Synthesis, Characterization and Process Optimization of Bone Whitlockite

nanomaterials

Article Synthesis, Characterization and Process Optimization of Bone Whitlockite

Sadaf Batool 1, Usman Liaqat 1 , Zakir Hussain 1,* and Manzar Sohail 2

1 School of Chemical and Materials Engineering (SCME), National University of Sciences & Technology (NUST), Sector H-12, 44000 Islamabad, Pakistan; [email protected] (S.B.); [email protected] (U.L.) 2 Department of Chemistry, School of Natural Sciences (SNS), National University of Sciences & Technology (NUST), Sector H-12, 44000 Islamabad, Pakistan; [email protected] * Correspondence: [email protected]; Tel./Fax: +0092-51-9085-5200

Received: 26 July 2020; Accepted: 20 August 2020; Published: 17 September 2020

Abstract: Whitlockite, being the second most abundant bio-mineral in living bone, finds huge applications in tissue regeneration and implants and its synthesis into its pure form has remained a challenge. Although precipitation of whitlockite phase has been reported recently in many publications, effects of various parameters to control such phase as well as conditions for the bulk preparation of this extremely important bio-mineral have not been investigated so far. In this work, we report the precipitation of pure whitlockite phase using common precursors. As reported in the literature, whitlockite is stable in a narrow pH range, therefore; optimization of pH for the stabilization of whitlockite phase has been investigated. Additionally, in order to narrow down the optimum conditions for the whitlockite precipitation, effect of temperature and heating conditions has also been studied. The obtained solids were characterized using powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, scanning electron microscopy (SEM) and thermogravimetric analysis (TGA). From PXRD analysis, it was observed that heating the precursor’s mixture at 100 ◦C with subsequent aging at the optimized pH resulted in the precipitation of pure whitlockite phase. These results were further confirmed by TGA, SEM and Raman spectroscopy analysis and it was confirmed that the conditions reported here favor whitlockite precipitation without formation of any secondary phase. These reaction conditions were further confirmed by changing all the parameters like aging, heating time, feed rate of precursors one by one. From PXRD analysis of these samples, it was concluded that not only pH but temperature, heating time, aging time and feed rate effect simultaneously on the precipitation of pure whitlockite phase and a subtle change in any of these parameters could lead to the formation of undesired stable secondary calcium phosphate phases.

Keywords: whitlockite; bone mineral; calcium phosphate; synthesis; process optimization

1. Introduction Bone is a composite of collagen ﬁbers reinforced with nano-sized apatites i.e., hydroxyapatite (HA) and whitlockite (WH) [1,2]. Hydroxyapatite is the major component of inorganic phase of bone [3], has a hexagonal crystalline structure and maintains bone strength. It was previously considered as the best bone alloplast but its poor solubility and fast degradation under the body’s acidic environment has limited its use. Some recent studies have shown that WH could be the best substitute for hydroxyapatite alloplast. WH has a rhombohedral crystalline structure [4,5], with R3C space group of alkali and alkaline earth metals that exist naturally in the form of Merrillite (Ca9MgNa (PO4)7)[6], β-tricalcium magnesium phosphate (β-TCMP, Ca2.86Mg0.14 (PO4)2) Stanﬁeldite (Ca, Mg)3(PO4)2 [7], β-tricalcium Phosphate (β-TCP, Ca3 (PO4)2) and magnesium or biological WH (Ca18Mg2H2 (PO4)14/Ca9Mg(HPO4)(PO4)6)[8].

Nanomaterials 2020, 10, 1856; doi:10.3390/nano10091856 www.mdpi.com/journal/nanomaterials Nanomaterials 2020, 10, 1856 2 of 14

Among various forms of WH, β-TCP and WH have been explored biologically since they are highly biocompatible with excellent osteogenic properties. β-TCP was once considered as bone WH due to its close structural resemblance with WH. However, recent powder X-ray diffraction (PXRD) and SEM analysis of bone have revealed that it is WH that exists naturally in the bone while β-TCP is merely a synthetic analog with similar structure [9–11]. Due to biological importance, presence of hydrogen and magnesium makes WH different from β-TCP. Magnesium maintains bone metabolism, stimulates growth during earlier stages of bone development and controls the biological functions of the body [12,13]. It exists in the form of octahedral chains of MgO6 and PO4H in the threefold axis of rhombohedral geometry [14,15] and its deficiency in the body can lead to osteoporosis along with malfunctions of other body parts such as cardiovascular failure. On the other hand, hydrogen helps magnesium in bone growth by camouflaging the immune system as well as gives stability to WH rhombohedral structure [7,16–19]. Synthesis of biological WH is not easy due to its poor thermodynamic stability and synthesis over a narrow pH and temperature range. For a long time, WH was considered as an intermediate of hydroxyapatite, magnesium brushite and monetite which converts into stable calcium phosphates (CaPs) under ambient conditions and to hydroxyapatite above pH 4.5 [20,21]. Thus, the ability to obtain WH with ideal chemical composition has remained a challenge. Previously, some researchers tried to synthesize WH by incorporating magnesium in hydroxyapatite and in β-TCP crystal lattice but ended up in mixed phases of WH and CaPs [22–25]. On the other hand, a lot of works have been reported to explore the exact Ca:Mg:P ratios for the synthesis of pure WH phase, however; resulting in mixed phases [26–29]. Similarly, different CaPs intermediates like monetite, brushite and calcium deficient apatites have also been used as precursors to synthesize pure WH [30,31]. Recently, Magalhaes et al., in 2018, reported that whitlockite phase is stable between narrow pH range of 5 and 6 [8] while in other works acid pH 5.6 and pH 6–7 allows precipitation of β-TCMP and other magnesium containing whitlockite using common precursors [32–34] as well as under high-temperature hydrothermal conditions [35] to synthesize pure WH phase. Shah et al., in 2017, did the crystallographic analysis of bone and reported that whitlockite formation in natural system occur in acidic conditions [36] while in another work done by Abdelkader et al. in 2001, basic pH 10 and large magnesium concentration were reported for biological whitlockite precipitation [32]. However, none of these works have reported the exact pH along with all relevant parameters such as heating and aging time for the synthesis of pure biological WH phase resulting in a big question mark on the reproducibility of pure WH phase. Therefore, due to huge potential of this material for biomedical applications, it was extremely essential to explore and report exact conditions for the formation of pure WH phase. Additionally, it was also essential to explore whether the synthesis process is reproducible and can it be optimized for the synthesis of WH at mass scale. Three batches of reactions were carried out to synthesize homogenous WH i.e., effect of heating conditions and temperature at pH 4 (batch one), effect of temperature and heating time at pH 5 (batch two) and effect of different parameters like aging time, heating time and feed rate on WH precipitation (batch three). Finally, the optimized parameters were checked for the reproducibility of the WH phase as well synthesis of material in grams scale quantity. To the best of our knowledge, we believe, this is the first detailed study on the synthesis of WH phase with comprehensive optimization of various parameters and extending the same for the mass production of this bio-mineral and demonstrates huge commercial value.

2. Materials and Method Orthophosphoric acid was purchased from Honeywell (Fluka, NC, USA), calcium hydroxide from GPR Rectapur and magnesium hydroxide from Duksan (Daejeon, SouthKorea). All the chemicals were used as received without any further processing. Nanomaterials 2020, 10, 1856 3 of 14

2.1. Synthesis of WH Here in, we have reported on the optimization of conditions for the precipitation of WH phase and demonstrated the scale-up capability of our synthesis method. In our method, a wet homogenous chemical precipitation procedure was employed and the effect of different pH conditions on the precipitant dissolution was also examined, starting from pH 13 to pH 1. As more data on acidic pH precipitation were reported therefore, first we added phosphoric acid till pH 1. The precipitates did not form at this pH and already formed precipitates dissolved in reaction mixture. Then we stopped acid addition at pH 2, but the same results as that of pH 1 were obtained. In next reaction, pH 3 was selected as final and it resulted in the formation of turbid liquid. All these samples were kept on aging at room temperature without any agitation for one month but none of the product was obtained. Therefore, we decided to check the stability of precipitates at pH 4 which resulted in formation of stable precipitates of magnesian whitlockite and β-TCMP. As the reported (Ca + Mg/P) ratios were used therefore, we decided to first to check the effect of pH 5 on biological whitlockite formation before changing other parameters. At pH 5, we obtained some precipitation of biological whitlockite in all the three batches while exact conditions were optimized in batch 2. It was observed that below pH 4, the precursor’s precipitates started dissolving with complete dissolution at pH 2.5 with no ≤ reappearance of precipitates even after aging for one month. For the synthesis of WH, Ca:Mg:P ratios were used as reported earlier [20]. Briefly, a 0.37 M solution of Ca(OH)2, 0.13 M solution of Mg(OH)2 and 0.5 M solution of H3PO4 were prepared. All the products obtained were filtered, washed thrice with deionized water and dried overnight at 50 ◦C. All the samples were aged at room temperature without any agitation. Table1 below shows the overall reaction conditions and formed products besides precipitation of WH phase. The overall approach used for the synthesis of WH is shown in Figure1.

Table 1. Eﬀect of various reaction parameters on whitlockite (WH) synthesis (WH-1 to WH-11).

Sr. # Sample Name pH Reaction Conditions Aging Time Product Ca pyrophosphate, Mg phosphate 1. WH-1 4 80 C/5 h/stirring 14 h ◦ hydroxide, Stanfieldite (Mg WH) Monetite, farringtonite 2. WH-2 4 Autoclaved/120 C/5 h 14 h ◦ (WH Phase), Ca phosphate oxide Mg phosphate, Monetite, 3. WH-3 4 Refluxed/100 C/5 h 14 h ◦ Ca-Mg mixed phosphates HA, β-TCP (WH Phase), 4. WH-4 5 80 C/10 h/stirring 14 h ◦ β-TCMP (WH Phase) Bone WH, Stanfieldite 5. WH-5 5 Autoclaved/120 C/10 h 14 h ◦ (WH Phase), β-TCMP (WH Phase)

6. WH-6 5 Reﬂuxed/100 ◦C/10 h 14 h Bone WH

7. WH-7 5 Refluxed/100 ◦C/12 h 14 h Stanfieldite, β-TCMP (WH-Phase) Bone WH, Stanfieldite 8. WH-8 5 Refluxed/100 C/10 h 18 h ◦ (WH-Phase), Monetite

9. WH-9 5 Refluxed/100 ◦C/10 h No aging Newberyite, Ca Phosphate Orthophosphoric acid feed Bone WH, Stanfieldite, β-TCP and 10. WH-10 5 14 h rate changed/refluxed/100 ◦C Ca-Mg phosphate

11. WH-11 5 WH-6/annealed/750 ◦C/6 h Bone WH Nanomaterials 2020, 10, 1856 4 of 14 Nanomaterials 2020, 10, x FOR PEER REVIEW 4 of 14

FigureFigure 1. Schematics 1. Schematics of approach of approach used for used WH for synthesis. WH synthesis. 2.1.1. Synthesis of WH at pH 4-Effect of Heating Conditions 2.1.1. Synthesis of WH at pH 4-Effect of Heating Conditions In the first batch, effect of heating conditions/temperature on the synthesis of WH phase at a Inconstant the first pH batch, of 4 waseffect investigated. of heating conditions/temperature Precursor’s solutions wereon the mixed synthesis and theof WH mixture phase was at stirreda constant pH of 4 was investigated. Precursor’s solutions were mixed and the mixture was stirred for 30 min at 45 ◦C followed by the addition of 0.5 M H3PO4 at 10 mL/5min to the reaction mixture for 30to min achieve at 45 pH°C followed 4. This mixture by the wasaddition divided of 0.5 into M threeH3PO parts4 at 10 as mL/5min WH-1, WH-2 to the and reaction WH-3. mixture The WH-1 to achieve pH 4. This mixture was divided into three parts as WH-1, WH-2 and WH-3. The WH-1 reaction mixture was kept stirring for 5 h at 80 ◦C, the WH-2 mixture was autoclaved at 120 ◦C for 5 h, reaction mixture was kept stirring for 5 h at 80 °C, the WH-2 mixture was autoclaved at 120 °C for 5 while WH-3 mixture was refluxed at 100 ◦C for 5 h. All reaction mixtures were aged for 14 h to achieve h, whilethe finalWH-3 products. mixture was refluxed at 100 °C for 5 h. All reaction mixtures were aged for 14 h to achieve the final products. 2.1.2. Synthesis of WH at pH 5-Effect of Heating Conditions 2.1.2. Synthesis of WH at pH 5-Effect of Heating Conditions In the second batch, effect of heating conditions/temperature on the synthesis of WH phase at Ina constantthe second pH batch, of 5 was effect investigated. of heating conditions In all such/temperature cases, addition on the ofphosphate synthesis of solution WH phase was at stopped a constantat pH pH 5. of This 5 was mixture investigated. was divided In all intosuch three cases, parts addition as WH-4, of phosphate WH-5 and solution WH-6. was The stopped WH-4 reaction at pH 5.mixture This mixture was kept was on divided stirring into for 10three h at part 80 ◦sC, as the WH-4, WH-5 WH-5 mixture and was WH-6. autoclaved The WH-4 at 120 reaction◦C for 10 h, mixturewhile was WH-6 kept on mixture stirring was for refluxed 10 h at 80 at °C, 100 the◦C WH-5 for 10 mixture h. All reaction was autoclaved mixtures at were 120 °C aged for for10 h, 14 h to whileachieve WH-6 mixture the final was products. refluxed at 100 °C for 10 h. All reaction mixtures were aged for 14 h to achieve the final products. 2.1.3. Synthesis of WH-Effect of Heating Time, Aging and Annealing 2.1.3. Synthesis of WH-Effect of Heating Time, Aging and Annealing In the third batch, effect of different parameters like heating time, aging, feed rate and annealing Inon the the third synthesis batch, of WHeffect phase of different was studied. parameters In the like case heating of WH-7, time, heating aging, time feed was rate increased and to annealing12 h whileon the in synthesis the case of of WH-8 WH agingphase timewas wasstud increasedied. In the to case 18 h. of In WH-7, addition, heating in the time case was of WH-9, increasedprecipitants to 12 h while were separatedin the casewithout of WH-8 aging aging while time inwas the increased case of WH-10, to 18 h. feedIn addition, rate of phosphoricin the case acid of WH-9,addition precipitants was changed. were Aseparated total of 10without mL of phosphoricaging while acid in the was case added of withoutWH-10, anyfeed constant rate of time phosphoricdifference. acid Finally,addition the was eff ectchanged. of annealing A total on of WH-610 mL precipitants of phosphoric was acid studied, was added and for without this purpose any the constantproduct time obtaineddifference. (WH-6) Finally, was the annealed effect of at annealing 750 ◦C for on 6 hWH-6 to achieve precipitants WH-11. was studied, and for this purpose the product obtained (WH-6) was annealed at 750 °C for 6 h to achieve WH-11. 2.2. Characterization of Materials 2.2. CharacterizationFor the crystallographic of Materials characterization of all prepared solids, Powder X-ray diffractometer Forof STOEthe crystallographic (Darmstadt, Germany) characterization and DRON-8 of all pr Bourevestnikepared solids, (Saint-Petersburg, Powder X-ray diffractometer Russia) were of used. STOEFTIR (Darmstadt, analysis Germany) was done usingand DRON-8 the PerkinElmer, Bourevestn SpectrumTM100ik (Saint-Petersburg, spectrophotometer Russia) were where used.KBr FTIR pellets analysiscontaining was done samples using werethe PerkinElmer, prepared for Spec the analysis.trumTM100 Raman spectrophotometer spectroscopic analysis where KBr was pellets done using containing samples were prepared for the analysis. Raman spectroscopic analysis was done using Nanomaterials 2020, 10, x FOR PEER REVIEW 5 of 14

Nanomaterials 2020, 10, 1856 5 of 14 BWS415-532S-iRaman manufactured by BW TEK INC (Newark, NJ, USA). TGA was done using TGA-5500 Discovery series(TA instruments, New Castle, DE, USA) under a nitrogen environment BWS415-532S-iRamanin the range of 100–1100 manufactured °C at a rate of by 10°C/min. BW TEK The INC size (Newark, and shape NJ, USA).of the prepared TGA was nanoparticles done using TGA-5500were observed Discovery using series(TA scanning instruments, electron microscopy New Castle, (SEM), DE, USA) NOVA under FEISEM-450(New a nitrogen environment York, NY, in theUSA). range of 100–1100 ◦C at a rate of 10 ◦C/min. The size and shape of the prepared nanoparticles were observed using scanning electron microscopy (SEM), NOVA FEISEM-450(New York, NY, USA). 3. Results and Discussion 3. Results and Discussion PXRD analysis of the first batch of products (WH-1, WH-2 and WH-3) is shown in Figure 2. MixedPXRD phases analysis of calcium of the first phosph batchates of productsappeared (WH-1, in XRD WH-2 diffractogram and WH-3) of is WH-1 shown i.e., in Figure calcium2. Mixedpyrophosphate phases of(JCDPS calcium 00-009-0345) phosphates [37], appeared magnesiu inm XRD phosphate diffractogram hydroxide of WH-1(JCDPS i.e., 00-047-0955) calcium pyrophosphate[38] and Stanfieldite (JCDPS (magnesian 00-009-0345) WH) [37], JCDPS magnesium 00-013-0404 phosphate [39]. hydroxide The XRD (JCDPS diffractogram 00-047-0955) of WH-2 [38] andshows Stanfieldite maximum (magnesian peaks of WH)Monetite JCDPS (JCDPS 00-013-0404 00-009-0080, [39]. The 01-070-0360) XRD diffractogram [40] with of some WH-2 peaks shows of maximumfarringtonite peaks (JCDPS of Monetite 00-033-0876) (JCDPS and 00-009-0080, 3rd calcium 01-070-0360) phosphate oxide [40] with (JCDPS some 01-070-1379). peaks of farringtonite While, the (JCDPSXRD spectrum 00-033-0876) of WH-3 and 3rd does calcium not show phosphate any WH oxide phase (JCDPS but peaks 01-070-1379). of magnesium While, thephosphate XRD spectrum (JCDPS of00-0025-1373), WH-3 does notMonetite show any (JCDPS WH phase01-070-1425) but peaks [40] of magnesiumand calcium phosphate magnesium (JCDPS mixed 00-0025-1373), phosphates Monetite(JCDPS 01-082-0503, (JCDPS 01-070-1425) 00-009-0396). [40] and Therefore, calcium magnesiumPXRD reflections mixed for phosphates this group (JCDPS demonstrate 01-082-0503, that 00-009-0396).probably pH Therefore, 4 does not PXRD favor reflections the precipitation for this group of WH demonstrate phase. Although, that probably after pH the 4 doesappearance not favor of theStanfieldite precipitation peaks of WHin WH-1, phase. Although,we were encouraged after the appearance to fine tune of Stanfieldite parameters peaks such in WH-1,as temperature we were encouragedand/or heating to fine conditions tune parameters to obtain such relatively as temperature pure WH and phase./or heating However, conditions such modifications to obtain relatively did not pureresult WH into phase. a pure However, WH phase such confirming modifications the didpH not4 as result not desired into a pure one WHto crystallize phase confirming the required the pHphase. 4 as not desired one to crystallize the required phase.

FigureFigure 2. 2.Powder Powder X-ray X-ray di diffractionffraction (PXRD) (PXRD) di diffractogramsffractograms of of the the first first batch batch of of products products (WH-1, (WH-1, WH-2, WH- WH-3)2, WH-3) (S for (S for Stanfieldite, Stanfieldite, F for F Farringtonite,for Farringtonite M, for M Monetitefor Monetite and and # for # mixedfor mixed CaP CaP phases). phases).

XRDXRD analysisanalysis ofof thethe secondsecond batchbatch ofof productsproducts (WH-4,(WH-4, WH-5WH-5 andand WH-6)WH-6) isis shownshown inin FigureFigure3 .3. β XRDXRD di diffractogramffractogram of of WH-4 WH-4 shows shows the the maximum maximum peaks peaks of hydroxyapatite of hydroxyapatite (JCDPS (JCDPS 00-009-0432), 00-009-0432),-TCP β- WHTCP (JCDPS WH (JCDPS 00-009-0169) 00-009-0169) [19,41] [19,41] and some and of some TCMP of (JCDPS TCMP 01-077-6692)(JCDPS 01-077-6692) [26,30]. However, [26,30]. However, in the XRD in ditheffractogram XRD diffractogram of WH-5, two of WH-5, peaks oftwo biological peaks of WH biological at 25.9 ◦WHand at 31.4 25.9°◦ (JCDPS and 31.4° 01-070-2064) (JCDPS 01-070-2064) are shown. Theare othershown. peaks The at other 46.7◦ peaksand 49.4 at◦ 46.7°, 42.2 ◦andare of49.4°, TCMP 42.2° (JCDPS are of 01-083-1888) TCMP (JCDPS while 01-083-1888) peaks at 51.2 while◦ and peaks 56.6◦ areat due51.2° to and the presence56.6° are ofdue the to Stanfieldite the presence phase of (JCDPS the Stanfieldite 00-013-0404) phase [8,26 (JCDPS]. Additionally, 00-013-0404) presence [8,26]. of few other peaks represents, calcium and magnesium phosphates mixed phases. In the case of WH-6, Nanomaterials 2020, 10, x FOR PEER REVIEW 6 of 14

Additionally,Nanomaterials 2020 presence, 10, 1856 of few other peaks represents, calcium and magnesium phosphates6 ofmixed 14 phases. In the case of WH-6, precipitation of pure biological WH (C18H2Mg2(PO4)14) matched with literature [20,30,42,43] as well as JCDPS (01-070-2064 and 00-042-0578). The XRD reflections of WH- 4 andprecipitation WH-5 demonstrated of pure biological the precipitation WH (C18H2Mg of2 (POmixed4)14 )WH matched phases with of literatureStanfieldite, [20, 30TCMP,42,43 and] as wellβ-TCP whichas JCDPS gave (01-070-2064 evidence that and WH 00-042-0578). precipitation The XRDcould reflections be possible of WH-4 at pH and 5 WH-5while demonstrateda slight change the in heatingprecipitation conditions of mixed could WH give phases pure of Stanfieldite,WH phase. TCMP The XRD and β -TCPreflections which of gave WH-6 evidence confirmed that WH this assumption.precipitation However, could be possiblethe PXRD at analysis pH 5 while of aWH-5 slight also change demonstrated in heating conditionsthat temperature could give above pure 100 WH phase. The XRD reflections of WH-6 confirmed this assumption. However, the PXRD analysis °C did not favor the formation of pure WH phase. Since magnesium replaces calcium ions of WH-5 also demonstrated that temperature above 100 C did not favor the formation of pure WH completely in the WH crystal lattice, it could lead to the◦ formation of slightly distorted structure phase. Since magnesium replaces calcium ions completely in the WH crystal lattice, it could lead to which is unstable under high pressure and temperature conditions (120 °C). Moreover, in order to the formation of slightly distorted structure which is unstable under high pressure and temperature further check the purity of WH-6, high-temperature treatment was carried out where PXRD results conditions (120 ◦C). Moreover, in order to further check the purity of WH-6, high-temperature treatment confirmed WH-6 as pure biological WH phase. If WH-6 has any secondary amorphous phases, it was carried out where PXRD results confirmed WH-6 as pure biological WH phase. If WH-6 has any wouldsecondary be converted amorphous to phases, other itmore would stable be converted crystalline to other phases more upon stable heat crystalline treatment phases [20]. upon Another heat evidencetreatment of [the20]. formation Another evidence of pure WH of the phase formation could of be pure associated WH phase with could the presence be associated of 0210 with peak the in WH-6presence and ofWH-11 0210 peak (discussed in WH-6 later), and WH-11 a characteristic (discussed later),peak of a characteristic WH. The reduction peak of WH. in d The-spacing reduction values (fromin d-spacing 2.60 to 2.58 values at 34.7°) (from 2.60of WH-6 to 2.58 and at 34.7WH-11◦) of also WH-6 confirmed and WH-11 the also precipitation confirmed theof pure precipitation WH phase [1,44–46].of pure WHMoreover, phase [1 ,the44– 46absence]. Moreover, of peaks the absence at the of001 peaks face at and the 001at 31.2° face andand at 31.8° 31.2◦ ofand β 31.8-TCP◦ ofand hydroxyapatiteβ-TCP and hydroxyapatite have demonstrated have demonstrated another evidence another of evidence the purity of theof WH-6 purity phase of WH-6 [47]. phase [47].

FigureFigure 3. 3. PXRDPXRD diffractograms diﬀractograms of of second second batch ofof productsproducts (WH-4, (WH-4, Wh-5, Wh-5, Wh-6) Wh-6) (S (S for for Stanﬁeldite, Stanfieldite, WW = =BiologicalBiological WH, WH, HA HA for for hydroxyapatite hydroxyapatite and ** forforβ β-TCMP-TCMP/TCP)./TCP).

PXRD analysis of the third batch of products (WH-7, WH-8 and WH-9, WH-10) is shown in PXRD analysis of the third batch of products (WH-7, WH-8 and WH-9, WH-10) is shown in Figure4. This batch of experiments was carried out to confirm whether the synthesis conditions of Figure 4. This batch of experiments was carried out to confirm whether the synthesis conditions of WH-6 are ideal for the precipitation of pure WH phase. In the case of XRD diffractogram of WH-7, WH-6 are ideal for the precipitation of pure WH phase. In the case of XRD diffractogram of WH-7, mixed phases of Mg WH i.e., Stanfieldite, β-TCMP (JCDPS 00-042-0578, 01-077-0692 and 01-070-2064) mixed phases of Mg WH i.e., Stanfieldite, β-TCMP (JCDPS 00-042-0578, 01-077-0692 and 01-070- are shown. Similarly, the XRD spectrum of WH-8 showed minor peaks of WH at 26.3◦, 36.0◦ and 2064) are shown. Similarly, the XRD spectrum of WH-8 showed minor peaks of WH at 26.3°, 36.0° 41.0◦ while other peaks are a mixture of Stanfieldite [48] as well as of Monetite (JCDPS 01-077-0692, and01-070-1425 41.0° while and other 00-042-0578). peaks are However, a mixture XRD of Stanfieldite diffractogram [48] of as WH-9 well did as notof Monetite show any (JCDPS peak of 01-077- WH 0692,phase, 01-070-1425 but instead and peaks 00-042-0578). of Newberyite However, [49] (JCDPS XRD diffractogram -00-035-0780) andof WH-9 calcium did phosphate not show (JCDPSany peak of01-082-0807, WH phase, 00-009-0169)but instead peaks could beof seen,Newberyite confirming [49] aging(JCDPS as -00-035-0780) one of the important and calcium factors phosphate for the (JCDPSprecipitation 01-082-0807, of WH 00-009-0169) phase along with could pH. be Furthermore, seen, confirming XRD di agingffractogram as one of of WH-10 the important showed peaks factors forof the WH precipitation at 22.4◦, 36.1◦ ,of 37.4 WH◦ (JCDP phase 00-042-0578) along with while pH. the Furthermore, rest of the peaks XRD corresponded diffractogram to Stanfieldite, of WH-10 showed peaks of WH at 22.4°, 36.1°, 37.4° (JCDP 00-042-0578) while the rest of the peaks corresponded to Stanfieldite, β-TCP and calcium magnesium phosphate (JCDPS 01-072-2042, 00- Nanomaterials 2020, 10, 1856 7 of 14

Nanomaterials 2020, 10, x FOR PEER REVIEW 7 of 14 β-TCP and calcium magnesium phosphate (JCDPS 01-072-2042, 00-012-0404, 00-09-0169, 00-020-0348). These012-0404, results 00-09-0169, have also 00-020-0348). demonstrated These the precipitation results have ofalso pure demonstrated WH phase which the precipitation was favored of by pure the slowWH additionphase which of phosphate was favored precursor. by the slow addition of phosphate precursor.

FigureFigure 4.4. PXRD didiffractogramsﬀractograms ofof thirdthird batchbatch ofof productsproducts (WH-7,(WH-7, WH-8,WH-8, WH-9,WH-9, WH-10)WH-10) (S(S forfor Stanﬁeldite,Stanfieldite, WW == Biological WH, M for Monetite, N N for for Newberyite, Newberyite, # # for for mixed mixed CaP CaP phases phases and and * *for for ββ-TCMP/TCP).-TCMP/TCP).

Finally,Finally, in in order order to to confirm confirm the the phase phase purity purity of of WH-6 WH-6 sample, sample, itit was was furtherfurther annealedannealed toto obtainobtain WH-11WH-11 (Figure(Figure5 ).5). XRD XRD di diffractogramffractogram ofof WH-11WH-11 showedshowed nono significantsignificant changechange inin peakpeak positionposition andand appearanceappearance ofof nono newnew peakpeak furtherfurther confirmedconfirmed WH-6WH-6 phasephase forfor purepure biologicalbiological WH.WH. PresencePresence ofof thethe 02100210 peakpeak asas thethe characteristiccharacteristic peakpeak ofof purepure WHWH phasephase inin WH-6WH-6 andand WH-11WH-11 hashas also also demonstrated demonstrated thethe phasephase puritypurity underunder givengiven conditions.conditions. Therefore,Therefore, XRDXRD analysisanalysis ofof thethe thirdthird batchbatch ofof productsproducts hashas demonstrateddemonstrated that that pure pure WH WH phase phase can can only only be be precipitated precipitated out out in a in narrow a narrow range range of temperature of temperature and pHand while pH agingwhile canaging also can be consideredalso be considered as one of theas essentialone of limitingthe essential factors limiting for the precipitationfactors for the of pureprecipitation WH phase. of pure WH phase. XRDXRD results have have clearly clearly demonstrated demonstrated the the format formationion of pure of pure WH WHphase phase in the in case the of case WH-6. of WH-6.Therefore, Therefore, this product this product was wasfurther further characterized characterized for forchemical chemical composition composition as as well asas morphologicalmorphological investigation.investigation. 1 TheThe FTIRFTIR analysisanalysis (Figure(Figure6 )6) of of WH-6 WH-6 showed showed a a peak peak at at 1029 1029 cm cm−−1 whichwhich isis thethe peakpeak ofofυ υ33 3 1 phosphatephosphate group group (PO (PO4 −4).3−). Peaks Peaks at 963at 963 and 606and cm606− couldcm−1 could be associated be associated with the with stretching the stretching vibration 3 (vibrationυ1) of the O-P-O(υ1) of bondthe O-P-O and the bond bending and vibrationthe bending (υ4 )vibration of the O-P-O (υ4) bondof the of O-P-O phosphate bond (PO of 4phosphate−) group. 3 1 1 The(PO asymmetric43−) group. The stretching asymmetric band ofstretching (PO4 −) groupband of can (PO be4 observed3−) group atcan 1120 be cmobserved− . The at peak 1120 at 872cm−1 cm. The− 2 ispeak the characteristicat 872 cm−1 is forthe HPOcharacteristic4 − group for [20 HPO,50].4 The2− group XRD [20,50]. results The were XRD verified results by thewere FTIR verified analysis by the of 1 2 theFTIR WH-6 analysis product. of the The WH-6 presence product. of a peak The atpresence 872 cm −ofconfirms a peak at the 872 presence cm−1 confirms of HPO 4the− which presence is the of onlyHPO peak42− which that di isff ersthe in only biological peak WHthat fromdiffers the in rest biol of theogical WH WH [20, 45from]. Moreover, the rest theof absencethe WH of [20,45]. peaks 1 1 atMoreover, 650 cm− theand absence 3570 cm −of confirmedpeaks at 650 that cm our−1 and samples 3570 were cm−1 freeconfirmed from the that secondary our samples phase were [7,31 ,43free]. 1 Thefrom peaks the secondary below 500 cmphase− are [7,31,43]. also present The peaks in the literaturebelow 500 and cm are−1 are unidentified also present [35 in,51 the]. The literature main FTIR and 1 1 regionare unidentified used for whitlockite [35,51]. The and main other FTIR CaPs region is between used for 800 whitlockite cm− to 1100 and cm other− wavelength. CaPs is between Therefore, 800 onlycm−1 thisto 1100 region cm is−1 more wavelength. focused. Therefore, Researchers only have this not region considered is more this focused. region [ 20Researchers,43]. have not considered this region [20,43]. NanomaterialsNanomaterials 2020 2020, ,10 10, ,x x FOR FOR PEER PEER REVIEW REVIEW 88 of of 14 14

FTIRFTIR resultsresults werewere alsoalso verifiedverified byby RamanRaman spectroscopicspectroscopic analysisanalysis asas itit givesgives moremore insightinsight aboutabout Nanomaterials 2020, 10, 1856 8 of 14 magnesiummagnesium additionaddition intointo thethe WHWH phasephase asas comparedcompared toto thethe XRD.XRD.

FigureFigure 5.5. PXRDPXRD diffractogramsdidiffractogramsﬀractograms ofof thirdthird batchbatch ofof productproduct (WH-11)(WH-11)(WH-11) through throughthrough annealing annealingannealing of ofof WH-6. WH-6.WH-6.

Figure 6. FTIR spectrum of WH-6 product (pure WH phase). FigureFigure 6.6. FTIRFTIR spectrumspectrum ofof WH-6WH-6 productproduct (pure(pure WHWH phase).phase). Nanomaterials 2020, 10, 1856 9 of 14

NanomaterialsFTIR results 2020, 10, were x FORalso PEER verified REVIEW by Raman spectroscopic analysis as it gives more insight about9 of 14 magnesium addition into the WH phase as compared to the XRD. The Raman Raman spectroscopic spectroscopic analysis analysis of of the the WH-6 WH-6 product product (Figure (Figure 7) 7showed) showed specific specific peaks peaks at 962 at 1 1 3 cm962−1 cm and− 586and cm 586−1 which cm− which are the are characteristic the characteristic peaks of peaks υ1 and of υυ14 POand43−υ ion4 PO while4 − ion the while peak theat 432 peak cm at−1 1 3 is432 a characteristic cm− is a characteristic peak of υ2 peakPO43− of [29].υ2 POTherefore,4 − [29]. both Therefore, Raman bothas we Ramanll as FTIR as wellresults as FTIRhave clearly results demonstratedhave clearly demonstrated the presence the of presenceWH phase of WHin WH-6 phase. The in WH-6. Raman The spectrum Raman spectrumof bone apatite of bone showed apatite 1 1 3 characteristicshowed characteristic peaks in peaks 959–975 in 959–975 cm−1 range cm− andrange at 432 and and at 432 578cm and− 578cm1 for υ−2 andfor υυ42 POand43−υ. 4ItPO also4 −showed. It also 1 3 peakshowed broadening peak broadening in the 959–975 in the cm 959–975−1 region cm −dueregion to a shift due in to PO a shift43− modes in PO 4which− modes occur which as a result occur asof 3 thea result deformation of the deformation of PO43− ionic of POstructure4 − ionic by structure surrounding by surrounding magnesium magnesiumions. The Raman ions. spectrum The Raman of hydroxyspectrum apatite of hydroxy and β apatite-TCP showed and β-TCP sharp showed peaks sharp in this peaks region in thisand regiona broadened and a broadened peak at 960 peak cm− at1, 1 confirming960 cm− , confirming the biological the biologicalWH [29,36,52]. WH [29,36,52].

Figure 7. RamanRaman spectrum of WH-6 product.

In order to study the degradation behavior/phase behavior/phase purity purity of of WH-6, WH-6, its its TGA TGA was was carried carried out. out. The TGA of of WH-6 WH-6 product product (Figure (Figure 8)8) showed showed a a cont continuousinuous weight weight loss loss till till 550 550 °C ◦whichC which could could be duebe due to the to adsorbed the adsorbed water water in crystal in crystal lattice latticeof solid. of Furthermore, solid. Furthermore, a slight tran a slightsition transition is also observed is also 2 afterobserved 550 °C after which 550 could◦C which be due could to breakage be due to of breakage Mg-HPO of42− Mg-HPObond. A 4sudden− bond. weight A sudden loss has weight been observedloss has been after observed 700 °C to after 1000 700 °C◦ Cwhich to 1000 represents◦C which the represents conversion the conversionof WH into of β WH-TCP into andβ other-TCP magnesiumand other magnesium phosphates. phosphates. Previous research Previous work research show worked that showed TCP becomes that TCP stable becomes up to stable 1400 °C up by to 1400the addition◦C by the of addition magnesium of magnesium in the crystal in the lattice crystal and lattice results and in results the formation in the formation of TCMP of and TCMP highly and stablehighly β stable-TCP [22,25].β-TCP [However,22,25]. However, our synthesized our synthesized WH-6 sample WH-6 degraded sample degraded at 1000 °C at which 1000 ◦ confirmsC which thatconﬁrms magnesium that magnesium is subsumed is subsumed completely completely into the Ca intoP crystal the CaP lattice crystal at C5 lattice and at C4 C5 position, and C4 position,forming (Caforming18Mg2 (Ca(HPO18Mg4)2(PO2(HPO4)12)4 rhombohedral)2(PO4)12) rhombohedral structure. structure.Since ionic Since radii ionic of magnesium radii of magnesium ions are ionssmaller are thansmaller calcium, than calcium, crystalline crystalline stability stability at a higher at a highertemperature temperature is decreased. is decreased. Moreover, Moreover, the weight the weight loss 2 afterloss after700 °C 700 could◦C could be attributed be attributed to the to change the change of HPO of HPO42− into4 − intoP2O7 P and2O7 POand4 and PO4 aand rapid a rapid weight weight loss afterloss after900 °C 900 could◦C could be associated be associated with witha unique a unique phenomenon phenomenon of WH of WHsince since it converts it converts into intoTCMP TCMP and βand-TCPβ-TCP after 700 after °C 700 [20,53,54].◦C[20,53 The,54 ].TGA The results TGA of results WH-6 of matches WH-6 matchesexactly with exactly the previous with the literature previous [18,45,46].literature [ 18,45,46]. Finally, in order to visualize the surface morphology as well as particle size of the WH phase, scanning electron microscopic images of the prepared WH-6 product were obtained. Figure9 shows the growth of rhombohedral microspheres with nanoparticles grown on their surfaces, similar to the bone nodules reported in the literature [11,18,36,42]. Nanomaterials 2020, 10, x FOR PEER REVIEW 10 of 14

Nanomaterials 2020, 10, 1856 10 of 14 Nanomaterials 2020, 10, x FOR PEER REVIEW 10 of 14

Figure 8. TGA graph of WH-6 product.

Finally, in order to visualize the surface morphology as well as particle size of the WH phase, scanning electron microscopic images of the prepared WH-6 product were obtained. Figure 9 shows the growth of rhombohedral microspheres with nanoparticles grown on their surfaces, similar to the bone nodules reported in theFigure literature 8. TGA [11,18,36,42]. graph of WH-6 product.

Figure 9. SEM images of WH-6 product with rhombohedral crystals. Figure 9. SEM images of WH-6 product with rhombohedral crystals. Finally, in order to investigate the reproducibility of the precipitation process to produce pure WH under the optimized conditions, reaction was repeated with larger quantities of the precursors. In this case, initially mixed phases of WH and CaPs appeared, which after annealing. for 6 h at 750 ◦C

Figure 9. SEM images of WH-6 product with rhombohedral crystals. Nanomaterials 2020, 10, 1856 11 of 14 resulted into pure WH phase, conﬁrmed again through the characterization techniques available. Moreover, in a single reaction with a ﬁnal reaction volume of 500 mL, we could achieve 25 g of the pure WH. Obtained product have clearly demonstrated the proof of concept for the bulk preparation of pure WH under the optimized conditions. Preparation of pure WH in grams quantities have clearly demonstrated its potential for commercial applications where HA is currently being explored.

4. Conclusions The optimum conditions for precipitation of pure biological WH phase are reported. WH is second abundant inorganic phase of bone having properties superior then that of hydroxyapatite (HA) and β-tricalcium phosphate (TCP). Synthesis of WH phase is not much explored and WH synthesis remained a challenge due to narrow range of parameters for WH stability. From our given data, it is concluded that there are several parameters which influence simultaneously on pure WH phase precipitation. pH is the most important parameter along with temperature and aging. Without aging no WH phase appears making it second most important parameter for WH precipitation. Other parameters such as feed rate, temperature and heating time also influence on the separation of pure phase. Slow addition and short time heating are favorable since magnesium replaces calcium in WH phase and the crystal structure produced is slightly distorted which probably could not maintain stability over long heating periods as well as slow addition favors magnesium to substitute calcium site completely. This work has great scientific importance since grams quantities of the pure WH phase can be produced under the optimized conditions and such material could help to imitate the exact inorganic phase of the bone containing all the essential minerals.

Author Contributions: Conceptualization, Z.H. and U.L.; methodology, S.B.; investigation, S.B.; data curation, S.B. and M.S.; resources, Z.H., U.L. and M.S.; visualization, Z.H. and U.L.; writing,—original draft preparation, S.B.; writing—review and editing, M.S.; supervision and project administration, Z.H. and U.L.; All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: This research was conducted at the School of Chemical and Materials Engineering (SCME), National University of Sciences and Technology, Sector H-12, Islamabad. ZH acknowledges the financial and administrative support from the SCME, NUST. ZH also acknowledges the support for SEM analysis of one of the samples provided by Irshad Hussain, Department of Chemistry and Chemical Engineering, LUMS, Lahore, Pakistan as well as for the Raman spectroscopic analysis of one sample by M. Aftab Akram, SCME-NUST, Pakistan. Conflicts of Interest: The authors declare no conflict of interest.

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