcium and Alkaline Phosphatase byColorimetry

Saos-2 cells were cultured (5 × 104) cells/well in mediumfree of serum containing 0.1% bovine serum albumin (BSA)in order to show the influence of the prepared RIS-loadedfoam on calcium accumulation and activity of alkalinephosphatase (29). Washing the cells with solution of Hanks’balanced salt was done after 10 days of incubation, wherecalcium accumulation was estimated by a colorimetric kit;Calcium Detection Kit (Sigma-Aldrich, catalog numberMAK022). Triton X-100 (0.5% in PBS) was used in orderto allow cells lysis in each well. In order to measure ALPactivity a 40 μL volume of lysate was tested. Activity of ALPwas assayed by a colorimetric kit; Alkaline PhosphataseAssay Kit; Detection Kit (catalog number ab83369, Abcam).After 10 days of treatment, Saos-2 cells lysate were used todetermine ALP activities by colorimetric assay as reportedpreviously by Schiller et al. (28). Each cell lysate wasincubated with 1 mg/mL 4-nitrophenyl phosphate (substrate)for about 30 min then the readings of absorbance weremonitored at 405 nm. Enzyme reaction specificity wasdetermined using substrate-free conditioned medium andconditioned medium co-incubated with Levamisole as ALPinhibitor. Bradford assay was used to estimate the proteinconcentration that used to normalize the concentration ofALP. Results were recorded as μmol alkaline phosphatase/min/mL.

RESULTS AND DISCUSSION

Statistical Analysis of the Factorial Design

Design expert® 7 software program was used tostatistically analyze the factorial design and test the signifi-cance of different formulation variables on the gelation time,% foamability, % porosity, in vitro degradation, injectability,and % drug released after 2 h (Q2h), and 24 h (Q24h). The

Table I. Composition of Different RIS-Loaded Foam Combinations

Formulations PF-127(w/v)

MC (w/v) Na-Alg(w/v) nHAP(mg/mL)

Gelationtime (sec)*

Percentagefoamability*

% Porosity* Flowrate* (sec −1)

Q2h* Q24h*

F1 15% – 2% 5 185.0 ± 5.0 33.5 ± 0.25 77.2 ± 1.52 0.33 ± 0.0 15.2 ± 1.5 50.8 ± 0.5F2 20 147.5 ± 2.5 16.8 ± 0.23 59.0 ± 1.68 0.13 ± 0.0 7.8 ± 0.3 40.7 ± 2.0F3 – 4% 5 120.0 ± 0.0 34.0 ± 0.35 81.6 ± 2.08 0.22 ± 0.0 14.0 ± 0.7 57.5 ± 6.3F4 20 92.5 ± 2.5 30.2 ± 0.29 64.8 ± 1.16 0.09 ± 0.0 9.3 ± 0.6 40.6 ± 0.6F5 2% – 5 305.0 ± 5.0 30.2 ± 0.28 49.6 ± 0.60 0.75 ± 0.3 13.8 ± 0.1 51.9 ± 2.4F6 20 247.5 ± 7.5 19.0 ± 0.22 42.1 ± 2.60 0.50 ± 0.0 7.6 ± 0.2 41.8 ± 1.4F7 4% – 5 25.0 ± 5.0 31.3 ± 0.14 54.8 ± 1.68 0.33 ± 0.0 14.4 ± 0.6 49.0 ± 0.7F8 20 30.0 ± 10.0 29.1 ± 0.53 50.6 ± 2.12 0.41 ± 0.1 8.3 ± 1.0 41.2 ± 7.6F9 20% – 2% 5 127.5 ± 7.5 50.5 ± 0.70 94.2 ± 1.88 0.41 ± 0.1 13.0 ± 1.3 49.7 ± 3.9F10 20 72.5 ± 7.5 37.7 ± 0.28 78.3 ± 1.48 0.16 ± 0.0 6.8 ± 0.3 42.2 ± 1.6F11 – 4% 5 5.0 ± 0.0 44.0 ± 0.35 70.3 ± 1.72 0.06 ± 0.0 12.7 ± 1.5 49.0 ± 3.6F12 20 5.0 ± 0.0 41.8 ± 0.30 65.4 ± 1.00 0.03 ± 0.0 7.0 ± 0.33 38.1 ± 3.4F13 2% – 5 15.0 ± 5.0 41.8 ± 0.23 97.0 ± 1.28 0.50 ± 0.0 12.6 ± 0.9 46.4 ± 1.0F14 20 30.0 ± 0.0 33.9 ± 0.28 71.3 ± 1.56 0.75 ± 0.3 7.0 ± 0.9 41.8 ± 3.4F15 4% – 5 10.0 ± 0.0 32.7 ± 0.29 55.0 ± 2.25 0.41 ± 0.1 14.3 ± 1.0 52.8 ± 1.8F16 20 7.5 ± 2.5 29.0 ± 0.38 51.9 ± 1.36 0.33 ± 0.0 7.6 ± 0.8 44.3 ± 0.3

*Data are mean values (n = 3 ± SD)

104 of 16 Morsi et al. (2019) 20:104

regression results of the measured responses are shown inTable II. The predicted R2 values were found to be in goodagreement with the adjusted R2 in all responses(approximately 0.2 difference between them) indicatingreliable models (30,31). Adequate precision measures thesignal-to-noise ratio to ensure that the model can be used toevaluate the design space (32). A ratio greater than 4 (thedesirable value) was observed in all responses.

Results of Gelation Time

Results of the gelation times of different RIS-loadedfoams are shown in Table I. The fastest gelation was achievedby foam formulations F11 and F12. This may be attributed tothe high concentration of Na-Alg (4% w/v) and PF-127(20%). On the other hand, the longest gelation time wasobtained by foam formulations F5 and F6 which contain lowconcentration of MC (2%) and PF-127 (15%). It is worthy tonote that formulations from F1 to F6 and F9 only becomemore viscous, but not completely gelled (still slightly flowwhen inverted). All other gelation time results were consid-ered reasonable for in situ gelation.

ANOVA results showed that both the concentration of PF-127 and the concentration of co-polymer had a significant effect onthe gelation time of the prepared foams (p< 0.0001) (Fig. 1(a)).Increasing the concentration of PF-127 resulted in decreasing thegelation time of the prepared foam formulations. This could beattributed to the lower gelation temperature (Tsol/gel) of thehigher PF-127 concentration, as there is an inverse relationshipbetween the Tsol/gel and the PF-127 concentration. As thetemperature increase, PF-127 copolymer molecules aggregate intomicelles, followed by gelation for sufficiently concentrated samples,and then at a certain point, micelles come into contact with eachother and no longer move. Micellar entanglements do not allowmicelles to separate easily from each other and that forms therigidity of the gel (33). Sol to gel transition temperature increaseswhen PF-127 concentration decreases and vice versa. Results alsoshow that increasing the concentration of the co-polymer signifi-cantly decreases the gelation time (Fig. 1(a)). This is attributed tothe increased viscosity of the higher polymer concentration. Similarresults were explained by El-Gawad et al (34) who reported thatincreasing the polymer concentration increases the viscosity, andthus decreases the gelation time and temperature. Similar resultswere also reported in the literature (35,36).

The concentration of PF-127 and the co-polymer (MC or Na-Alg) had a significant synergistic effect on the gelation time of theprepared foams (p= 0.0038). Increasing the co-polymer concen-tration (from 2% to 4%) resulted in much faster gelation at 20%PF-127 than that at 15% PF-127 (Fig. 1(a)). This could be

attributed to the synergistic viscosity increasing nature of bothpolymers. Similar results were obtained by Rarokar et al. (37) whostated that the addition of co-polymers such as; methylcellulose,carbopol, and hydroxypropylmethylcellulose to PF-127, wouldincrease the viscosity, and thus decrease the gelation temperatureand time as well.

Results of % Foamability Test

Results of the % foamability of the RIS-loaded foamsare shown in Table I. ANOVA results showed that all thetested factors (the concentration of PF-127, type, andconcentration of the co-polymer and the amount of nHAP)had a significant effect on the % foamability. Increasing theconcentration of PF-127 significantly increases the %foamability (p < 0.0001), owing to the surfactant nature ofPF-127, which can stabilize the air pockets of the foam.Similar results obtained by Unosson et al. (38) who stated thatsurfactants stabilize foams at the water-air interface, bylowering the energies required to maintain the largerinterfacial area associated with the formation of air bubbles.The type of co-polymer also significantly affected the %foamability of the prepared foams, where RIS-loaded foamscontaining Na-Alg had significantly higher % foamabilitythan those containing MC (p < 0.0001). Na-Alg tends to forma stronger gel than MC and thus can stabilize the foam to agreater extent compared to MC (39). ANOVA results showedthat increasing the concentration of the co-polymer resultedin significantly higher % foamability (p = 0.0255). This is maybe due to increasing the foam stability upon increasing the co-polymer concentration due to higher viscosity. This increasein viscosity would hinder gravitational separation of the liquidfrom the foam (19). Increasing the amount of nHAP in theprepared foams significantly decreases the % foamability(p < 0.0001). This is may be due to the formation of a densermatrix due to higher solid content, which is unable to befoamed upon increasing nHAP concentration (40).

The interaction between the tested variables also had asignificant effect on the % foamability of the prepared foams.Figure 1(b, i) shows a synergistic significant effect betweenPF-127 concentration and the co-polymer type on increasingthe % foamability (p < 0.0001). Increasing PF-127 concentra-tion (from 15% to 20%) resulted in much higher %foamability in presence of Na-Alg than that in the presenceof MC. This may be attributed to the ability of Na-Alg toform very strong gel (41) which could stabilize the foam morethan the MC. Figure 1(b, ii) shows that increasing PF-127concentration (from 15 to 20%) at 2% co-polymer increasesthe % foamability more than that at 4% co-polymer

Table II. Regression Results of the Mesured Responses

Regressionparameters

Gelationtime (Y1)

% Foamability (Y2) % Porosity (Y3) % weight remainingafter 7 days (Y4)

Flowrate (Y5)

% RIS releasedafter 2 h (Y6)

% RIS releasedafter 24 h (Y7)

R2 0.8483 0.9844 0.9244 0.9167 0.7422 0.9470 0.7558Adjusted R2 0.7761 0.9770 0.8883 0.8771 0.6011 0.9218 0.6325Predicted R2 0.6478 0.9638 0.8244 0.8066 0.4015 0.8769 0.4395Adequate precision 11.392 44.350 18.086 13.953 7.184 15.056 7.305


(p < 0.0001). Similar results were reported by Alfredo et al.(42) who found that at low polymer concentration, thesurface tension is about 16% lower than that of a puresurfactant solution, indicating a strong adsorption of thepolymer to the air/liquid interface. This low surface tensionexplains the high foamability at these polymer concentra-tions. As the polymer concentration increases, the surfacetension increases progressively, increasing the energy associ-ated with the air/liquid interface and thus reducing thefoamability of the solutions.

Figure 1(b, iii) shows that increasing co-polymer concen-tration (from 2 to 4%) in presence of Na-Alg as a co-polymersignificantly increases the % foamability (p = 0.0006), whereasincreasing co-polymer concentration with MC slightly de-creases the % foamability. Increasing Na-Alg concentrationleads to stabilization of the foam whereas the presence of MCresulted in enhanced compactness of the blends, decreased

porosity and so foamability (43). Figure 1(b, iv, v) shows thatincreasing nHAP concentration (from 5 to 20 mg/mL)significantly decreases % foamability at both PF-127 concen-trations, and both co-polymers (p = 0.0428 and 0.0123,respectively). This may be attributed to the higher densityof foam resulting from increasing nHAP concentration (40).Figure 1(b, vi) shows that increasing the co-polymer concen-tration (from 2% to 4%) at low nHAP concentration (5 mg/mL) slightly decreases the % foamability while increasing co-polymer concentration at high nHAP concentration (20 mg/mL) increases the % foamability. This may be attributed tothe fact that at low nHAP concentration, the % foamability isalready high and could not be affected by increasing the co-polymer concentration, while at high nHAP concentration,the % foamability is much lower and the presence of the co-polymer which act as foam stabilizer significantly enhance the% foamability (44).

Design-Expert® Software

Gelation time

C- 2.000

C+ 4.000

X1 = A: Concentration of PF 127

X2 = C: Concentration of co-polymer

Actual Factors

B: Type of co-polymer = Na alginate

D: Amount of nHAP = 12.50

C: Concentration of co-poly mer

15.00 16.25 17.50 18.75 20.00

Interaction

A: Concentration of PF 127

Ge

latio

n tim

e

0

77.5

155

232.5

310


Foamability

B1 Na alginate

B2 Methyl cellulose


X2 = B: Type of co-polymer

Actual Factors

C: Concentration of co-polymer = 3.00


B: Ty pe of co-poly mer

15.00 16.25 17.50 18.75 20.00

Interaction


Fo

am

ab

ility

16

24.75

33.5

42.25

51


Foamability

C- 2.000

C+ 4.000



Actual Factors

A: Concentration of PF 127 = 17.50



Na alginate Methyl cellulose

Interaction


Fo

am

ab

ility

16

24.75

33.5

42.25

51

b i b ii

b iii b iv

b v b vi

a


Foamability

C- 2.000

C+ 4.000



Actual Factors




15.00 16.25 17.50 18.75 20.00

Interaction


Fo

am

ab

ility

16

24.75

33.5

42.25

51


Foamability

D- 5.000

D+ 20.000


X2 = D: Amount of nHAP

Actual Factors



D: Amount of nHAP

15.00 16.25 17.50 18.75 20.00

Interaction


Fo

am

ab

ility

16

24.75

33.5

42.25

51


Foamability

D- 5.000

D+ 20.000



Actual Factors



D: Amount of nHAP

2.00 2.50 3.00 3.50 4.00

Interaction


Fo

am

ab

ility

16

24.75

33.5

42.25

51


Foamability

D- 5.000

D+ 20.000



Actual Factors



D: Amount of nHAP

Na alginate Methyl cellulose

Interaction


Fo

am

ab

ility

16

24.75

33.5

42.25

51

Fig. 1. a Interaction plot showing the effect of PF-127 concentration and co-polymerconcentration on the gelation time. b Interaction plot showing the effect of differentfactors on the % foamability (i) PF-127 concentration and type of co-polymer, (ii) PF-127 concentration and co-polymer concentration, (iii) type of co-polymer and co-polymer concentration, (iv) PF-127 concentration and nHAP amount, (v) type of co-polymer and nHAP amount, and (vi) co-polymer concentration and nHAP amount

104 of 16 Morsi et al. (2019) 20:104

Results of % Porosity Test

Results of the % porosity of the prepared foams areshown in Table I. ANOVA results showed that all the testedfactors (the concentration of PF-127, type and concentrationof the co-polymer and the amount of nHAP) had a significanteffect on the % porosity (Fig. 2(a–c)). Results are also ingood agreement with the results of % foamability, where thesame factors that increased the percentage foamability alsoincreased the percentage porosity, owing to the stabilizationof air pockets within the foam (38).

Results showed that increasing the concentration of PF-127 significantly increases the % porosity (p < 0.0001). This isattributed to increasing the foaming ability of PF-127 uponincreasing its concentration and as a result the porosityincreases. Results also showed that RIS-loaded foams con-taining Na-Alg had significantly higher % porosity(p < 0.0001) compared to those containing MC, owing tohigher foaming ability of the system (45). The co-polymerconcentration also significantly affected the % porosity

(p < 0.0001), where increasing the co-polymer concentrationdecreases the % porosity, owing to the formation of a densermatrix with higher viscosity (46). Increasing the nHAPamount significantly decreases the % foamability and there-fore decreases the % porosity as well (p < 0.0001), owing toincreasing the solid content within the foam (40).

Significant interaction between the concentration of PF-127, and the concentration of co-polymer on the % porositywas observed (p < 0.0001). Figure 2(a) shows that increasingPF-127 concentration (from 15% to 20%) at low co-polymerconcentration (2%) significantly increases the % porosity,owing to the higher foamability of the higher surfactantconcentration. On the other hand, increasing the PF-127concentration from (15% to 20%) at high co-polymerconcentration (4%) significantly decreases the % porosity.This could be due to the fact that higher porosity expectedupon increasing the surfactant PF-127 concentration wascounter-balanced by the decrease in porosity generated bythe higher co-polymer concentration, due to increasedviscosity. Therefore, the increase in porosity with increasing


Porosity

C- 2.000

C+ 4.000



Actual Factors




15.00 16.25 17.50 18.75 20.00

Interaction


Po

rosity

100

150

200

250

300


Porosity

B1 Na alginate

B2 Methyl cellulose



Actual Factors




15.00 16.25 17.50 18.75 20.00

Interaction


Po

ro

sity

100

150

200

250

300


Porosity

D- 5.000

D+ 20.000



Actual Factors



D: Amount of nHAP

2.00 2.50 3.00 3.50 4.00

Interaction


Po

ro

sity

100

150

200

250

300


in-vitro degradation(% wt remaining after 7 days)

B1 Na alginate

B2 Methyl cellulose



Actual Factors




15.00 16.25 17.50 18.75 20.00

Interaction


)s

ya

d7

retf

ag

nini

am

er

tw

%(

noit

ad

ar

ge

do

rtiv

-n i

9

26

43

60

77



C- 2.000

C+ 4.000



Actual Factors




15.00 16.25 17.50 18.75 20.00

Interaction


)s

ya

d7

retf

ag

nini

am

er

tw

%(

noit

ad

ar

ge

do

rtiv

-ni

3

21.5

40

58.5

77

a b

c

d e

Fig. 2. Interaction plot showing the effect of a the PF-127 concentration and co-polymerconcentration, b the PF-127 concentration and co-polymer type, c the co-polymer concentrationand nHAP amount on %porosity, d interaction plot showing the effect of concentration of PF-127and co-polymer type on the in vitro degradation and e interaction plot showing the effect ofconcentration of PF-127 and co-polymer concentration on the in vitro degradation


the PF-127 concentration due to increased foamability wasovercome by the decrease in porosity with higher co-polymerconcentration (4%) due to high viscosity that may hinder thefoam formation. Similar results were obtained by Tan et alwho reported that the pore size of the 3D network decreasesas the Na-Alg concentration increases (47). Figure 2(b) showsthat at low PF-127 concentration (15%) formulations con-taining Na-Alg has significantly higher % porosity than thosecontaining MC (p = 0.0048), while at high PF-127 concentra-tion (20%), formulations containing Na-Alg has slightlyhigher % porosity than those containing MC. This may beattributed to the fact that at high PF-127 concentration (20%)the % foamability and thus the % porosity is already high andmight not be affected by changing the co-polymer type, whileat low PF-127 concentration, the % foamability and thus the% porosity is much lower. Therefore, the presence of Na-Algwhich tends to form a stronger gel than MC and thus canstabilize the foam to a greater extent compared to MC, leadsto much higher porosity. Figure 2(c) shows that increasing theco-polymer concentration from (2% to 4%) in foamscontaining low concentration of nHAP (5 mg/mL) has nosignificant effect on the % porosity of the foams. On the otherhand, at higher concentration of nHAP (20 mg/mL), increas-ing the co-polymer concentration (from 2 to 4%) resulted insignificantly lower % porosity (p = 0.0014), owing to theformation of denser matrix of high solid content (40).

Results of In vitro Degradation Test

Results of the in vitro degradation of RIS-loaded foamformulations over a period of 7 days are graphicallyillustrated in Fig. 3. Results showed that the % weightremaining after 7 days for F11, F12, F15, and F16 was thehighest among the tested RIS-loaded foams. This result is in agood correlation with the gelation time test results, wherethose formulations (F11, F12, F15, and F16) showed the leastgelation times among the tested RIS-loaded foams.

ANOVA results showed that both the type and theconcentration of the co-polymer had a significant effect on the% weight remaining of RIS-loaded foams after 7 days.Results showed that RIS-loaded foams containing Na-Alg asa co-polymer had higher % weight remaining after 7 dayscompared to those containing MC. This may be attributed tothat Na-Alg tends to form a stronger and more durable gelthan MC, especially in the presence of calcium released fromdegradation of the nHAP. A previous study by Kathleen etaal (48) on the preparation of Na-Alg/MC scaffold by 3Dplotting reported that, after 7 days of incubation under cellculture conditions, MC has been almost completely releasedfrom the plotted scaffolds leaving porous Na-Alg scaffold forthe remaining 28 days of incubation. The composite wasstabilized by calcium which induced crosslinking of Na-Alg;however, MC was not crosslinked under these conditions andtherefore the majority of MC was released over time.Another study by Witek et al (49) reported that nHAP servesas a supply of calcium and phosphate ions, where calcium ionslead to cross-linking of Na-Alg. Thus RIS-loaded foamscontaining MC degraded more rapidly than those containingNa-Alg. ANOVA results also showed that increasing the co-polymer concentration (from 2 to 4%) significantly increasesthe % weight remaining after 7 days (p < 0.0001). This result

is in good accordance with the results of porosity testing,where the percentage porosity decreased upon increasing co-polymer concentration. This hinders the penetration of waterand subsequent degradation of the matrix. As a consequence,the foam containing the lower concentration of the co-polymer degraded much faster than those containing thehigher concentration of the co-polymer. Significant interac-tions between the tested formulation variables were alsoreported (Fig. 4(a, b)). Figure 4(a) showed that increasing PF-127 concentration (from 15 to 20%) in RIS-loaded foamscontaining Na-Alg had a synergistic significant effect onincreasing the percentage weight remaining after 7 days(p < 0.0001). This was explained as in the effect of the co-polymer type where Na-Alg has high gel strength and formmore durable gel than MC due to Alg crosslinking with theCa2+ released from nHAP degradation.

A synergistic effect between the concentration of PF-127and the concentration of co-polymer on the % weightremaining of the foam after 7 days was also observed (Fig.4(b). Increasing both the concentration of PF-127 and co-polymer resulted in significantly lower porosity of thelyophilized foam and lower degradation in buffer, and hencehigher % weight remaining after 7 days (p = 0.0004). Thismay be attributed to the stronger gel structure at high PF-127and higher co-polymer concentration with lower porosity.

Results of Injectability Evaluation

Table I shows the results of the flow time and flow rate of1 mL sample of different RIS-loaded foams at 4°C. All foamformulations show flow time less than 1 min, which is

0102030405060708090

100

0 2 4 6 8

% W

eigh

t rem

aini

ng

Time (days)

F1 F2 F3 F4 F5 F6 F7 F8

0102030405060708090

100

0 2 4 6 8

% W

eigh

t rem

aini

ng

Time (days)

F9 F10 F11 F12 F13 F14 F15 F16

Fig. 3. The percentage weight remaining for thermosensitive foams.Results are mean values n = 3 ± S.D.

104 of 16 Morsi et al. (2019) 20:104

reasonable for injection. RIS-loaded foam (F12) showed thelongest flow time and the lowest flow rate compared to otherformulations, owing to its high Na-Alg concentration (4%)and high nHAP amount (20 mg/mL).

ANOVA analysis was used to evaluate the level ofsignificance of the tested foam variables on the flow rate ofthe prepared RIS-loaded foams. Results showed that both thetype and the concentration of the co-polymer had significanteffect on the flow rate. Results showed that RIS-loaded foamscontaining MC had significantly higher flow rate compared tothose containing Na-Alg (p < 0.0001). This may be attributedto the thermosensitive behavior of the MC, where it remainsin sol state at low temperature (4°C) at which the test wasdone, whereas Na-Alg is not affected by temperature (50).Results also showed that increasing the co-polymer concen-tration (from 2 to 4%) significantly decreases the flow rate(p = 0.0007). This may be attributed to increasing the viscosityupon increasing the co-polymer concentration (37).

Results of the In vitro Drug Release of the RIS-LoadedFoams

The release profiles of RIS from RIS-loaded foams aregraphically illustrated in Fig. 5. Controlling the release of ahighly water-soluble drug like RIS is a great challenge, as it issoluble within the aqueous foam formulation prepared.Results showed that all formulations succeeded to achievesustained release of the hydrophilic drug (RIS) over 72 h. Thepercentage RIS released after 2 h, and 24 h are presented inTable I for Q2h, and Q24h, respectively. ANOVA resultsshowed that PF-127 concentration and nHAP concentrationhad a significant effect on Q2h, whereas only nHAP concen-tration had a significant effect on Q24h.

Increasing the concentration of PF-127 resulted insignificantly lower Q2h (p = 0.0014) (Fig. 6(a)). This could beattributed to the increased viscosity of the formulation uponincreasing the PF-127 concentration, creating a highly viscousgel layer, which hinders the drug release. This result is ingood accordance with the result of gelation time, whereincreasing the concentration of PF-127 resulted in fastergelation preventing the rapid release of the hydrophilic drug(RIS) from the foam. Similar results were obtained by

Rangabhatla et al (43) in their study on the use of PF-127 andMC for etidronate delivery and their application for osteo-genesis. They reported that the higher concentration of thePF-127 in the blends produced a denser internal structure,which retarded release of etidronate.

Results also showed that increasing the concentration ofnHAP (from 5 to 20 mg/mL) significantly decreased Q2h, andQ24h (Figure 6(b, c)) (p < 0.0001) respectively. This result is ingood agreement with the result of porosity testing, where thepercentage porosity decreased upon increasing nHAP con-centration owing to the increased solid content, and hencehindering the diffusion of the drug to the release medium.Similar results were obtained by AL-Sokanee et al (51) intheir study on a drug release study of Ceftriaxone fromporous hydroxyapatite scaffolds. They reported that theincrease of the drug amount released from the scaffoldreflects the increase in porosity.

Statistical Optimization of the Results

Design expert7® was used to choose the optimizedformulation that could achieve gelation time less than 3 min,highest % foamability, % porosity, and % weight remainingafter 7 days, with the lowest % drug released after 2 h and24 h. Optimization results revealed that a new formulationprepared using 20% PF-127, 2.98% Na-Alg, 20 mg/mL nHAPwas suggested as an optimum formulation, with the highestdesirability factor (0.678). This optimized formulation (F) wasprepared and evaluated, and the results of the predicted andactual responses were reported in Table III. The differencebetween the predicted and estimated values calculated usingthe following equation:

%Difference ¼ predicted value−estimated valuepredicted value

� 100

All estimated results were found to be within 10%difference from the predicted value.



B1 Na alginate

B2 Methyl cellulose



Actual Factors




15.00 16.25 17.50 18.75 20.00

Interaction


)s

ya

d7

retf

ag

nini

am

er

tw

%(

no it

ad

ar

ge

do

rtiv

-ni

9

26

43

60

77



C- 2.000

C+ 4.000



Actual Factors




15.00 16.25 17.50 18.75 20.00

Interaction


)s

ya

d7

retf

ag

nini

am

er

tw

%(

noi t

ad

ar

ge

do

rtiv

-ni

3

21.5

40

58.5

77

Fig. 4. Interaction plot showing the effect of (a) the PF-127 concentration and co-polymer type (b) the PF-127 concentrationand co-polymer concentration on the in vitro degradation


-10

10

30

50

70

90

110

0 100 200 300 400

% R

IS re

leas

ed

Time (hr)

F1 F2 F3 F4

-10

10

30

50

70

90

110

0 100 200 300 400

% R

IS re

leas

ed

TIME (hr)

F5 F6 F7 F8

-10

10

30

50

70

90

110

0 100 200 300 400

% R

IS re

leas

ed

Time (hr)

F9 F10 F11 F12

-10

10

30

50

70

90

110

0 100 200 300 400

% R

IS re

leas

ed

TIME (hr)

F13 F14 F15 F16

Fig. 5. Release profiles of RIS from thermosensitive RIS-loaded foams. Results are mean values n = 3 ± S.D.

a b

c

Fig. 6. a Line chart showing the effect of PF-127 concentration on Q2h. b Line chart showing the effect ofnHAP concentration on Q2h. c Line chart showing the effect of nHAP concentration on Q24h

104 of 16 Morsi et al. (2019) 20:104

Differential Scanning Calorimetry

DSC is a conventional tool to investigate the physicalproperties of the material and to give an insight into the meltingand recrystallization behavior of crystalline materials (52).Figure 7 shows the DSC thermograms for RIS, nHAP, PF-127,Na-Alg, and the optimized RIS-loaded freeze-dried foam (F).TheDSC thermogramofRIS showed several endothermic peaks,as RIS monosodium may be one of three forms: an anhydrate, alattice monohydrate, a hemi-pentahydrate consisting of manytypes of water of hydration; channel and lattice-type waters ofhydration, and a variable channel hydrate that under mildhumidity conditions would be expected to exist as a 4–6 Mhydrate. Figure 7 shows the thermogramof the hemipentahydrateform which is the equilibrium form at room temperature and at37°C, in the presence of water (53,54). The DSC thermogram ofnHAP showed only an endothermic peak at temperature of300°C due to dehydration (55). The DSC thermogram of PF-127showed an endothermic peak at 55°C due to its melting point, aswell as an endothermic peak at 312°C due to dehydration (56).The DSC thermogram of Na-Alg showed an endothermic peakclose to 100°C due to dehydration, then an exothermic peak at245°C owing to the decomposition of the polymer (57). The DSCthermogram of RIS-loaded foam optimized formulation (F)indicates the absence of any unwanted interactions, since no

new peaks have appeared. Only the intensity of peaks has beendecreased due to the dilution occurring during formulation.

Fourier Transform Infrared Spectroscopy

FT-IR spectra were obtained for the optimized formulationas well as for individual components (Fig. 8(a–e)). The IR chart ofnHAP shows bands at 1000–1180 cm−1 and 490–610 cm−1 whichare related to internal vibrations of the phosphate (PO4) group.This tetrahedral PO4 is assigned to as symmetric stretch, bendingmode, and anti-symmetric stretching vibrations. The bands at3444 and 3568 cm−1 are related to the stretching of OH groupsvibration corresponds to Ca(OH)2. The presence of band at1419 cm−1 is related to CO3

2− ion (58) (Fig. 8(a)). The IR chart ofRIS shows the absorption band at two distinct OH stretchingbands at 3564 and 3618 cm−1, representative to two differentwater populations inside the crystal lattice. The shoulderobserved at 3363 cm−1 is attributed to a CO-H vibrationcorresponding to OH groups not related to water but attachedto the central carbon. Hydroxyl vibrations from OH groupsassociated with theO=P-OHof the phosphonate group producedbroad bands in the spectral region between 1658 and 2692 cm−1

for the RIS. The characteristic IR bands of the phosphonategroup (PO3) were observed between 1000 and 1150 cm−1 for theRIS, overlapping with pyridine ring vibrations (59) (Fig. 8(b)).

Table III. The Predicted and Estimated Values of Different Responses

Gelationtime (sec)

Flowrate (sec−1)

%Foamability %Porosity % weight remainingafter 7 days

%RIS releasedQ2h

% RIS releasedQ24h

Predicted 42 0.11 39.4 70 45 7 40Measured 40 0.1 37.5 76 48.3 7.5 41% Difference 4.7 10 4.8 7.8 7.3 6.66 2.5

-5

0

5

10

15

20

25

30

0 50 100 150 200 250 300 350 400 450

DSC

(mV)

Temperature (ºC)

nHAP PF-127 RIS Na-Alg F

Fig. 7. DSC thermograms for RIS, nHAP, PF-127, Na-Alg and the selected RIS-loaded freeze-dried optimized foam formulation (F)


The IR chart of Na-Alg showed mannuronic acid functionalgroup at wavenumber 891.1 cm−1 and the uronic acid atwavenumber 948.9 cm−1, OH functional group at wavenumber3200–3600 cm−1, and CH2 stretching at wavenumber 2932 cm−1

(60) (Fig. 8(d)). The IR chart of Pf-127 showed characteristicpeaks at around 3000 cm−1 and around 1100 cm−1. Theprincipal absorption bands (stretching) at 3487.3 cm −1

correspond to the following functional groups; (O-H),2885.51 cm−1 (CH), 1111 cm−1 (C-O). Our spectra for PF-127were comparable with the previously recorded FT-IR spectraof PF-127 (61) (Fig. 8(c)). The FT-IR spectrum for theoptimized RIS-loaded foam formulation (F) (Fig. 8(e)) didnot show any shifts in the peaks of both the drug and thepolymers but the intensity of peaks slightly decreased whichmay be due to the dilution of the drug with the polymerscompared with drug alone. Thus, it can be concluded thatbased on FT-IR spectra there is no interaction between thedrug and polymer in the optimized formulation.

Results of Sterilization by Gamma Radiation on the PhysicalStability of the Optimized Foam Formulation

The gelation time and in vitro RIS release weredetermined before and after sterilization. No significantchange was observed in the gelation times after sterilizationof the optimized foam formulation (40, and 38 s for sterilizedand unsterilized foam formulation respectively). In addition,the release of drug from the optimized foam formulation wasnot affected after sterilization (results of Q2h were 7%, 7.2%before and after sterilization, respectively). These resultsconfirm the physical stability of the foam formulation afterexposure to gamma radiation at the dose of 10 KGy. Thus,gamma radiation can be used in sterilization of this formula-tion safely with minimal changes in property. Similar resultswere obtained by EL-Bagory et al. (62) who found that lowirradiation doses (15 and 20 kGy) was safe and did notsignificantly affect the properties of the prepared gel.

a

c

d

e

b

Fig. 8. FTIR spectrum of nHAP (a), RIS (b), PF-127 (c), Na-Alg (d), and the optimizedfoam formulation (e)

104 of 16 Morsi et al. (2019) 20:104

Saos-2 Cell Line Cell Viability and Osteogenic MarkersEvaluation

Effect of Hydrogels on Saos-2 Cell Viability

Since the prepared RIS-loaded foam formulations have apromising role in bone tissue engineering, their influence on theosteoblast-like Saos-2 cells viability was investigated. The growthand accumulation of Saos-2 cells on the foam formulation wasdetermined byMTTassay method, which relies on estimating themitochondrial dehydrogenases activity, where its activity wassupposed to be related to cell number and cell viability (63).

Evaluating the results of cell viability of cells treated withthe drug solution (D) and the optimized RIS-loaded foamformulation (F) revealed that the growth level of the Saos-2cells on the foam formulation was significantly higher thanthat of the drug solution (RIS only) (p < 0.05), confirming thatthis optimized foam formulation was more preferable for thecells growth, owing to the synergistic effect of nHAP togetherwith RIS in the osteogenesis process (64,65). This supportsthe efficient role of the bisphosphonate drug (RIS), andnHAP cement, on enhancing osteoblastic growth and devel-opment (53). Therefore, the addition of both nHAP and RISshowed an effective role in the cellular growth.

Growth Pattern of the Cell and Interaction with Hydrogels

Adhesion to the cell is a complicated pattern of physicochem-ical reactions, which influenced by various factors like the behaviorof the cell, surface properties of the cell such as (hydrophobicity,chemical, and physical composition, texture) (66). Also, theadhesion of the cell to foam surface is important in affecting thewhole behavior of cell with respect to their proliferation,expansion, differentiation, sending signals, extracellular matrixdisplacement and transmission (67). The cell layers adhesion to thefoam’s surface shows that foam has affinity to the cells (68). Thecells/foam interaction was evaluated by carrying a fixed cell countwhich is suspended in medium, onto foam surface, the co-culturewas kept incubated for 1 week, and soon after acridine orange dyewas used to stain the foam (which is aDNAdye that could stain thenuclei to bright green color of both live and dead cells).

Results showed in (Fig. 9(a)) reveal that the existence of theRIS and nHAP in the optimized foam formulation enhanced cellproliferation, transmission towards, and collecting around the foamfragments in an organized pattern. This induced and clearly

propagated cell behavior was recorded within 1 week of treatment.Thehighly propagation and attachment of the cells in the optimizedformulation was clearly due to the bone-building effect of thiscomposite foam which contained RIS as a bone-building drug andnHAP as the main mineral of natural bone tissue (69) (70).Moreover, the fluorescence microscope images (Fig. 9(b)) showedthat the cells handled with the drug solution had started to grow,however in an unorganized manner, within 1 week of treatment.This result together with the previous cell viability results was ingood correlation, where the drug solution only revealed lower cellviability values, compared to the optimized foam.

Alkaline Phosphatase Activity

Osteogenic differentiation of cell have many markers,one of the early released important markers is ALP, whereduring active bone formation its level was markedly elevated(71). Function of ALP is essential in the initial process ofbone mineralization, as increasing activity of ALP levels arecorrelated with increasing Ca2+ uptake, and thus boneformation. ALP is important in the osteogenic processes,where it is a main role in the mineralization process,modulation of bone cell differentiation and Ca2+ binding(72). Hence, the level of its production was evaluated in theSaos-2 cells to estimate the tested foam co-cultured cellsbioactivity. The optimized foam formulation containing bothRIS and nHAP resulted in cell differentiation, as showed bythe elevation of ALP activity significantly (p < 0.05) than thedrug solution (RIS only). This revealed that this foamformulation has the ability to enhance the osteoblast primaryfunctional activity. This improvement proved that the pres-ence of the RIS, together with nHAP in the foam had anadditive effect and resulted in more effective expression ofthe osteoblastic phenotype.

Calcium Deposition

Upon determining the activity of the osteoblasts, bonemineralization is an important mark for bone regenerationand should be evaluated with any bone tissue engineeringmaterial. Results recorded with measuring the Ca2+ bindingto the cells, were in good correlation with that of the ALPactivity, since the optimized foam formulation containingboth RIS and nHAP showed higher Ca2+ deposition on thefoam than that with the drug solution only (5.6 ± 0.05 mg/

a b

Fig. 9. Fluorescence microscope images for Saos-2 cells at magnification × 100: a drug solution, boptimized RIS-loaded foam formulation


mL for drug solution and 7.8 ± 0.2 mg/mL for theoptimized formula).

These data prove that RIS-loaded foam can obviouslyenhance osteoblast cells Ca2+ mineralization, thus the cellsperform full functionality after dealing with this foam for only1 week. This indicates the significant cytocompatibility of theRIS and nHAP containing foam.

Results of cell viability study together with the activity ofALP and Ca2+ accumulation were correlated to each other, asthe optimized foam formulation containing both RIS andnHAP, induced the high cell viability. This is attributed to theaction of nHAP, and RIS as a bisphosphonate drug onpromoting the osteoblastic differentiation (73).

This result suggests that the RIS-loaded foam promote greatbone regeneration biocompatibility. The foam biocompatiblenature results into the growth of cells that will support withmineral deposition on the foam. This is important for the cellgrowth and for tissue regeneration as well. Thus, this foam canenhance various biological activities such as adhesion, prolifera-tion, and differentiation of osteoblastic cells.

CONCLUSION

In the present study, injectable thermosensitive foamusing PF-127/Na-Alg, loaded with both bone-building drug(RIS), and synthetic bone cement (nHAP) was successfullyformulated and evaluated for their foamability, porosity,in vitro degradation and in vitro drug release, andinjectability. The obtained results revealed that using 20%(w/v) PF-127 with 3% (w/v) Na-Alg solution and 20 mg/mLnHAP loaded with RIS 10 mg/mL, produced thermosensitivefoam with the most sustained drug release profiles and cellgrowth, cell adhesion, and mineralization. The presence ofnHAP clearly enhanced the Saos -2 cell adhesion and cellproliferation on the foam surface. Thus, this RIS-loaded foamcould be used as suitable alternative for applications of bonetissue engineering. Also, this RIS-loaded foam succeeded toreach the desired biological in vitro characteristics withoutaddition of any other growth factor, or bone osteogenicmaterials. This property together with being less invasivenonsurgical treatment makes this thermosensitive foam apromising alternative for surgical ones.

Publisher’s Note Springer Nature remains neutral with regard tojurisdictional claims in published maps and institutional affiliations.

REFERENCES

1. Studart AR, Gonzenbach UT, Tervoort E, Gauckler LJ.Processing routes to macroporous ceramics: a review. J AmCeram Soc. 2006;89(6):1771–89.

2. Tozzi G, de Mori A, Oliveira A, Roldo M. Composite hydrogelsfor bone regeneration. Materials. 2016;9(4):267.

3. Assaker R. Minimal access spinal technologies: state-of-the-art,indications, and techniques. Joint Bone Spine. 2004;71(6):459–69.

4. Wang W, Yeung KW. Bone grafts and biomaterials substitutesfor bone defect repair: a review. Bioactive Mater. 2017;2(4):224–47.

5. Habib M, Baroud G, Gitzhofer F, Bohner M. Mechanismsunderlying the limited injectability of hydraulic calcium phos-phate paste. Acta Biomater. 2008;4(5):1465–71.

6. Constantz BR, et al. Skeletal repair by in situ formation of themineral phase of bone. Science. 1995;267(5205):1796–9.

7. Ginebra M-P, Traykova T, Planell JA. Calcium phosphatecements as bone drug delivery systems: a review. J ControlRelease. 2006;113(2):102–10.

8. Xu HH, Quinn JB, Takagi S, Chow LC. Synergistic reinforce-ment of in situ hardening calcium phosphate composite scaffoldfor bone tissue engineering. Biomaterials. 2004;25(6):1029–37.

9. Ginebra MP, Delgado JA, Harr I, Almirall A, del Valle S,Planell JA. Factors affecting the structure and properties of aninjectable self-setting calcium phosphate foam. J Biomed MaterRes A. 2007;80(2):351–61.

10. Del Valle S, et al. In vivo evaluation of an injectablemacroporous calcium phosphate cement. J Mater Sci MaterMed. 2007;18(2):353–61.

11. De Groot AS, Scott DW. Immunogenicity of protein therapeu-tics. Trends Immunol. 2007;28(11):482–90.

12. Altuntaş E, Yener G. Formulation and evaluation of thermore-versible in situ nasal gels containing mometasone furoate forallergic rhinitis. AAPS PharmSciTech. 2017;18(7):2673–82.

13. Alexandridis P, Hatton TA. Poly (ethylene oxide) poly (propyl-ene oxide) poly (ethylene oxide) block copolymer surfactants inaqueous solutions and at interfaces: thermodynamics, structure,dynamics, and modeling. Colloids Surf A Physicochem EngAsp. 1995;96(1–2):1–46.

14. Abdi SIH, Choi JY, Lee JS, Lim HJ, Lee C, Kim J, et al. In vivostudy of a blended hydrogel composed of pluronic F-127-alginate-hyaluronic acid for its cell injection application. TissueEng Regener Med. 2012;9(1):1–9.

15. Gan T, Zhang Y, Guan Y. In situ gelation of P (NIPAM-HEMA) microgel dispersion and its applications as injectable3D cell scaffold. Biomacromolecules. 2009;10(6):1410–5.

16. Kim JH, Lee SB, Kim SJ, Lee YM. Rapid temperature/pHresponse of porous alginate-g-poly (N-isopropylacrylamide)hydrogels. Polymer. 2002;43(26):7549–58.

17. Tang Y, Wang X, Li Y, Lei M, du Y, Kennedy JF, et al.Production and characterisation of novel injectable chitosan/methylcellulose/salt blend hydrogels with potential applicationas tissue engineering scaffolds. Carbohydr Polym.2010;82(3):833–41.

18. Talaat WM, Haider M, Kawas SA, Kandil NG, Harding DRK.Chitosan-based thermosensitive hydrogel for controlled drugdelivery to the temporomandibular joint. J Craniofac Surg.2016;27(3):735–40.

19. Montufar E, et al. Foamed surfactant solution as a template forself-setting injectable hydroxyapatite scaffolds for bone regen-eration. Acta Biomater. 2010;6(3):876–85.

20. Huang Y, Zhang X, Wu A, Xu H. An injectable nano-hydroxyapatite (n-HA)/glycol chitosan (G-CS)/hyaluronic acid(HyA) composite hydrogel for bone tissue engineering. RSCAdv. 2016;6(40):33529–36.

21. Li F, Liu Y, Ding Y, Xie Q. A new injectable in situ forminghydroxyapatite and thermosensitive chitosan gel promoted byNa 2 CO 3. Soft Matter. 2014;10(13):2292–303.

22. Leroux L, Hatim Z, Frèche M, Lacout JL. Effects of variousadjuvants (lactic acid, glycerol, and chitosan) on the injectabilityof a calcium phosphate cement. Bone. 1999;25(2):31S–4S.

23. Shamma RN, Elkasabgy NA, Mahmoud AA, Gawdat SI,Kataia MM, Abdel Hamid MA. Design of novel injectable in-situ forming scaffolds for non-surgical treatment of periapicallesions: in-vitro and in-vivo evaluation. Int J Pharm.2017;521(1):306–17.

24. Paul S, Egbert JE, Walsh AW, Hoey MF. Pressure measure-ments during injection of corticosteroids. Med Biol EngComput. 1998;36(6):729–33.

25. Kumbhar D, Pokharkar VB. Engineering of a nanostructuredlipid carrier for the poorly water-soluble drug, bicalutamide:physicochemical investigations. Colloids Surf A Physiochem.2013;416:32–42.

26. Nafee N, Zewail M, Boraie N. Alendronate-loaded biodegrad-able smart hydrogel: a promisimg injectable depot formulationfor osteoporosis. J Drug Target. 2017;26(7):563–75.

27. Hansen MB, Nielsen SE, Berg K. Re-examination and furtherdevelopment of a precise and rapid dye method for measuringcell growth/cell kill. J Immunol Methods. 1989;119(2):203–10.

104 of 16 Morsi et al. (2019) 20:104

28. Gohel A, McCarthy M-B, Gronowicz G. Estrogen preventsglucocorticoid-induced apoptosis in osteoblasts in vivo andin vitro. Endocrinology. 1999;140(11):5339–47.

29. Kalpana B, Lakshmi PK. Transdermal permeation enhance-ment of Tolterodine Tartrate through invasomes and iontopho-resis. Pharm Lett. 2013;5(6):119–26.

30. Quinten T, Gonnissen Y, Adriaens E, Beer TD, Cnudde V,Masschaele B, et al. Development of injection moulded matrixtablets based on mixtures of ethylcellulose and low-substitutedhydroxypropylcellulose. Eur J Pharm Sci. 2009;37(3–4):207–16.

31. Basalious EB, El-Sebaie W, El-Gazayerly O. Application ofpharmaceutical QbD for enhancement of the solubility anddissolution of a class II BCS drug using polymeric surfactantsand crystallization inhibitors: development of controlled-releasetablets. AAPS PharmSciTech. 2011;12(3):799–810.

32. Lima DL, Calisto V, Esteves VI. Adsorption behavior of 17α-ethynylestradiol onto soils followed by fluorescence spectraldeconvolution. Chemosphere. 2011;84(8):1072–8.

33. Talasaz AH, et al. In situ gel forming systems of poloxamer 407and hydroxypropyl cellulose or hydroxypropyl methyl cellulosemixtures for controlled delivery of vancomycin. J Appl PolymSci. 2008;109(4):2369–74.

34. El-Gawad A, et al. Formulation and physical characterization ofa novel sustained-release ophthalmic delivery system forSparfloxacin: the effect of the biological environment.Ophthalmol Res Int J. 2013;1(1):1–22.

35. Fakhari A, Corcoran M, Schwarz A. Thermogelling propertiesof purified poloxamer 407. Heliyon. 2017;3(8):e00390.

36. Gioffredi E, Boffito M, Calzone S, Giannitelli SM, Rainer A,Trombetta M, et al. Pluronic F127 hydrogel characterization andbiofabrication in cellularized constructs for tissue engineeringapplications. Procedia CIRP. 2016;49:125–32.

37. Rarokar NR, Saoji SD, Khedekar PB. Investigation of effec-tiveness of some extensively used polymers on thermoreversibleproperties of Pluronic® tri-block copolymers. J Drug DeliverySci Technol. 2018;44:220–30.

38. Unosson J, Montufar EB, Engqvist H, Ginebra MP, Persson C.Brushite foams—the effect of Tween® 80 and Pluronic® F-127on foam porosity and mechanical properties. J Biomed MaterRes B Appl Biomater. 2016;104(1):67–77.

39. Shimoyama T, Itoh K, Kobayashi M, Miyazaki S, D’EmanueleA, Attwood D. Oral liquid in situ gelling methylcellulose/alginate formulations for sustained drug delivery to dysphagicpatients. Drug Dev Ind Pharm. 2012;38(8):952–60.

40. Perut F, Montufar EB, Ciapetti G, Santin M, Salvage J,Traykova T, et al. Novel soybean/gelatine-based bioactive andinjectable hydroxyapatite foam: material properties and cellresponse. Acta Biomater. 2011;7(4):1780–7.

41. Hegge AB, Andersen T, Melvik JE, Kristensen S, TønnesenHH. Evaluation of novel alginate foams as drug deliverysystems in antimicrobial photodynamic therapy (aPDT) ofinfected wounds—an in vitro study: studies on curcumin andcurcuminoides XL. J Pharm Sci. 2010;99(8):3499–513.

42. Cervantes-Martínez A, Maldonado A. Foaming behaviour ofpolymer–surfactant solutions. J Phys Condens Matter.2007;19(24):246101.

43. Rangabhatla ASL, Tantishaiyakul V, Oungbho K, Boonrat O.Fabrication of pluronic and methylcellulose for etidronatedelivery and their application for osteogenesis. Int J Pharm.2016;499(1–2):110–8.

44. Raharitsifa N, Genovese DB, Ratti C. Characterization of applejuice foams for foam-mat drying prepared with egg whiteprotein and methylcellulose. J Food Sci. 2006;71(3):E142–51.

45. Ceccaldi C, Bushkalova R, Cussac D, Duployer B, Tenailleau C,Bourin P, et al. Elaboration and evaluation of alginate foam scaffoldsfor soft tissue engineering. Int J Pharm. 2017;524(1–2):433–42.

46. Bakeri G, Ismail AF, Shariaty-Niassar M, Matsuura T. Effect ofpolymer concentration on the structure and performance ofpolyetherimide hollow fiber membranes. J Membr Sci.2010;363(1–2):103–11.

47. Tan R, Feng Q, Jin H, Li J, Yu X, She Z, et al. Structure andbiocompatibility of an injectable bone regeneration composite. JBiomater Sci Polym Ed. 2011;22(14):1861–79.

48. Schütz K, Placht AM, Paul B, Brüggemeier S, Gelinsky M, LodeA. Three-dimensional plotting of a cell-laden alginate/

methylcellulose blend: towards biofabrication of tissue engi-neering constructs with clinically relevant dimensions. J TissueEng Regen Med. 2017;11(5):1574–87.

49. Witek L, Shi Y, Smay J. Controlling calcium and phosphate ionrelease of 3D printed bioactive ceramic scaffolds: an in vitrostudy. J Adv Ceram. 2017;6(2):157–64.

50. Lin H-R, Sung K, Vong W-J. In situ gelling of alginate/pluronicsolut ions for ophthalmic del ivery of pi locarpine.Biomacromolecules. 2004;5(6):2358–65.

51. Al-Sokanee ZN, et al. The drug release study of ceftriaxonefrom porous hydroxyapatite scaffolds. AAPS PharmSciTech.2009;10(3):772–9.

52. Balestrieri F, Magrì AD, Magrì AL, Marini D, Sacchini A.Application of differential scanning calorimetry to the study ofdrug -exc ip ien t compat ib i l i t y. Thermoch im Acta .1996;285(2):337–45.

53. Gong T, Chen Y, Zhang Y, Zhang Y, Liu X, Troczynski T, et al.Osteogenic and anti-osteoporotic effects of risedronate-addedcalcium phosphate sil icate cement. Biomed Mater.2016;11(4):045002.

54. Redman-Furey N, Dicks M, Bigalow-Kern A, Cambron RT,Lubey G, Lester C, et al. Structural and analytical characteri-zation of three hydrates and an anhydrate form of risedronate. JPharm Sci. 2005;94(4):893–911.

55. Elfalaky A, Hashem H, Mohamed T. Bone-like material,synthesis, optimization and characterization. Nanosci Nanoeng.2014;2(1):10–6.

56. El-Badry M, et al. Performance of poloxamer 407 as hydrophiliccarrier on the binary mixtures with nimesulide. Farmacia.2013;61(6):1137–50.

57. Soares J, et al. Thermal behavior of alginic acid and its sodiumsalt. Eclética Química. 2004;29(2):57–64.

58. Khajuria DK, Disha C, Vasireddi R, Razdan R, Mahapatra DR.Risedronate/zinc-hydroxyapatite based nanomedicine for oste-oporosis. Mater Sci Eng C. 2016;63:78–87.

59. Errassifi F, Sarda S, Barroug A, Legrouri A, Sfihi H, Rey C.Infrared, Raman and NMR investigations of risedronateadsorption on nanocrystalline apatites. J Colloid Interface Sci.2014;420:101–11.

60. Aprilliza, M. Characterization and properties of sodium alginatefrom brown algae used as an ecofriendly superabsorbent. InIOP Conference Series: Materials Science and Engineering.2017. IOP Publishing.

61. Fathalla ZM, et al. Poloxamer-based thermoresponsive ketorolactromethamine in situ gel preparations: Design, characterisation,toxicity and transcorneal permeation studies. Eur J PharmBiopharm. 2017;114:119–34.

62. El-Bagory IM, et al. Effect of gamma irradiation on pluronicgels for ocular delivery of ciprofloxacin: in vitro evaluation.Aust J Basic Appl Sci. 2010;4(9):4490–8.

63. Stockert JC, Blázquez-Castro A, Cañete M, Horobin RW,Villanueva Á. MTT assay for cell viability: intracellular locali-zation of the formazan product is in lipid droplets. ActaHistochem. 2012;114(8):785–96.

64. Wang H, Li Y, Zuo Y, Li J, Ma S, Cheng L. Biocompatibilityand osteogenesis of biomimetic nano-hydroxyapatite/polyamidecomposite scaffolds for bone tissue engineering. Biomaterials.2007;28(22):3338–48.

65. Elkasabgy NA, Mahmoud AA, Shamma RN. Determination ofcytocompatibility and osteogenesis properties of in situ formingcollagen-based scaffolds loaded with bone synthesizing drug forbone tissue engineering. Int J Polym Mater Polym Biomater.2017;67(8):494–500.

66. Tang L, Thevenot P, Hu W. Surface chemistry influencesimplant biocompatibility. Curr Top Med Chem. 2008;8(4):270–80.

67. Hunt NC, Grover LM. Cell encapsulation using biopolymer gelsfor regenerative medicine. Biotechnol Lett. 2010;32(6):733–42.

68. Chang, H.-I. and Y. Wang, Cell responses to surface andarchitecture of tissue engineering scaffolds, in Regenerativemedicine and tissue engineering-cells and biomaterials 2011,INTECH.

69. Pleshko N, Boskey A, Mendelsohn R. Novel infrared spectro-scopic method for the determination of crystallinity of hydroxy-apatite minerals. Biophys J. 1991;60(4):786–93.


70. Ma X, He Z, Han F, Zhong Z, Chen L, Li B. Preparation ofcollagen/hydroxyapatite/alendronate hybrid hydrogels as poten-tial scaffolds for bone regeneration. Colloids Surf B:Biointerfaces. 2016;143:81–7.

71. Cui J, Liang J, Wen Y, Sun X, Li T, Zhang G, et al. In vitro andin vivo evaluation of chitosan/β-glycerol phosphate compositemembrane for guided bone regeneration. J Biomed Mater ResA. 2014;102(9):2911–7.

72. Clarke B. Normal bone anatomy and physiology. Clin J Am SocNephrol. 2008;3(Supplement 3):S131–9.

73. Jeong HM, Jin YH, Choi YH, Chung JO, Cho DH, Chung MY,et al. Risedronate increases osteoblastic differentiation andfunction through connexin43. Biochem Biophys Res Commun.2013;432(1):152–6.

104 of 16 Morsi et al. (2019) 20:104

risedronate-loaded macroporous gel foam enriched with ... risedronate-loaded macroporous gel foam...

Documents