the biocompatibility of bone cements: progress in

The biocompatibility of bonecements: progress in methodological approachCarlo Dall’Oca,1 Tommaso Maluta,1Gian Mario Micheloni,1Matteo Cengarle,1Giampaolo Morbioli,2 Paolo Bernardi,3Andrea Sbarbati,3Daniele Degl’Innocenti,3 Franco Lavini,1Bruno Magnan1

1Department of Surgery, Orthopaedicand Traumatology Clinic, University ofVerona2CIRSAL - Interdepartmental Center ofScientific Research on LaboratoryAnimals, University of Verona3Department of Neuroscience,Biomedicine and Movement, HumanAnatomy and Histology Section,University of Verona, Italy

AbstractThe ideal bone graft substitute should

have certain properties and there are manystudies dealing with mixture of polymethyl-metacrilate (PMMA) and β-tricalci-umphospate (β-TCP) presenting the bestcharacteristics of both. Scanning ElectronMicroscopy (SEM), for ultra-structuraldata, resulted a very reliable in vivo modelto better understand the bioactivity of acement and to properly evaluate its suitabil-ity for a particular purpose. The presentstudy aims to further improve the knowl-edge on osteointegration development,using both parameters obtained with theEnvironmental Scanning ElectronMicroscopy (ESEM) and focused histologi-cal examination. Two hybrid bone graftsubstitute were designed among ceramicand polymer-based bone graft substitutes.Based on β-TCP granules sizes, they werecreated with theoretical different osteocon-ductive properties. An acrylic standardcement was chosen as control. Cementswere implanted in twelve New ZealandWhite (NZW) rabbits, which were sacri-ficed at 1, 2, 3, 6, 9 and 12 months aftercement implantation. Histological sampleswere prepared with an infiltration processof LR white resin and then specimens werestudied by X-rays, histology andEnvironmental Scanning ElectronMicroscopy (ESEM). Comparing the result-ing data, it was possible to follow osteointe-gration’s various developments resultingfrom different sizes of β-TCP granules. In

this paper, we show that this evaluationprocess, together with ESEM, provides fur-ther important information that allows tofollow any osteointegration at every stageof develop.

IntroductionMore than 500,000 bone grafting proce-

dures are performed yearly in the UnitedStates, to fill bone defects in orthopaedicsurgery, neurosurgery and dentistry.1,2 Bonegraft is the second most common transplan-tation tissue, with blood being by far thecommonest.3 Bone graft substitutes caneither replace autologous bone graft orexpand an existing amount of autologousbone graft. Autologous bone remains thegold standard for stimulating bone repairand regeneration. However there are severallimitations, especially if there is massivesegmental bone loss or a very large cancel-lous void.4 The ideal bone graft substituteshould be biocompatible, bioabsorbable,osteoconductive, osteoinductive, structural-ly similar to the bone, easy to use and costeffective.5,6 There are many studies dealingwith mixture of polymethylmetacrilate(PMMA) and β-tricalciumphospate (β-TCP) and presenting the best bone graftsubstitution characteristics of both.7-9 Infact, β-TCPs are reabsorbed through cell-mediated processes, including osteoclastactivity.10,11 As a consequence of this mech-anism resembling bone remodelling, β-TCPwill not disappear until new bone is beingformed.12 Therefore by mixing PMMA andβ-TCP, a bone void filler can be createdwith osteoconductive properties i.e. proper-ties of bioactivity.13,14 In this case bioactivi-ty, as the interaction between a biomaterialand the surrounding tissue, describes theinfluence of a material on bone forma-tion.13,15-17 To better understand the bioactiv-ity of a cement and to properly evaluate thesuitability of cements for a particular pur-pose, Dall’Oca et al.13 have demonstratedthe usefulness of the ultrastructural dataobtained by Scanning Electron Microscopy(SEM) and proposed a new procedure totest the bioactivity of acrilic cements in vivomodels. This approach improves the visual-ization of the bone-cement interface, addingnew data on the behavior of an importantcomponent of treatment.13 Porous cementsmade of β-TCP/PMMA could be consideredfor vertebroplastic or augmentationorthopaedics uses,14,18-20 but further studiesare necessary in order to evaluate the in vivoproperties of this material. The presentstudy aims to further improve the proposedprocedure, using parameters obtained with

the Environmental Scanning ElectronMicroscopy (ESEM).

Materials and Methods

Bone cementsA hybrid bone graft substitute was

designed between ceramic and polymer-based bone graft substitutes. It is made upof a mixture of PMMA and β-TCP to beosteoconductive, to facilitate osteointegra-tion of the bone void filler. By mixingPMMA with granules and powder of β-TCP, bone void fillers with different osteo-conductive properties were created. Poresgive bone the opportunity to grow in, result-ing in better fixation of the void filler whichremains implanted permanently. For controlgroup it was used a PMMA cement withoutβ-TCP(Mendec Spine® Tecres SpA,Sommacampagna, VR, Italy), named Ccement. For the experimental groups, twocements presenting different characteristicsof porosity were realized, with the purposeof studying the behavior of bone tissuealong the time. One using β-TCP in powder(granules of 53 μg) named P cement(Porosectan® I, Tecres SpA) and the secondone composed by β-TCP in powder (gran-ules of 53 μg) and granules (100-300 μg),named PG cement (Porosectan® II, TecresSpA). The β-TCP powder used in thiscement meets the medical grade standard

European Journal of Histochemistry 2017; volume 61:2673

Correspondence: Carlo Dall’Oca, Departmentof Surgery, Orthopaedic and TraumatologyClinic, University of Verona, Ospedale BorgoTrento, Piazzale A. Stefani 1, 37126 Verona,Italy. Tel. +39.045.8123542 - Fax: +39.045.8027470. E-mail: [email protected]

Key words: Plymethylmetacrilate; calciumphosphate cement; osteointegration; biocom-patibility; Environmental Scanning ElectronMicroscopy.

Acknowledgments: we thank the companyTecres S.p.A., Sommacampagna, VR, Italy,for providing the cements used in this trial.

Received for publication: 28 April 2016.Accepted for publication: 7 March 2017.

This work is licensed under a CreativeCommons Attribution-NonCommercial 4.0International License (CC BY-NC 4.0).

©Copyright C. Dall’Oca et al., 2017Licensee PAGEPress, ItalyEuropean Journal of Histochemistry 2017; 61:2673doi:10.4081/ejh.2017.2673

[European Journal of Histochemistry 2017; 61:2673] [page 1]

[page 2] [European Journal of Histochemistry 2017; 61:2673]

described by ASTM 1088 4a (2010)“Standard Specification for Beta-Tricalcium Phosphate for SurgicalImplantation”. This was carefully chosen tobe aligned with available studies on poresize.21 The specimens of the implantedmaterial, supplied by the Company TecresSpA, were made up of cylinders of 2 mm indiameter and 6 mm in length; their compo-sition is shown in Table 1.

All experiments were performedaccording to the following standards:UNI EN ISO 10993-6 :2007: Test of localeffects after implantation;UNI EN ISO 10993-1 :2004: Biologicalevaluation of medical devices. Part 1Evaluations and testing;UNI EN ISO 10993-12 :2005: Sample andreference materials.

AnimalsCements were implanted in twelve New

Zealand White (NZW) rabbits, which weresacrificed at 1, 2, 3, 6, 9 and 12 months afterimplantation of the cement. The animalsused were female rabbits weightingbetween 3 and 4.5 kg before the beginningof the study. The rabbits were reared andoperated at CIRSAL (CentroInterdipartimentale per la RicercaScientifica su Animali da Laboratorio -Interdepartmental Center of ScientificResearch on Laboratory Animals,University of Verona). Breeding conditionswere in accordance with the EuropeanDirectives (CE Directive 86/609) and UNIEN ISO 10993-2; (2006): Animal welfarerequirement. They were bred in an individ-ual metallic cage (Tecniplast® according toD.Lgs 116/02) and fed with complete gran-ular diet for rabbits, ad libitum. Water wasfreely accessible. Temperature was between15 to 21°C and room relative humidity wasbetween 30% to 70%. The operative staffwas qualified and educated. Experimentswere approved by Ethics Committee ofVerona University and performed in accor-dance with the Guideline for AnimalExperimentation of the Italian Departmentof Health, under the supervision of a veteri-

narian, and organized to minimize stressand the number of rabbits used.

SurgeryAfter trichotomy of concerned area,

each rabbit was brought into operatoryroom and positioned lateral on the operato-ry table. General anesthesia induction wascarried out by a veterinarian with intramus-cular injection of an association of tileta-mine hydrochloride and zolazepamhydrochloride (Zoletil 100®) at a dose of 20mg/kg and Xylazione (Rompun®) at a doseof 5 mg/kKg. A local anesthesia with lido-caine was also injected in the site of the sur-gical incision.

After washing, disinfection and prepa-ration of sterile operatory field, we madesmall skin incisions on lateral surface ofboth femur. The femur was drilled with ster-ile electrical drill (diameter 2 mm, depth 6mm), in femoral distal and proximal meta-physis on the right femur and in proximalmetaphysis on the left one. After drilling,we washed and cleaned the holes to removeevery bone fragments created. We filled thefemoral cavities with cement cylinders, inparticular P cement distally and PG cementproximally in the right femur, C cement inthe proximal methaphysys of the left femur.We did skin suture with not reabsorbablespin Prolene® n. 3-0. After final disinfec-tion, we made an elastic adhesive bandage.Analgesic therapy with Altadol® IM (4mg/Kg) was administrated to the rabbits,after that the animals were located underUV lamp for ten minutes, before returns intheir cage.

Post-operative clinical follow-up andanimal sacrifice

Animals were observed daily to detectclinical abnormalities and antibiotic therapywas administrated for four days (Baytril®

2.5% SC 5 mg/Kg). Medications withiodate antiseptic (Braunol®) were done, andcutaneous stitches were removed 10 daysafter surgery. The rabbits’ state of well-being was assessed with physiological ele-ments (feeding, stool and urine), physical

elements (functions of operated limb andcoat growth) and behavior elements, like“open field”. This test, known as “escapetest”, is an indication of fear response (rab-bit is prey animal, with avoidance reactions,like fear of new situations and human pres-ence).

Rabbits were sacrificed through phar-macology euthanasia with a lethal injectionof Tanax® after 1-2-3-6-9-12 months. Bothfemurs were extracted from thigh, bonecurettage from soft tissues was carried outand then all bones were put inside a trans-parent sterile envelope.

Macroscopic and radiographicexamination, MRI observation

After the removal of the femurs, macro-scopic observations and digital pictures ofthe implant areas were performed. A radi-ographic examination was carried out postmortem using a Philips Practix 360 mobileradiography system, to define the area ofsamples: it consisted of antero-posterior andlateral side of left and right femur of eachrabbit.

Magnetic resonance imaging (MRI)was acquired the using a Bruker BioSpectomograph for animals (4.7 T), equippedwith self-shielded gradients and BGA9BGA20 (Bruker, Bremen, Germany). Thebones were positioned above the coil flatsurface and then into the magnet. T1w andT2w sequences were acquired respectivelywith techniques Spin Echo and RARE(Rapid Acquisition with RelaxationEnhancement). The acquisition and geomet-ric parameters (number of virtual slices,slice thickness, field of view, the matrix)have been optimized for the bone tissue.

Histological samples preparationThe freshly explanted femurs were

cleaned from soft tissue and rinsed in salinesolution, then fixed in buffered formalde-hyde (pH=7) for a minimum time of 20days at 4°C. With reference to the analysisof X-ray and MRI, it was possible to accu-rately dissect the femur close to the cementcylinders. Each section was progressively

Technical Note

Table 1. Tested bone cements composition.

C cement P cement* PG cement°

Cylinder composition Polymethylmethacrylate 67.5% Polymethylmethacrylate 52.9% Polymethylmethacrylate 40.7% Barium sulphate# 30.0% b-tricalcium phosphate§ 26.1% b-tricalcium phosphate§ 33.3% Benzoyl peroxide 2.5% Barium sulphate^ 7.0% b-tricalcium phosphate granule$ 8.3% Benzoyl peroxide 1.0% Benzoyl peroxide 1.0% Saline solution 13.0% Saline solution 16.7%

*No granule of b-tricalcium phosphate, only powder; °with granule and powder of b-tricalcium phosphate; #barium sulphate powder; §b-tricalcium phosphate powder 53 micron; ^barium bulphate gran-ule 200-400 micron; $b-tricalcium phosphate granule 100-300 micron.


dehydrated by immersion in increasing con-centrations of ethanol (30%, 50%, 80%,95%, 100%), leaving the sample steeped forat least 48 h for each concentration, and per-forming three passages in absolute ethanol.The infiltration with LR white resin wasperformed at room temperature with 3 pas-sages, each of 72 h, in oxygen-poor envi-ronment by placing the samples under vac-uum (with glass bell) to promote the pene-tration of the resins. Polymerization tookplace by UV radiation at room temperaturefor at least 3 days in oxygen-poor environ-ment. Once cured, specimens were sec-tioned with a diamond saw blade mountedon a Leitz microtome according to a planeperpendicular to the femur axis. For eachsample, sections were obtained with a thick-ness of about 100 micron, then manuallythinned down to a thickness of 30 μm ±10with fine abrasive paper (P paper water-proof 1000 WSC BMA) immersed in coldwater at 4° C approximately to prevent sur-face overheating. The obtained sectionswere stained by immersion in a toluidineblue 1% solution for 5 min, rinsed in run-ning water, immersed in a fuchsine acid 2%solution for 3-5min, rinsed in runningwater, immersed in a 0.02% solution ofacetic acid for 1 minute, stained with fastgreen (Diapath) for 5 min and finally rinsed.The stained sections were put on slides

holders with aqueous upright and observedunder an optical microscope OlympusBX51 both bright field and in fluorescence.The digital images were acquired with highresolution camera Quicam connected toDell PC using software image analysisImageproplus v.7 (MediaCybernetic,Bethesda, MD, USA).

Evaluations of biocompatibility (localtolerance) and osteointegration

The local tolerance was evaluatedaccording to microscopic criteria. A semi-quantitative analysis was defined in accor-dance with the UNI EN ISO 10993-6:2009.Particular attention was given to the inflam-matory response, the behavior of the boneand the nature of the contact bone/implant.Each parameter was evaluated by scoringfrom 0 (absent) to 4 (severe) allowing anaccurate assessment of the phenomena ofinflammation and/or foreign body reaction,bone repair and degradation of the material.The biocompatibility evaluation scale isreported in Table 2. To evaluate the osteoin-tegration of the cements, we defined a scaleof evidences according to the gradingdescribed in Table 3.

Ultrastructural analysisIn order to obtain samples for the investi-

gation with the ESEM, longitudinal cuts have

been performed until the exposure of the sur-faces of the plants grafted. In order to betterunderstand the phenomenon of osteointegra-tion, we proceeded to the observation withelectron microscope ESEM equipped withEnergy Dispersive Spectrometry (EDS)microanalytical Probe. The combined use ofESEM with EDS allows to provide the chem-ical composition of a point of interest on thesample surface (microanalysis). The sectionsobtained, with a thickness of about 2-3 mm,were put on aluminum stubs with sticky car-bon and observed with the electron micro-scope ESEM XL 30 FEI Philips low vacuumequipped with EDAX® probe for micro-analysis.

Results

Post-operative clinical follow-upThe surgery procedure was well-tolerat-

ed and the animals managed to feed himselfsince the day after the operation. Within afew days, they managed to move againinside the cage. Neither signs of localswelling or inflammation, nor systemicsigns of cement-toxicity or allergic reac-tions were found. All the animals subjectedto the operation survived.

Technical Note

Table 2. Biocompatibility evaluation scale.

Cell reaction Definitions

Score 0 1 2 3 4Flogosis Absent Slight Moderate Intense SeverePolymorphonuclear cells 0 Rare, 1-5 / phf 5-10 / phf Intense infiltration FullLymphocytes 0 Rare, 1-5 / phf 5-10 / phf Intense infiltration FullPlasmacells 0 Rare, 1-5 / phf 5-10 / phf Intense infiltration FullMacrophages 0 Rare, 1-5 / phf 5-10 / phf Intense infiltration FullGiant cells 0 Rare, 1-2 / phf 3-5 / phf Intense infiltration LaminaeNecrosis Absent Minimum Slight Moderate SevereNeovascularization Absent Minimum capillary Groups of 4-7 capillary Wide band of capillary vessels proliferation, vessels with fibroclastic vessels with focal, 1-3 bud structure of support fibroclastic structure of support Wide band of capillary vessels with fibroclastic structure of supportFibrosis Absent Thin band Moderately thick band Thick band Thick bandFat infiltration Absent Minimum Several layers of Stretched and wide Wide accumulation amount of fat fat and fibrosis accumulation of fat of fat completely associated with cells around the implant surrounds the implant fibrosis Evaluation criteria Score Irritation degree 0.0 – 2.9 Not irritating 3.0 – 8.9 Slightly irritating 9.0 – 15 Moderately irritating > 15 Severely irritating

Macroscopic and radiographicexamination, MRI observation

The visual appearance of implants hadthree kinds of features: some with an evi-dent trace of the plant (Figure 1A), somewith a bulge as a consequence of bone cal-lus (Figure 1B) and others with an homoge-neous surface where the implant was notidentifiable (Figure 1C). These differentvisual appearances were detected in allthree types of cements and in every periodwithout any relationship between themacroscopic and the microscopic findings.

Radiographic analysis (Figure 2)showed a good adjustment and maintenanceof the samples in the original implant areas,in all different times and for all three typesof cements. In the samples of the PGcement, x-ray images showed a weaker sig-nal compared to C and P cements. In partic-ular, for PG cement the radiographic imagesat 6 months do not show sufficiently theimplant. Because of that a more efficientidentification was carried out by magneticresonance imaging. With the MRI, theimplant of the PG cement at 6 months wasevident in its shape and position (Figure 3).Newetheless, it was well visible at 12months (Figure 2I).

Evaluations of biocompatibility(local tolerance) and usteointegra-tion

The results of the semi-quantitativeevaluation of local tolerance (biocompati-bility) are reported in Table 4. In the earlytimes (1st month) at the periphery of theimplants the formation of a bony capsule ofvariable thickness is visible. In these areasthere are aspects of cellular suffering of

Technical Note

Table 3. Description of grading.

Grading Description

G0 No penetration of organic material inside the resin.G1 Presence of organic material fuchsin positive into part of the sampleG2 Presence of organic material fuchsin positive in most of the sampleG3 Presence of cells in part of the sampleG4 Presence of cells in the majority of the sampleG5 Presence of osteoid formations within the sampleG6 Training osteonic spread over the whole sample

Table 4. Biocompatibility evaluation results.

Score Month 1 Month 2 Month 3 Month 6 Month 9 Month 12

Cement C P PG C P PG C P PG C P PG C P PG C P PGFlogosis 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0Polymorphonuclear cells 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0Lymphocytes 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0Plasmacells 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0Macrophages 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0Giant cells 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0Necrosis 2 2 2 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0Neovascularization 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0Fibrosis 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2Fat infiltration 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0Total score 5 4 4 2 3 3 2 2 2 2 2 2 2 2 2 2 2 2Irritation degree Slight Slight Slight Not SlightSlight Not Not Not Not Not Not Not Not Not Not Not Not

Figure 1. Macroscopic images of some femurs. Visual appearance of implants: evidenttrace of the implant (A), bulges resulting in bone callus (B) and implant with externalhomogenous surface (C). Scale bar: 1 cm.



modest degree, not significantly differentbetween the cements. These issues tend todisappear in a longer time with bone reac-tion thickening around the implant. None ofthe cement resulted in necrotic or inflam-matory reactions of relief. There were sig-nificant differences between the materialsin terms of biocompatibility parameters onhistological survey.

In histological preparations, the bonematrix is indicated by the presence of mate-rial fuchsin positive (f+). Histological sam-ples of C cements did not show the presenceof f+ material at any month neither in theperipheral areas nor inside the implant asshown in Figure 4 A,B,C.

Histological samples of P cementshowed the presence of f+ material in theperipheral areas staring from 1st month(Figure 4D). At the 2nd month we witnessedthe entry of f+ material inside the newlyformed cavities in the implant; in the fol-lowing months, no further progress wasnoticed and it remain constant until 12th

month (Figure 4 E,F).Histological samples of PG cement at

the 1st month showed the presence of f+material in the peripheral areas (Figure 5A).From the second month, we observed a pro-gressive diffusion of f+ material over thewhole sample. From the first 2 months onwe observed the generation of a pattern ofmicro internal cavity which was rapidlyinvaded by organic f+ material probable offibrinoid nature. At month 3, the cement PGshowed an initial deposit of bone matrix inthe peripheral portion of the sample, partic-ularly in areas with a greater density of f+material (Figure 5B). After 3 months ithighlights the appearance, inside the plant,of cellular elongated elements of fibroblas-toid morphology. In the following months,these phenomena tend to spread fromperipheral areas to the entire system. At the6th month, we observed the presence ofosteoid material on the entire sample(Figure 5C, red arrows). In the built up larg-er blocks, you had cellular elements withingaps. The osteoid material forms within thecentral areas of the sample in which the fib-rillar f+ material was organized to form realwell cellulate connective areas (Figure 5C,yellow arrows). After the sixth month, thef+ material filled in the previous months, isflooded by fibroblastoid cellular elementslikely of bone marrow origin. In fact, at theperiphery of the sample is sometimes visi-ble anatomical continuity between the inter-nal areas of connective and bone marrowitself (Figure 5C, blue arrow). At theperiphery of these connective areas, at theinterface with the polymer, may yet be visi-ble a layer of acellular fibrillar f+ material.

Technical Note

Figure 2. X-rays: antero-posterior and lateral side of femurs of three treatments (C cementin top row, P cement in middle row, PG cement in the bottom row) to 1 month (left col-umn), 6 months (middle column) and 12 months (right column). Radiographs showedgood images and maintenance of the samples in the original implant areas (red circles) inall different times and for all three types of cements. Only the radiographic images of PGcement at 6 months do not show sufficiently the implant (image H). Scale bar: 1 cm.

Figure 3. MRI of PG cement at 6 months. The implant is clearly visible in its shape andposition (red arrow).


In larger areas we witnessed the depositionof bone matrix that presents isolated stepsof not osteocytic gaps, and others that areorganized into osteons. These areas mayhave a diameter <300 μm (Figure 8A, 8B,red circles). At the 9th month, the process ofcavitation in the sample is very large as wellas the deposition of bone matrix within thespace. Indeed, also aspects of bone deposi-tion are visible in the central portions of thesample (Figure 5D, red arrows). At the 12th

month, the process of formation of bonematerial shall be borne by the connectiveinterior spaces that appear in almost all sitesof deposition of calcified matrix. The newlyformed areas assume a morphology withosteoid formation of gaps and drafts of cel-lular osteons-like systems (Figure 5E, redarrows).

Results about osteointegration arereported in Figure 6. In the C cement thescore was 0 at each time, showing a lack ofpenetration of the organic material withinthe polymer. The phenomena of osteointe-gration appears in this case due to eventsinvolving the external interface of the poly-mer with the tissues and bone marrow. Inthis regard, it was noted that the stability ofthe sample did not significantly alter itscharacteristics even in the observations car-ried out up to 12th month. P cement hadscore 1 at 1st month and score 2 at 2nd

month, which remained the same till the endof the trial. The material belonging to thegroup P is able to generate a lattice insteadof the internal micro-cavity that spreadsrapidly in the whole polymer during thefirst two months. This lattice is rapidlyinvaded by organic f+ material probable offibrinoid nature. It was noted that also inthis case the remarkable stability of the pat-tern that does not appear to change signifi-cantly up to 12th month.

PG cement increased progressively itsscore from 1/2 of the 1st month up to 5 of the12th month showing the best results betweenthe cements. PG cement has instead showna consistent and progressive increase of thephenomena of osteointegration.

Ultrastructural analysis of cementwith ESEM

At ESEM investigation before theimplant, C cement’s surface morphologyshows a rare presence of pores. There is nosubstantial difference of the surface mor-phology in the samples of the C cementgroup from the first to the last month. It ischaracterized by large circular areas sur-rounded by widespread granular deposition.Throughout the whole samples sequence(from 1st to 12th month) there are not evi-dences of formation of bone matrix. The

Technical Note

Figure 4. Histological samples of C cement (A,B,C) and P cement (D,E,F), at 1st (A,D),6th (B, E) and 12th month (C,F). f+ material is visible at peripheral areas of the implant(red arrows). Scale bars: 500 µm.

Figure 5. Histological samples of PG cement at 1st (A), 3rd (B), 6th (C), 9th (D) and 12thmonth (E). f+ material is visible at peripheral areas of the implant (red arrows). Scale bars:500 µm.


microanalysis results are similar to the resinbefore the implant (Figure 7 A,B). Circularareas show main peaks attributable to car-bon (C) and oxygen (O). The surroundingsurfaces with granular deposition havehighlighted peaks attributable to sulfur (S)and barium (Ba).

The surface morphology in the sampleof P cement shows a widespread presenceof 100 µm pores. From the 2nd to the 6th

month, morphological analysis of P cementsamples highlight some areas, inside thematerial, with irregular formations ofdeposits of granular appearance. Theymatch with the areas weakly f+ at the histo-logical survey. In these areas the micro ana-lytical survey detects high peaks due to cal-cium (Ca) and phosphorus (P); the neigh-bouring area showed only peaks attributableto C and O due to the resin. On the 9th and12th month P cement shows irregular areaswith granular appearance but with low den-sity of granules inside (Figure 8 A,B). Themicroanalysis shows significant peaks ofCa and P (Figure 8C). There were no rele-vant aspect of areas with sketches ofosteons.

Technical Note

Figure 6. Comparison of osteintegration grading at 1, 2, 3, 6, 9 and 12 months. Thehorizontal axis shows the time, on the ordinate the osteointegration grading.

Figure 7. C cement, ESEM microscopy of C cement cylinder before the implant (A) and at 12 months (B). The microanalysis of thesample before the implant end after 12 months shows similar peaks of C, O, S and Ba. Scale bars: A) 50 µm; B) 50 µm.


The surface morphology in the sampleof PG cement shows a widespread presenceof 200-300 µm pores. Morphological analy-sis of PG cement inside the cylinder at 1st

and 2nd month, highlighted several areaswith deposits of granular appearance(Figure 9 A,B). These areas match withweakly f+ areas of histological survey. Onthese areas, microanalysis detected highpeaks attributable to Ca and P. The neigh-boring areas showed peaks attributable to Cand O because of its resin composition. Atthe 3rd month PG cement, in addition to thevarious areas of granular appearance, fur-ther areas begin to appear with the look ofsketches of osteons (Figure 9C). Theseareas match with the fast green+ areas ofhistological survey. On all these areas,microanalytical probe detects high peaksattributable to P and Ca. Otherwise, the sur-rounding area showed only peaks attributa-ble to C and O. At the 6th month, in PGcement we observed a decrease of the gran-ular looking areas and larger number ofareas of a clearer osteoid form (Figures 9Dand 10 A,B). On these areas microanalyticalprobe detected high peaks attributable to Caand P (Figure 10 g,o). At 9th and 12th month,in PG cement the areas with osteoid lookingsketches appear to be more widespread thanthe granular areas which are significantlyreduced. They correspond to the fast green+ areas of histological survey. Such aspectsare not found in adjacent areas, consist ofresin alone.

DiscussionHistological analysis showed good

osteointegration of the samples. Theosteointegration was mediated by the for-mation of a bone layer of variable thicknesson the boundary of the implant. This areahas no inflammatory or degenerativeaspects but the newly formed tissue andappears constantly on the outside of theimplanted material. Apart from this feature,we observed significant differencesbetween the three cements. C cement hasnot occurred to any significant penetrationof the tissues inside of the resin at anytimes. The others cements had significantphenomena of osteointegration not limitedto the outer surface but extended within theimplant. For both cements these phenomenawere characterized by the penetration of acellular matrix (f+) in the first two monthsof implantation. While for the P cementthere have been no further developments inlater times, it appears to further progress inPG cement only. In PG cement, from thethird month, we witnessed a gradual onset

Technical Note

Figure 8. P cement, optical (A) and ESEM (B) microscopy at 12 months. The microanaly-sis of the sample (C) shows peaks of C, O, and significant peaks of P and Ca. Scale bar:A) 200 µm; B) 100 µm.


of cellular elements. We saw the appearanceof elongated cellular elements of fibroblas-toid morphology, inside the internal cavitiesof the implant. In the following monthsthere is an evolution of phenomena thattend to spread from peripheral areas to theentire system. The newly formed areas pro-gressively assume a morphology withosteoid formation of gaps and drafts of cel-lular osteonic-like systems. By ESEM andmicroanalysis we could closely follow theevolution of the three cements within awhole year of trial. As expected, in the sam-ples of C cement from the first to the lastmonth (1-12), we didn’t observe a substan-tial difference of the surface morphology ofthe cylinder, characterized by large circularareas surrounded by areas with the presenceof a widespread granular deposition. Themicroanalysis on the circular areas, per-formed in all periods, showed the mainpeaks attributable C and O, due to the

TMMA composition and it was very similarto the microanalysis of the cylinder beforethe implantation. Differently, samples per-formed in the P cement, from the 2nd to the12th month, show a substantial uniformity ofdiffusion of C and O peaks except smallscattered fragments attributable to Ca and P.On 9th and 12th month, P cement showedirregular areas with granular appearancewhich presented significant peaks of Ca andP. However, they did not form any osteoidstructure. The mapping with microanalysisof PG cement have revealed and made evenmore explicit the different chemical natureand morphology of the areas analyzed.From the 1st month inside the implant pro-gressively increased several deposits of tri-calcium phospate with high peaks attributa-ble to Ca and P. At the 3rd month begin toappear sketches of osteons which becomeof a clearer osteoid form and greatlyincrease the number in the following

months, replacing the granular areas. Theseareas were phased out by growths of bone.Because of this development PG cement,consisting of acrylic resin-based porouspolymethacrylate and tricalcium phosphatehas characteristics of particular interest inthe process of osteointegration; especiallyas regards the deposition of calcified matrixwithin the polymer. Besides the different ofbarium sulphate content, the main differ-ence between P and PG consists of the sizeof the granules21,23 (granules of 53 μm onlyfor P cement and also granules of 100-300μm for PG cement) and pores (pores ofabout 100 μm for P cement and 100-200μm for PG cement), we can assume that Pcement not evolved in bone tissue inside theimplant in the following months probablyfor the inability of the internal lattice to cre-ate the environmental conditions able toallow the evolution to the next generation oftrue tissue areas within the polymer.

Technical Note

Figure 9. PG cement, ESEM microscopy at 1, 2, 3, 6, 9, 12 months (panels A, B, C, D, E and F, respectively). The areas identified by the letters‘g’ indicate the formation of granular appearance while those identified with letters ‘o’ indicate the osteoid formations. Scale bars: 100 µm.


Hypothetically, we might assume that thisnon-vascular and cellular invasion is due tomechanical phenomena (pore size withinsufficient access as consequence ofusing), rather than toxic factors, since therewere no cytotoxic aspects linked to thepolymer in the interface with the tissues ofthe host bone marrow. Granules of 100-300μm and pores of 100-200 μm allow the pen-etrations of cells from the surrounding tis-sue creating a tridimensional scaffold thatsupply fluid penetration inside the matrix17

as demonstrated in vitro23 and in vivo13 stud-ies. In a previous work of Dall’Oca et al.13

it was shown the importance of the use ofLR white resin and the use of SEM to makecomplete and objective assessment of thebiocompatibility of porous vs non porous

bone cements. In this paper, we show thatthis evaluation process, together with themicroanalysis, provides further importantinformation that allows to follow anyosteointegration at every stage of develop.

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Technical Note

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Technical Note

the biocompatibility of bone cements: progress in

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