to be biocompatible and osteoconductive [38–40]. Furthermore, it was demon-strated that some compositions are able to induce bone in nonosseous sites (e.g., in muscle). The surface structure of these materials plays a key role [41] in this bone induction.
It was demonstrated how the microstructure of resorption pits is organized [35] and that the structure itself (Fig. 4.6b) is able to influence osteocyte precursors to attach, differentiate, and secrete specific noncollagenous proteins and collagen. It was furthermore demonstrated that it can alleviate mineraliza-tion of the fibers and the interfiber space with apatite. The mineralizamineraliza-tion of ECM was taken as a template for the development of simulated body fluid [42].
Molecular biology provides methods, findings, and explanations that have to be applied to the interpretation of cellular and tissue reactions as known from histology and cytology. It is expected that these new methods can contribute to better interpretation of existing implants and their outcome. It is also expected that they can help predict the behavior of new materials intended to be used as biomaterials for implantation and in extracorporeal devices.
There have been attempts (1) to analyze gene activation (i.e., DNA up- or-down-regulation using a microarray technique); (2) follow the path of tran-scription and analyze mRNA (transcriptome) after reverse trantran-scription for production of cDNA (complementary DNA); and (3) control translation (i.e., the structure and function of proteins—proteome— intracellularly and extra-cellularly). There have also been studies of cluster analysis and gene expression profiles using cDNA microarray systems. An example compares human dental pulp stem cells with human mesenchymal stem cells for tissue engineering and for understanding biomaterials’ interactions [43].
Table4.1Selectedinvitrostudiesoncalciumphosphatecoatings Invitrocellcultureconditions,cellline, materialObjectivesResultsReference MousepreosteoblastMC3T3-E1.Wet chemicalprecipitation,poorlycrystalline calciumphosphateapatitecrystals(PCA)on TCPSorglass Cellproliferation,cellcycle, cellattachment,cell signalingpathway Decreasedcellproliferation, entryinS-phaseofcellcycle delayed,reducedcell attachment,impairedsignal transduction
Leeetal.Biomaterials, 27,2006,3738–3744 Humanosteoblast-likecellsfrompatients. Magnetronco-sputteringofsilicon containinghydroxyapatite(HA),as depositedorheat-treatedtoincrease crystallinityontitanium
Cellattachment,cellgrowth upto56daysHighestcellgrowthand mineralizationonheat treatedSi-HAfilms
Thianetal.Biomaterials, 26,2005,2947–2956 Humanprimaryosteoblastsonoctacalcium phosphate(OCP)andMn-dopedcarbonate HAMn-CHA)ontitaniumsubstrateby pulsedlaserdeposition(PLD)
Proliferation,viability, alkalinephosphatase activity,TGFß Goodproliferation,and viability,alkaline phosphatasehigherthanin controlTi,TGFßlowat 3and7daysbuthigherat 14and21days Bigietal.Biomaterials, 26,2005,2381–2389 Humanfetalosteoblastcellline(hfob),and fibroblasts,onoctacalciumphosphatethin filmbypulsedUVlaserdeposition(PLD)
Cellproliferation,DNA replication,apoptosis,cell morphology,celladhesion Fibroblastsandosteoblasts adhere,normal morphologyand proliferation,nosignsof toxicity
Socoletal.Biomaterials, 25,2004,2539–2545 OsteoblastscelllineMC3T3-E1orprimaryrat calvarialosteoblastsonpoorlycrystalline calciumphosphateapatitecrystals(PCA) wetchemicalfromsupersaturatedsolution onTCPS
Osteoblasticcellsadhesion, proliferation,expressionof genemarkers,calcification Attachment,proliferation, expressionofgenemarkers, calcifiedmatrixpresent Hongetal.Biomaterials, 24,2003,2977–2984 (continued)
Table4.1(continued) Invitrocellcultureconditions,cellline, materialObjectivesResultsReference C3T3-E1preosteoblastsonconventional apatite,precursorapatitespheres,large plate-likeapatite(LPA),Smallplate-like apatite(SPA),TCPS,biomimeticfromSBF 1,1.5,and5
Differentapatitein microenvironmenton preosteoblastspreading, viability,proliferation,gene expression After4or14daysTCPSwas bestforcellproliferation; apatiteinhibitedcell growth.LPAwasbestfor osteocalcin,bone sialoprotein,and osteopontinexpression
Chouetal.Biomaterials, 26(2005)285–295 Ratbonemarrowcells.Calciumphosphateon Ti6Al4Vbyelectrostaticspraydeposition (ESD)orradiofrequencymagnetronsputter coating(rf-msc).Thicknessofcoatings2mm each
Twoexperimentswithsixfold samples.Cellproliferation 4,8,and14days,ngDNA forproliferation,alkaline phosphatase,osteocalcin, RT-PCRmRNAcollagenI andosteocalcin—SEM Cellproliferationdifferentin 1stand2ndrun.Cells confluentonboth substrateswithECMand mineralizedglobules.Slight differencesforalkaline phosphataseand osteocalcinbetween coatings Siebersetal.Biomaterials 25(2004)2019–2027 TGFb,transforminggrowthfactor-b;UV,ultraviolet;TCPS,tissueculturepolystyrene;Si-HA,silicon-substitutedhydroxyapatite;RT-PCR,reverse transcriptionpolymerasechainreaction;ECM,extracellularmatrix;SEM,scanningelectronmicroscopy
hypothesis. Nevertheless, it must be pointed out that this positive outcome is a small nail to pin down a working hypothesis. Because in vivo there are many cell types cooperating in certain environments and at a determined time, the pre-dictions can be erroneous. Furthermore, in vivo the turnover of the interstitial fluid plays an important role for the accumulation or dilution of substances.
This influence cannot be simulated appropriately in conventional tissue culture conditions.
In many cases, the characterization of surfaces coated with calcium phos-phates is not comparable. In many publications the figures show different roughness, structure of the surface (e.g., some polished smooth surfaces, surfaces resembling hobnails, displaying clefts at grain boundaries, openings to microporous structures below the main surface), needle-like crystals, plate-like crystals, and amorphous areas at which shear forces can hardly resist. This variety in surface structure is enlarged by variations in chemical composition and solubility of components that are partially related to the variations in crystallinity.
Apparently, the technique of coating influences the various mentioned para-meters. It is not the task of this chapter to deal with coating techniques in detail (for this see Chapters 3, 4 and 6); it should be mentioned, however, that in the cited publications (Tables 4.1, 4.2) there are biomimetic calcium phosphate deposition (e.g. using simulated body fluid), wet chemical techniques with precipitation from supersaturated solution, magnetron co-sputtering, and sub-sequent heat treatment to increase crystallinity, pulsed laser deposition, elec-trostatic spray deposition, radiofrequency magnetron sputter coating, and additional heat treatment or surface modification by grinding or polishing.
Therefore, it is not astonishing that the results of most of the publications cannot be compared. Nevertheless, some papers are mentioned.
The mouse preosteoblast MC3T3-E1cells were cultured on poorly crystalline calcium phosphate apatite (PCA) and compared to the behavior on tissue culture polystyrene or uncoated glass coverslips for up to 6 days. MC3T3-E1 cells on PCA displayed reduced proliferation, delayed cell cycle progression, underdeveloped stress fiber and tubulin expression, reduced focal adhesion, and decreased activation of tyrosine receptor (RTK)-Ras–extracellular signal-regu-lated kinase (ERK). Therefore, it was assumed that weak adhesion and reduced signaling was responsible for the reduced proliferation of MC3T3-E1 cells on PCA [44].
Human osteoblast-like cells from hip bone were cultured on: (1) pure tita-nium; (2) 0.8% silicon containing hydroxyapatite (Si-HA) as deposited by Magnetron co-sputtering; (3) 0.8% Si-HA heat treated at 7008C for 3 hours, thickness of the coating 600 nm. Time for culture was up to 56 days. The heat-treated Si-HA stimulated cell growth, deposition of ECM, attachment of cells to the substratum as shown by vinculin attachment points, and development of cytoskeleton shown by actin and deposition of calcium phosphate in the ECM [45].
Table4.2Selectedinvivostudiesoncalciumphosphatecoatings Invivoanimalspeciesimplantation siteMaterialsResultsReference NewZealandwhiterabbits, femoralcondyles—9daysImplantsofTi6Al4Vwithtwoorthree layersofbeadscoatedwitheither inorganicororganicroute carbonatedHAthinfilms(1mm). Uncoatedimplantsforcontrol BothHAcoatingsenhancedearly boneingrowthandfixationversus uncoatedimplants
Ganetal. Biomaterials, 25,2004,5313–5321 NewZealandwhiterabbits, tibia—2weeksPorousTi6Al4V,thincalcium phosphatesol-gelcoating(1mm) versusuncoatedsurfaces, morphometry
Coatedimplantsdisplayfasterbone ingrowthandmorebone,more completefillofthepores
Nguyenetal. Biomaterials, 25,2004,865–876 NewZealandwhiterabbits, back—1,4,8,12weeks0.1,1.0,4.0mmCaPonTiO2discs,RF magnetron-sputteredHeattreatment atpresentandupto4weeks
After1week,0.1and1.0mmCaP disappeared.AmorphousCaP(4mm) replacedbyO3-apatite Wolkeetal. Biomaterials, 24,2003,2623–2629 TexcelXcontinentalsheep, femoralcondyle—6and 12weeks
PureHASi-HA0.8and1.5wt%Si Granulesandpowders,nocoatingSEM,high-resolutionTEMHAvoids after12weeks1.5wt%Si-HA> 0.8wt%Si-HA>HAdissolution,Si increasesbioactivityofHA Poreretal. Biomaterials 24,2003,4609–4620 Sprague-Dawleyrats,medullary cavityofthefemur—7,28, 56days
TitaniumKirschnerwire,diameter 1.4mm,sandblastedand electrochemicallycoatedwithHA, thicknessofthecoating2mm Coatedmaterialshearstrength27MPa, uncoated8MPa.Histomorphometry HAp-coatedboneimplantcontact 68%,uncoated26%
Schmidmaieretal. J.Biomed.Mater. ResB(Appl. Biomater.)63,2002, 168–172 BeagledogsHAcoateddental implantsinthemandible—3 and15weeks
Dentalimplants,plasmaspraying, controlscalcititecoatingwith10% solublephasesversusheat-treated 95%crystallineHAcoating Lessdirectboneappositionatsurfaces withhighcrystallinecoatingandless pulloutforcesforhighcrystalline coatings.MacrophageswithHA particlesatfibrousinterfaces Burgessetal.Clin.Oral Implant.Res.10, 1999,257–266 (continued)
Table4.2(continued) Invivoanimalspeciesimplantation siteMaterialsResultsReference MaleJapanesewhiterabbittibia— 4,8,16weeksCeriastabilizedtetragonalzirconia alumina,foursurfacepreparations: (a)Uncoatedpolished (b)Uncoatedmicroporous (c)SubmicronHA-coatedmicroporous withSBF (d)4mmHA-coatedmicroporous withSBF Bestresultsindetachmenttest>25N failureloadfor(d)and(c) Bone-bondingabilityfor(d)and(c)
Takemotoetal.J. Biomed.Mater.Res. A78,2006,693–701
Human primary osteoblasts, donated by patients, were expanded and cultured on three substrates and tissue culture polystyrene. Pulsed laser deposition of octacalcium phosphate (OCP) or Mn doped (0.55%), carbonate (5%), or HA (Mn-CHA) was done. The thickness of the coatings was about 800 nm for Mn-CHA and 1 mm for OCP. The surfaces were slightly different in surface roughness and morphology. Proliferation of osteoblasts was best on Mn-CHA and OCP, as was the development of alkaline phosphatase and collagen type I up to 21 days. Transforming growth factor-b1 (TGFß1) level was highest for Mn-CHA and a little less for OCP after 14 and 21 days of culture. The presence of TGFß1 indicated stimulation of growth and differentiation of osteoblasts [46].
Human fetal osteoblast-like cell line (hFOB 1.19) and murine fibroblast cell line L929 were cultured on Si wafers or titanium (Ti, polished or etched) that had been coated with OCP by pulsed UV laser deposition. No differences were found for adhesion of hFOB between control tissue culture polystyrene (TCPS), Ti, and OCP but lower hFOB on OCP compared with TCPS; there were no differences with uncoated Ti. DNA replication and caspase-1 activity of hFOB were not affected by the materials. It was concluded that after 3 days L929 fibroblasts and hFOB adhere with normal morphology, proliferate, and remain viable on OCP coatings [47].
In an additional study, poorly crystalline calcium phosphate apatite crystals (PCA) were formed at low temperature by precipitation on TCPS, giving some granular structure and a rather smooth surface. For up to 25 days, primary rat calvaria osteoblasts and MC3T3-E1 osteoblasts were cultured on the PCA thin film on TCPS or on the culture dish material (control). After 1 hour there were more cells attached to the PCA surfaces compared to TCPS. However, after 3 and 6 days there was statistically no difference between PCA and TCPS.
Both cell types expressed alkaline phosphatase. Calcification nodules devel-oped with calvarial and MC3T3-E1 cells. There were more nodules at the surface of PCA with osteocalcin than on TCPS. MC3T3-E1 cells expressed osteoblast marker genes such as alkaline phosphatase, osteocalcin, osteonectin, and osteopontin [48].
These in vitro studies and others mentioned in Table 4.1 [49] were mainly performed using osteoblast-like cells or cells with some ability to express features of osteoblasts. The rationale for this procedure is to some extent unclear as the first cells that control a foreign surface are not osteoblasts but macrophages. Therefore, it would be much more realistic if the primary reac-tions of macrophages were studied. There are two arguments in this context:
(1) According to the rule elaborated in 1969 by Frost [34], the sequence of events in bone remodeling comprises (a) activation, (b) resorption, and (3) forma-tion—the so-called ARF rule. (2) In Fig. 4.7a,b, the early phase of a cellular reaction to an implant can be seen: the appearance of macrophages. In the presented case, macrophages are on the surface of bone bonding glass ceramic (Ceravital). The question arises as to why these macrophages appear after 3 days postoperatively and disappear and leave room for osteoblasts after less than 7 days? Do they move away, or do they disappear owing to apoptosis? Do
they leave remnants of cell membrane, receptors of cell organelles at the surface, to condition the ground for osteoblasts or fibroblasts? There is impressive documentation of osteoclast activity at the surface of implants at an early stage after implantation and even later, as well as in vitro coupling of resorption and formation [25, 35].
Thin CaP coatings on metals, alloys, and polymers (e.g., TCPS, glass) show better osteoblast growth (i.e., more viable cells) than uncoated substrates (Table 4.1). There is, however, one exception: When the coating displays poorly crystalline calcium phosphate, cell proliferation is reduced, the cell cycle phase is delayed, and cell attachment and signal transduction are impaired [44]. Other coatings with less amorphous phases display positive results (i.e., increased cell proliferation and differentiation). One important parameter might be a suffi-ciently stable ground for cells. It seems that osteoblasts need some firm ground on which to attach and to express stress fibers of actin. Up to now it has been unclear whether the amorphous calcium phosphate areas attract more and more active macrophages or resorbing multinuclear cells (osteoclasts or mineraloclasts).
Some in vitro studies (Table 4.1) [49, 50] with thin CaP coatings controlled the expression of proteins or substances that are known to be features of the osteoblast lineage, such as alkaline phosphatase, osteocalcin, osteonectin, bone sialoprotein, osteopontin, collagen I. These markers and mineralizing nodules are basic for an acceptable coating intended for clinical studies or application in human patients. The question arises, however, whether this expression of osteoblast markers predicts early bone bonding and the load-bearing ability of the coated material in a specific implantation site. The current conclusion is that this question cannot be answered on the basis of in vitro tests with osteo-blasts. It would be necessary to investigate more, prognostically relevant cell lines that contribute to tissue formation at the interface of coated implants. It should be determined which genes are activated in what cells when substances are released from coatings. When a bone fracture or a surgical bone trauma is healing, the release of material from the coating layer should interfere posi-tively, not negaposi-tively, with differentiation of the cells. A positive step in this direction was published [51]. The release of ions from the coating acted bene-ficially for the adhesion and development of osteoblasts [51]. It was found that the release of Mg and carbonated HA coated on Ti6Al4V up-regulated key intracellular signaling proteins (Shc, Ras/Mapkinase pathway, c-fos) in human bone-derived cells (HBDCs) and that attachment and differentiation of HBDC was stimulated. Results from those investigations can improve the guidelines for manufacture of second-generation biomaterials in this field.
There are other thin CaP coatings that were doped with molecules other than just calcium and phosphate (e.g., silicon, Mn-doped carbonate apatite, octa-calcium phosphate, structured apatite with small or large plate-like apatite).
Interestingly there are also reports on inhibitory effects of released material from coatings (Table 4.1).