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[Masanori KIKUCHI](https://orcid.org/0000-0002-9451-8147)

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[Developments of calcium phosphate-based bone regenerating materials utilizing interfacial interactions between inorganic–organic substances](https://mdr.nims.go.jp/datasets/9a769fd7-2412-47bf-9758-40e7dafece5e)

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Developments of calcium phosphate-based bone regenerating materials utilizing interfacial interactions between inorganic–organic substancesSPECIAL ARTICLEThe 74th CerSJ Awards for Academic Achievements in Ceramic Science and Technology: ReviewDevelopments of calcium phosphate-based bone regenerating materialsutilizing interfacial interactions between inorganic­organic substancesMasanori KIKUCHI1,³1Bioceramics Group, National Institute for Materials Science, 1–1 Namiki, Tsukuba, Ibaraki 305–0044, JapanCalcium phosphate-based bone regenerating materials were developed by utilizing interfacial interactionbetween inorganic­organic substances. Although apatitic calcium phosphates, hydroxyapatite (HAp) and ¢-tricalcium phosphate (¢-TCP), have unique affinity to organic substances, and utilization of the affinity requiresappropriate surrounding conditions. The author and his colleagues control the surrounding conditions to realizeporous HAp ceramics with high porosity, interconnectivity and compressive strength, composite membrane of ¢-TCP and polylactide-based biodegradable polymers for guided bone regeneration, and bone-like nanocompositeof HAp and type-I atelocollagen (HAp/Col). Electrostatic interactions between calcium phosphates andpolymers in these composites were presented by reflection infrared spectra. They also examined in vitro andin vivo and demonstrate good bone regeneration properties. Particularly, the HAp/Col exhibits completelyincorporation into bone remodeling process that is the first in the world for synthetic materials. Three of thesematerials are also commercialized and used in medical and dental fields and contribute to human health.©2020 The Ceramic Society of Japan. All rights reserved.Key-words : Calcium phosphates, Interfacial interaction, Binder, Organic polymer, Bone tissue reaction[Received April 24, 2020; Accepted May 25, 2020]1. IntroductionApatitic calcium phosphates, hydroxyapatite [Ca10-(PO4)6(OH)2, HAp] and tricalcium phosphate, [Ca3(PO4)2,TCP] of ¡- and ¢-phases, are widely used as bone voidfillers due to their excellent biocompatibility, so-calledbioactivity, a property to bond directly with bone. Further,these calcium phosphates have a unique property to adsorbbiomolecules, nucleic acids, proteins, amino acids, sac-charides and lipids. This property have been utilized asadsorbent of liquid chromatography and toothpaste toremove saccharides and other organic molecules fromtooth surface effectively. However, not so many reportswere found in utilization the affinity, in other words, inter-facial interaction between organic substances and apatiticcalcium phosphates to develop new materials.In this review, research and development of three newbone regenerating materials by utilizing the interfacialinteractions are described.2. Porous HAp ceramicsIn the 20th century, artificial bone void fillers composedof HAp have low porosity up to 65% due to the loss ofmechanical strength by increasing their porosity. In fact,orthopedic surgeons frequently push the bone void fillersinto bone defect; thus, weak materials are not preferredby them. Many researchers tried to increase porosity tomore than 75% without losing mechanical strength but didnot succeed. The authors focused on the following twopoints to strengthen a porous body: 1. Good sinterability toachieve high mechanical strength between granules aswell as low pores (crucks) formation between granules,2. Ignoring cruck formation during burning of porogens.The second requirement denied using polymeric porogenwhich inflates during burning process, and the expansionstretches pore wall to create crucks. Accordingly, foamcasting is chosen for the preparation. The first requirementis divided into two parts. One is starting HAp powder andthe other is a binder molecule. From the author’s experi-ences, HAp prepared by a wet method using own-madeCa(OH)2 from highly pure CaCO3 (e.g., Alkaline analysisgrade, Wako Pure chemical, Osaka, Japan) and H3PO4(e.g., Reagent grade, Wako Pure chemical, Osaka, Japan)as starting materials shows good sinterability more than99.5% of relative density when appropriate organic binder(e.g., polyvinyl alcohol) is used. Therefore, finding appro-priate binder to prepare foam and crosslinker to fix aporous structure is important issue for fabricate novelporous bone void filler having 75% porosity with fullyinterconnected pores and the same compressive strength asthe conventional bone void fillers, approximately 10MPa.³ Corresponding author: M. Kikuchi; E-mail: KIKUCHI.Masanori@nims.go.jpJournal of the Ceramic Society of Japan 128 [8] 547-554 2020DOI http://doi.org/10.2109/jcersj2.20093 JCS-Japan©2020 The Ceramic Society of Japan 547This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by-nd/4.0/),which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.http://doi.org/10.2109/jcersj2.20093https://creativecommons.org/licenses/by-nd/4.0/This work was performed in collaboration with ToshibaCeramics Co. ltd (at that time, CoorsTek Inc. in present)that have good procedure to fabricate foam-casting ofalumina ceramics. After the transfer of the HAp symthesismethod from the author, they still faced a problem insinterability of HAp by using their conventional binderand crosslinker. The author hypothesized that the problemcould be attributed to the interaction between a binder andthe HAp surface. Generally, the HAp synthesized by ourmethod and calcined at 800 °C has a negative surfacecharge. Thus, a binder with positive functional group, suchas amino and imino groups, could interact to the HApsurface and could play a role of good binder. A binder andcrosslinker chosen by the hypothesis worked as expectedand the authors succeeds to prepare HAp porous ceramicswith 75% porosity, high compressive strength and highinterconnected porous structure.1) This porous HAp wasthen moved to animal experiments and clinical trial collab-orated with Department of Orthopedic Surgery of OsakaUniversity Medical School, National Institute of AdvancedIndustrial Science and Technology and MMT Co. Ltd. (atthat time, Aimedic MMT Co. Ltd. in present). The porousHAp was then commercialized as Neoboneμ in 2003.Similar approach was also applied to fabricate uniaxialporous body of HAp using ice crystal growth in HApcontaining hydrogel, which was sold as Regenosμ.2)3. Composite with syntheticbiodegradable polymersAlthough biocomaptibility and bioactivity of apatiticcalcium phosphates are excellent in comparison to othermaterials, a high elastic modulus, cause of stress shielding(reason of inhibition of bone maturation), and brittleness,cause of fracture, are still problems on practical use.Ceramics with higher porosity, as mentioned in the section2, reduce those risks but remaining of ceramics with differ-ent mechanical properties to bone have a potential risk offractures in the materials and/or on the interface betweenbone and ceramics. The author considered that this prob-lem can be solved by the following two ways. One is usinga inorganic/organic composite that has a similar mechani-cal properties, and the other is using a biodegradable mate-rial that degrades with formation (regeneration) of novelbone tissue; however, both ways have respective prob-lems. Big problem on the former one is fatigue of materialwhich reduce mechanical properties and increase a riskof fracture. In fact, HAPEXμ, a composite of HAp andpolyethylene, developed by Bonfiled and his colleaguesdemonstrated bone-like mechanical properties and bio-activity3) but is only applied to non-load-bearing site4) toreduce the risk of fracture caused by the material. As forthe latter one, the most famous biodegradable ceramics atthat time in Japan is Osferionμ, the first commercialized ¢-TCP bone void filler in Japan, from Olympus Corp. (at thattime, Olympus Terumo Biomaterials Corp. in present). Itgenerally needs about a year for complete degradation inpractice; therefore, it may remain some risk of fractureafter removing fixator.Therefore, many researchers tried to prepare inorganic/organic composite with biodegradability. At that date,many researchers had worked on a combination of syn-thetic biodegradable polymer and HAp, but degradationbehavior of HAp in those kinds of composite is still un-clear. On the other hand, poly ¡ hydroxy acids representedby poly-L-lactic acid degrades by hydrolysis reaction atcarbonyl group on its chain, and its degradation ratio isaccelerated by humidity, temperature, basic and acidicconditions. This means that degradation of polymer is self-accelerated by acid from degraded polymer, especially forthe polymer of which decomposition products are lowmolecular weight, e.g., glycolic acid and lactic acid. Inaddition to that, acidic conditions induced by polymerdegradation will cause of inflammation of surroundingtissues. Including this problem, poly ¡ hydroxy acid hasthe following problems when it used for bone void filler,no osteoconductivity and low elastic modulus. The lattertwo issues had already considered by researchers studiedon composites using HAp, acid problem had been littlefocused, at least no descriptions in any paper at that time.The author considered all above problems and focusedon combination of synthetic biodegradable polymer and ¢-TCP, obvious biodegradable ceramics. That is to say, ¢-TCP “ceramics” increases elastic modulus, ¢-TCP “bio-active (a material directly bond with bone)” ceramics sup-plements osteoconductivity (bone grows on the surface ofa material) and ¢-TCP “biodegradable” bioactive ceramicscompensates acidification, because ¢-TCP is a substancecomposed of weak acid and strong base. As syntheticpolymers, the author had chosen two type of poly-L-lactide based copolymers to control mechanical propertiesand degradation rate. One is copolymerized poly-L-lactidewith sebacic acid (CPLA) and the other is poly-L-lactide-co-glycolide-co-¾-caprolactone (PLGC). Essentially, thesetwo polymers demonstrate similar results in physicochem-ical and biological properties of composites.General preparation method for polymer/ceramics com-posite is a mixing of ceramics with a polymer dissolved innonpolar solvent and volatilize solvent after casting. Thisis of course good method to reduce a risk of decom-position of poly ¡ hydroxy acid. However, interactionbetween “polar” ceramics and “nonpolar” polymer is diffi-cult to form by this method, even though HAp and ¢-TCPhave a good affinity to some organic substances includinglipids. In fact, gap is formed between ¢-TCP particles andthe above polymers by the solvent mixing method.5) Thisproblem is solved by surface treatment of ceramics toimprove affinity to nonpolar substances in structural com-posite, carbon and glass fiber reinforced plastic, butsupplementation of the third substance should avoid aspossible if the material will be used as a biomaterial.Therefore, the author changed the preparation way to aheat-kneading which mix ¢-TCP with molten poly ¡hydroxy acids melt at higher than melting points undervacuum.5),6) High temperature may decompose polymerbut decomposition rate will be reduce by avoiding waterin air and on ceramics surface. After the preparation,Kikuchi: Developments of calcium phosphate-based bone regenerating materials utilizing interfacial interactions between inorganic–organic substancesJCS-Japan548obtained composites were evaluated physicochemical andbiological properties ex and in vivo.Three-point flexural strengths of the for TCP/CPLAcomposites demonstrate in the range of 40­50MPa at dif-ferent mixing ratios as shown in Fig. 1.6) These values areslight weaker than pure CPLA. Mass mixing ratio of 75%of ¢-TCP is very near to equal volume ratio between ¢-TCP and CPLA (precise mass ratio for that is 72­3%) andshows the highest strength in trend, and as a comparison, aresult of HAp/CPLA at the same mixing ratio and thesame preparation method also is indicated in Fig. 1. Inter-estingly, three-point flexural strength of the HAp/CPLAdrastically decreases to 70% in comparison to the TCP/CPLA, because the weight average molecular weight ofCPLA for the HAp/CPLA was much decreased from 150to 60.3 kDa than that of the TCP/CPLA of 110 kDa. Thereason for the difference is considered to be the presence ofhydroxyl groups in HAp, which would acted as a catalystfor polymer degradation during kneading at high tem-perature and promoted polymer degradation even undervacuum.6) The TCP/CPLA and TCP/PLGC compositesdemonstrate good thermoplastic property above glass tran-sition temperature of CPLAs6) and PLGCs.5) Further, scan-ning electron micrograph of fracture surface of the TCP/CPLA shown in Fig. 2 exhibits homogeneous dispersionof ¢-TCP particles and good wettability between ¢-TCPparticles and CPLA6) or PLGC,5) which suggests a pres-ence of physicochemical interaction between them.The physicochemical interaction between ¢-TCP andCPLA was investigated by a reflection method usingFourier-transformed infrared (FTIR) spectrometer at 60,75 and 85° of reflection angle. Mixing ratios of the ¢-TCPand CPLAwere 50, 75, 85 and 90%. The reflectance spec-tra of FTIR were converted to absorbance spectra by theKramers-Kronig method. The reflectance of particle fillertype composite mirrors multiple reflection of interfacebetween particle fillers and polymer. The energy for inter-action has to be supplied from both substances; therefore,presence of physicochemical interaction is indicated as ared shift of absorption band of functional group in polymerreflecting energy consumption for the physicochemicalinteraction. The results summarized in Fig. 3 illustratestretching band of carbonyl group in the CPLA observedaround wave number of 1770 cm¹1. Spectra of lower reflec-tion angle, 60°, indicate no significant red shift due to smallmultiple reflection numbers; however, spectra of higherangles, 75 and 85°, depict red shift of stretching band ofcarbonyl group, including splitting in two bands in somecase.7) Unfortunately, this interaction which improvesmechanical strength and thermoplasticity could becomedisadvantage in bioactivity, i.e., osteoconductivity, because¢-TCP particles are mostly covered with the CPLA.Changes in pH of physiological saline and phosphatebuffered saline in which the composite was soaked demon-strate similar results.5),8) As expected at material design,pH maintains neutral during polymer decomposition.No cytotoxic signs are found in cell culture tests of thecomposites.9) After a preliminary animal experiment, sig-nificantly lower osteoconductivity than HAp and ¢-TCP isconfirmed, even though the composite presents good apa-tite forming ability in simulated body fluid than HAp andFig. 1. Changes in three-point flexural strength as a function ofmixing ratio of ¢-TCP in composite. Fig. 2. Fracture surface of TCP/CPLA composite.Fig. 3. Reflection FTIR spectra of TCP/CPLA composite.Journal of the Ceramic Society of Japan 128 [8] 547-554 2020 JCS-Japan549¢-TCP.10) Therefore, in vivo tests using dogs to apply thecomposite membrane for guided bone regeneration (GBR)were performed. A GBR is one of guided tissue regen-erations. Small tissue defect of human is generally filledwith regenerated original tissue; however, defect sizeexcesses tissue-dependent limit of regeneration, most ofthe defect is filled with scar tissue. This is a protectivereaction of human body performed by invasion of fibro-blasts, collagen fiber producing cells. Therefore, inhibitionof fibroblasts migration by membrane supports originaltissue reconstruction.The authors tried to regenerate large bone defect onmandible and tibia of beagles. Buccal-lingual defects of10 © 10 © 10mm3, i.e., whole bone removal in the direc-tion, were created on both sides of the alveolar bone of themandible. One side was GBR treated by covering defectwith a TCP/CPLA membrane fitted to the surroundingalveolar shape by warming in physiological saline of 50 °Cand the other side was a negative control, i.e., creation ofdefect without the GBR treatment, and both sides wereclosed by suturing the gingiva. Full segmental tibial defectof 20mm in length was created after an application ofexternal fixator and was GBR treated by the similar pro-cedure to mandible. Other dog was used for negative con-trols, with the GBR treatment with the CPLA or withoutthe GBR treatment of the same-sized tibial defect. All theanimal tests described in the present review paper are con-formed with “The Guidelines of Tokyo Medical and DentalUniversity for Animal Care” based on “The NIH Guide-lines for the Care and Use of Laboratory Animals” (NIHPublication #85­23 Rev. 1985). All treatments were per-formed by veterinarians, whereas animal maintenance wasperformed by veterinarians and animal health technicians.In mandible defects, the experimental group exhibitsquite good regeneration that at least 90% of bone height isrecovered at the center of defect after 12 weeks postopera-tion. Further, no sign of remaining of TCP/CPLA mem-brane is observed. Tibial defect GBR model also demon-strates good results that complete bridging between distaland proximal edges is observed with remaining of mem-brane at 12 weeks after operation; whereas GBR treatmentwith pure CPLA membrane and no GBR treatment do notachieve bone bridging at all.8) The similar results are foundfor GBR treatment with the TCP/PLGC membrane.5)4. Bone-like nanocomposite4.1 Composite toward real boneVertebrates bone is a typical nanocomposite composedof approximately same volume of calcium deficientcarbonate-containing HAp as an inorganic substance andtype-I atelocollagen (Col, collagen that antigenic telope-pitdes are enzymatically removed) as a dominant organicsubstance. In addition to that, bone has a nanostructure inwhich c-axes of HAp nanocrystals aligns elongation direc-tion of Col fibers, the same as Col long axis. Therefore,these main components, HAp and Col are of course maintarget to fabricate composite for bone void fillers as well aselucidating bone formation mechanism for biomimeticsynthesis of materials. For instance, Du et al., implantedmixture of nano-HAp and Col in marrow cavity.11) Further,so-called “biomimetic” approaches are also performed bymany researchers, i.e., HAp nanocrystals are formed onprefabricated Col fibers in sponges or membranes com-posed of Col. However, they have no bone-like nanostruc-tures and demonstrates no similar bone tissue response toautologous bone transplantation, incorporating into boneremodeling process. The bone remodeling process is alsoobserved in both remodeling old bone into new bone andbone maturation at bone fracture repair, and is osteoclastic“old” and/or “broken” bone resorption followed by osteo-blastic new bone formation. The osteoclastic bone resorp-tion is based on the following pure chemical reactions:acidification of a resorption site by supplying proton todissolve HAp nanocrystals in bone and decomposition ofCol by supplying enzymes. Bone formation by osteoblastsis also sequential chemical reactions: 1. Secretion of Col asprotocollagen, 2. Fibrillogenesis of Col and 3. Depositionof calcium phosphate (HAp or its precursors). On the con-trary, ex vivo the “biomimetic” precipitation of HAp onCol fiber only showed limited formation of HAp in Colfibers and limited orientation of HAp nanocrystals on Colfibers when HAp mass ratio in composites becomes closeto that in bone. In 2007, improved biomimetic calcificationprocess, polymer-induced liquid precursor (PILP)” wasproposed by Gower’s group who works on biomimeticcalcification processes of hard tissues, i.e., Olszta et al.,fabricated bone-like composite of HAp and Col by precip-itation of HAp in and on Col fibers utilizing polylysine asa liquid precursor.12) The method is good to elucidatebiomimetic process but not suit for mass production ofbone void fillers.4.2 Simultaneous titration method to fabri-cate bone-like nanocompositePrior to Olszta’s work, the author and his colleaguesreported a synthesis method and bone tissue reaction ofbone-like nanocomposite of HAp and Col (HAp/Col)having bone-like nanostructure and chemical compositionin 2001.13) The author has not considered that osteoblastsalign HAp nanocrystals and Col directly by their tentacles.In fact, size relation between HAp nanocrystals in bone,20­40 nm in long direction, and cell tentacles is like rela-tion of that between sand particles and human finger.Obviously, we cannot align sand particles in one crystallo-graphic direction along sewing threads by our fingers;hence, cells must not be able align HAp nanocrystals andCol fibers by their tentacles, either. Nevertheless, HAp andCol are align each other in bone, supplying raw materialsand controlling surroundin condition osteoblasts’ role isenough to realize bone-like nanostructure. Therefore, theauthors proposed the simultaneous titration method, a verysimple system to mimic osteoblast roles. Briefly, raw mate-rials, calcium hydroxide suspension and orthophosphoricacid solution containing Col, are simultaneously titratedthrough each tube pump into a reaction vessel in whichthe same amount of pure water as calcium hydroxideKikuchi: Developments of calcium phosphate-based bone regenerating materials utilizing interfacial interactions between inorganic–organic substancesJCS-Japan550suspension is previously added to measure reaction pH andmaintain reaction temperature from start of the reaction.Reaction temperature is controlled and maintained with theouter water bath. Reaction solution pH is measured withthe pH meter equipped with electricity outlet to turn onand off power of tube pumps to control pH of reactionsolution.After several synthesis conditions were examined, pH 9and 40 °C of outer water bath temperature (reaction solu-tion temperature of approximately 37 °C) were found tobe the best conditions to form the HAp/Col composites.Typical transmission electron micrograph and selected areaelectron diffraction are shown in Fig. 4. Although thealignment of HAp nanocrystals are not complete, this levelof alignment is observed in immature bone. Consequently,the authors considered that the HAp/Col has bone-likenanostructure. Chemical compositions of the HAp/Col areeasily controlled by raw material ratios, i.e., calcium, phos-phate and collagen ratios.4.3 Influence of concentration of rawmaterials14)Generally, concentration in a reaction vessel controlssize of crystals formed in it. The author hypothesized thatthis analogue can be applied to the HAp/Col formation.The same amount of HAp/Col was synthesized under dif-ferent starting material concentration. The synthesis con-ditions are summarized in Table 1. Obtained quantity ofthe HAp/Col was 10.05 g, and HAp/Col mass ratio was4:1. As described above, water amount in the reactionvessel differed according to calcium hydroxide suspensionamount; therefore, raw material concentration in the reac-tion vessel is automatically changed in this system.Photographs of the HAp/Col fibers obtained from dif-ferent conditions are shown in Fig. 5. As the hypothesis,fibers, more precisely fibrous aggregates of the HAp/Colnanofibers, grow with decreasing in concentration in thereaction solution. Changes in calcium ion concentrationmeasured with calcium ion selective electrode during theHAp/Col synthesis at 50, 100 and 200mM of starting Caconcentration are illustrated in Fig. 6. Starting Ca con-centration of 100mM without adding collagen is measuredas a control. As seen in Fig. 6, Ca concentrations duringpseudo-plateau area, e.g., 400­1400 s, are very similar, butfluctuations of Ca ion concentration are quite different.Considering the standard deviation as an index of fluctua-tion, results are summarized in Table 2. Starting concen-trations that allow long fiber growth, 50 and 100mM,demonstrate very small fluctuations, instead standard devi-ation for short fiber grown conditions, 200mM, shows halfof the reaction concentration. Further, starting concentra-tion of 100mM without Col demonstrates higher fluctua-tion than that with Col. This result suggests Col influenceson HAp formation on its surface.4.4 Mechanism for nanostructure formationAs the same as composite with the synthetic biodegrad-able polymers, the HAp/Col demonstrates an electrostaticinteraction between Ca2+ on HAp and C-O 1.5 fold bondFig. 4. Transmission electron micrograph of HAp/Col withselected area electron diffraction pattern.Table 1. Synthesis conditions for HAp/Col for examination ofinfluence of concentrationCa(OH)2Conc./mM 50 100 200 400Quant./cm3 1600 800 400 200H3PO4Conc./mM 15 30 60 120Quant./cm3 3200 1600 800 400Collagen/g 2.01H2O Quant./cm3 1600 800 400 200Fig. 5. HAp/Col fiber synthesized from different startingmaterial concentrations.Fig. 6. Changes in calcium ion concentration during the HAp/Col synthesis.Journal of the Ceramic Society of Japan 128 [8] 547-554 2020 JCS-Japan551of carboxyl group on Col in reflection FTIR.13) Sato et al.reported that HAp is formed onto on Langmuir­Blodgett(LB) monolayer terminated by carboxyl group but not onthat terminated by amino group.15) This results suggestcarboxyl group on the Col also plays a role of nucleationcenter for HAp nanocrystal. They also revealed that HApcrystals close to substrate LB monolayer terminated by thecarboxyl groups demonstrate c-axis orientation along withcarboxyl group alignment,16) i.e., aligned carboxyl groupsinteract with Ca2+ on HAp crystals and align HAp crystalsin one direction.Carboxyl groups on Col molecule of course havealignment almost the same direction to molecule itself;therefore, HAp crystals mostly align along Col moleculesin the HAp/Col. This phenomenon suggests that HApnanocrystals in bone also grow epitaxially from solution.This means that even though precursor model, calciumhydrogen phosphate dihydrate or octacalcium phosphatewas formed before HAp formation in bone, is correct,dissolution-reprecipitation process is necessary to growHAp epitaxially as seen in bone.4.5 Fabrication of biomaterialsThe HAp/Col is a fibrous nanocomposite, and simplelyophilization of the HAp/Col gives a cotton-like softmaterial, therefore needs to fabricate shapes to suit forbiomedical use. The first shape is dense body13) which isshaped by compression squeezing of water from the HAp/Col fiber in a specially designed mold. The dense body hasa maximum 3-point flexural strength of approx. 40MPaand does not have enough strength for bone void fillerswithout a fixator. The second shape is a porous body usingice crystal grown from collagen hydrogel containing theHAp/Col as porogens.17) The HAp/Col porous body(sponge) is a rigid in dry condition; however, it demon-strates sponge-like viscoelasticity in wet condition, a prac-tical use condition, as exhibits in Fig. 7. The third one ispaper-like membrane with a translucency.18) After that,injectable self-setting paste with anti-decay property19) andcoating on metal20) are also fabricated.4.6 Influences on cells in vitroOsteoblastic cell line, MG63 derived from human osteo-sarcoma, was used for cytocompatibility and influences onosteoegenic cell functions of the HAp/Col paper-likemembrane18) and sponge.21) The cells are well proliferatedon the HAp/Col membrane as the same as the tissue cul-ture polystyrene (TCPS) and demonstrate no difference inshape in optical microspcopic observation after Giemsastaining. Alkaline phosphatase (AlkP), an early-stagemarker of osteogenesis, gene expression of MG63 culturedon the HAp/Col membrane shows more than three timeshigher than that on the TCPS both with and withoutaddition of osteogenic supplements, 50¯g/ml L-ascorbicacid phosphate magnesium salt and 10 nM ¢-glycerophos-phate.18) Pressure/perfusion culture was performed for theHAp/Col sponge using a Col sponge as a control.21) Initialcell proliferation on the HAp/Col porous body indicatesthe similar number to that of the Col sponge, but later cellnumbers on the HAp/Col sponge are greater than those onthe Col sponge. The Col sponge has a high affinity to cellsand proliferates cells very well; however, exchanges ofmedium from inside of sponge to outside and vise-versaare limited due to its small pore size, and cells inside thesponge move out to peripheral of the Col sponge. Con-trarily, the HAp/Col sponge has larger pore size, and cellsmigrate into inside of the HAp/Col sponge. This is thereason why the HAp/Col demonstrates higher prolifer-ation at latter periods. Gene expression of AlkP is greaterfor cells on the Col sponge at 7, 10 and 14 days after incu-bation, because AlkP is upregulated when the cell densityis high and it is higher on the peripheral of the Col spongethan the HAp/Col sponge. Osteocalcin, the latter-stagemarker, gene expression of cells on the HAp/Col and Colsponge showed no significant differences. However, cellsinside of the HAp/Col sponge only form bone nodules at21 days after incubation. From these results, the HAp/Colimproves osteogenic activity to cells in vitro.Osteoclastic differentiation of bone marrow cells on theHAp/Col was examined by co-culture of primary osteo-blasts and bone marrow cells isolated from C57BL/6mice22) following to Irie’s paper.23) Bone marrow cells inthe method differentiate through cytokines secreted fromosteoblasts upregulated by osteoclast differentiation sup-plements, 10 nM of 1,25-Dihydroxyvitamin D3 and 1¯Mof prostaglandin E2. Bone marrow cells cultured on con-trol groups, dentin, glutaraldehyde-crosslinked dentin, sin-tered HAp and TCPS differentiate after addition of thesupplements and dose not differentiate without addition ofthem, except for slight differentiation observed for cellson dentin. The HAp/Col demonstrated osteoclastic dif-ferentiation of bone marrow cells with or without additionof the supplements. Even after gene expression analysis,the author and his colleague cannot specify the HAp/Colrole to the osteoclastic differentiation of bone marrowcells. Possible roles of the HAp/Col are: 1. Absorption ofosteoprotegerin, OPG, an inhibitor of receptor activator ofNF-jB ligand, RANKL, which bind to its receptor, RANK,Table 2. Pseudo-plateau concentration (average) and standarddeviation as an index of fluctuationStarting Ca concentration/mM 50 100 200 100 w/o ColConcentration/ppm 7.69 10.2 13.0 11.4Standard deviation 0.58 0.75 6.50 1.88Fig. 7. Sponge-like viscoelasticity of the wet HAp/Col sponge.Kikuchi: Developments of calcium phosphate-based bone regenerating materials utilizing interfacial interactions between inorganic–organic substancesJCS-Japan552of osteoclasts and their precursors to regulate osteoblasticdifferentiation and activation of osteoclasts, and/or 2.Upregulation of osteoclast-associated immunoglobulin-like receptor, OSCAR, which is considered as an initiatorof osteoclast differentiation, by specific collagen confor-mation in the HAp/Col.4.7 Bone tissue reactions13),19),20),24)–33)The HAp/Col dense body with a several holes paral-lel and perpendicular to distal-proximal direction wasimplanted into beagle’s tibial full segmental defect of 2 cmwith external fixation.13) At 3 months after implantation,the HAp/Col is almost disappeared. Hematoxylin-eosin,AlkP and tatrate-resistant acid phosphatase (TRAP, amarker of osteoclast) stained sections demonstrated thatthe disappearance of the HAp/Col is resorption by osteo-clasts followed by new bone formation by osteoblasts, i.e.,bone remodeling process. Actually, body fluid is super-saturated to HAp and cannot dissolve HAp, not like ¢-TCP, thus, HAp has to be decomposed biochemically, e.g.,resorbed by osteoclasts or phagocytosed by macrophages.Hydroxyapatite particles in generally used HAp ceramicsis too large to be decomposed completely by these bio-chemical reactions. This is the reason why HAp ceramicsis practically non-biodegradable. Contrarily, HAp in theHAp/Col is a nanocrystal and can be decomposed bio-chemically; accordingly, the HAp/Col become the worldfirst synthetic material that completely incorporated intobone remodeling process to substitute with new bone. Theauthors also revealed that resorption rate is easily con-trolled by crosslinking to Col in the HAp/Col.29) Consid-ering cell and tissue penetration, the HAp/Col sponge waschosen for practical use as a commercialized product. In2013, the HAp/Col sponge, ReFitμ, was start to be soldfrom HOYA Technosurgical Co. The HAp/Col spongedemonstrates excellent bone substitution property in bonesin its clinical trial34) and practical use. Further, earlier boneformation of the HAp/Col for anterior cervical discectomyand fusion using Ti cage than ¢-TCP is also reported.35)Recently, the authors found that Ti dip-coated with theHAp/Col implanted into subperiosteum of rat craniuminduces trice faster osseointegration (gaps between mate-rial and bone are not observed with naked-eye and opticalmicroscope but observed with electron microscope) thanpure and biomimetic HAp coated Ti.20) The detailedmechanism is under investigation but it will be applied todental and medical prostheses.The authors, as mentioned above, developed injectableself-setting HAp/Col paste with anti-decay property using(3-glycidoxypropyl)trimethoxysilane.19) The HAp/Colpaste shows no local and systemic toxic effect includingirregular inflammation after direct injection to tibial hole ofpigs. At 3 month postimplantation, the HAp/Col paste iscompletely substituted with newly formed bone.5. SummaryThe author has focused on interfacial interactionbetween inorganic and organic substances to fabricatebone regenerating materials based on calcium phosphateceramics in collaboration with many colleagues in materialscience, biochemical, veterinary, medical and dental fields.This concept allows high interconnected porous and poros-ity ceramics with sufficient mechanical strength for bonevoid fillers, thermoplastic and biodegradable compositewith sufficient mechanical properties for GBR treatment,and bone-like nanocomposite of HAp and Col which iscompletely incorporated into bone remodeling process.The HAp/Col demonstrates many potentials to use forbone void filler in porous and injectable paste, coating onmetal to accelerate osseointegration. These researches anddeveloped materials contribute to human health.Acknowledgement The author expresses deep appreci-ate to co-authors in the referred papers, especially Prof.Emeritus Junzo Tanaka, Drs. Yasushi Suetsugu, Sun-BaekCho, Kimiyasu Sato, Ms. Ikuko Akahane in the same group inNIMS with the author at that time, and Profs. Hiroo Miyairi,Kenichi Shinomiya, Kazuo Takakuda, Keiji Moriyama,Soichiro Itoh, Yoshihisa Koyama, Shinichi Sotome,Masayoshi Uezono of Tokyo Medical and Dental University.The author also thanks to all students researched under myguidance. The studies in the paper were supported in part byCREST, JST and Research Promotion Bureau, Ministry ofEducation, Culture, Sports, Science and Technology, Japanunder the contract No.17-83. Lastly, the author dedicates thepaper to animals sacrificed for the researches described in.References1) M. Kikuchi, Y. Suetsugu, J. Tanaka, K. Imura, H.Umemoto, N. Ninowa, M. Kinoshita, A. Hojo and H.Yamazaki, “Calcium phosphate porous sintered bodyand production thereof”, JP3400740, US6,340,648,GB2348872A.2) Y. Suetsugu, Y. Hotta, M. Iwasashi, M. Sakane, M.Kikuchi, T. Ikoma, T. Higaki, N. Ochiai and J. Tanaka,Key Eng. 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Ae, A. Okawa, M. Ishizuki, H. Morioka,S. Matsumoto, T. Nakamura, S. Abe, Y. Beppu and K.Shinomiya, J. Orthop. Sci., 21, 373­380 (2016).35) N. Kikuchi, Y. Ohara, Y. Tomita, H. Matsuoka andJ. Mizuno, Spinal Surg., 31, 257­261 (2017).Masanori Kikuchi is a Group Leader of Bioceramics Group, National Institute forMaterials Science (NIMS) in Tsukuba, Japan from 2007. He received his B.E. (1990), M.E.(1992) and Ph.D. (1995) from Waseda University, Japan. He started his job in 1995 as aVisiting Researcher of Waseda University, then moved and has worked at National Institutefor Research in Inorganic Materials (NIRIM, presently NIMS) from 1995. He also workedon international standardization from 2004 and is currently a chairperson of the Japanesecommittee of ISO/TC 150 from 2020 and a convener of ISO/TC 150/SC 1/WG 3 from2011. He established the first international standard for tissue engineering medical prod-ucts, ISO 19090. He also has worked as a Visiting Professor of Hokkaido University from2011 and Professor of University of Tsukuba from 2016.Kikuchi: Developments of calcium phosphate-based bone regenerating materials utilizing interfacial interactions between inorganic–organic substancesJCS-Japan554