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Implantomics: A Paradigm Shift in Implantology

Implantomics: A Paradigm Shift in Implantology Current Directions in Biomedical Engineering 2019;5(1):13 3 1-13 6 Herbert P. Jennissen* Abstract: Implantomics is the science of the implantome. The adsorption is the first fundamental interaction between the implantome is a blend of the two terms implant and human body and the surface of a prosthesis on implantation. proteome. The proteome is defined as the protein Every implant poses a non-specific molecular recognition complement of the genome. The term proteome also implies surface in the body with high affinity for the spontaneous the mass screening of proteins for the determination of all adsorption of human proteins. The adsorbed protein layer is proteins – and indirectly of all genes – involved in a certain thus a result of the protein composition in the environment tissue or organ response. In this sense the term proteome is (periimplant protein pool) and the respective surface with employed here in a new way to specify the totality of proteins affinity for proteins. As will be shown the proteomic associated with a foreign body inserted into the human body. approach (for definition see [5]) necessitates a reevaluation of It will be addressed, why the determination of the implant- present models in implant science. In a sandwich model of tome is important and which role the implantome may play in the bone-implant interface the implant proteome can be the bone-implant interface. viewed as the sandwich-spread in-between. It's understanding may be the key to master biocompatibility and implant integration. Keywords: implant proteome, bone-implant interface, biocompatibility, osseointegration, peri-implant bone healing, 2 Materials and Methods https://doi.org/10.1515/cdbme-2019-003 4 All methods related to the hip implant proteome are described in ref. [4], together with the primary proteome data, available as supplementary Table 1 for download [4] 1 Introduction allowing third party evaluations. Briefly [4] the in situ femoral stems were retrieved 2 min after implantation, The term "paradigm Shift" was coined by Kuhn in 1962 washed with saline, quick frozen in liquid nitrogen and stored at -80 °C. Proteins were by solubilized by SDS and reducing [1]. Three steps are involved: "crisis", "reform" and agents at room temperature and analyzed by tandem LC- emergence of a "new paradigm". The crisis appears to be, MS/MS with the Proteome Discoverer software (Thermo that after ca. 35 years of research biocompatibility and Scientific) by Mosaique GmbH (D-30659 Hannover) [4]. osseointegration are still only poorly understood (see Biocontact hip implants (Braun Aesculap, D-78532 comprehensive reviews [2,3]). Published conclusions of Tuttlingen) with a rough plasmapore TPS surface and a protein adsorption being only of minor importance in smoother glass pearl blasted surface were employed. implantology are up for revision. A radical reform of this thinking has been triggered by a new technology, i.e. LC- Table 1 MS/MS, allowing the complete, simultaneous identification Suggested Nomenclature for implant proteome research* of all initial proteins layered on a human implant [4]. A new Blended Connotation paradigm is suggested as the emergence of a proteomic scale Terms new understanding of protein function in implantology. Implantomics Science that deals with the proteome on implants in general Biocompatibility is primarily a function of the implant or Explantomics Science that deals with the explant proteome biomaterial surface. Corresponding research is Genome- and host-based in situ protein layer on implants Implantome multidisciplinary involving e.g. surface physics, surface during physiological peri-implant healing chemistry and surface biochemistry, terms which could be Genome- and host-based in situ protein layer on explanted Explantome implants, due to pathological peri-implant events. condensed to one word, epiphanostics (from greek Genome- and host-based physiological steady-state "epiphaneia" = surface). On the biochemical level protein Integratome protein layer on a tissue-integrated implant in situ *implantome genesis is the dynamic product of a foreign-body ______ reaction of the host. The blended terms are fully free for scientific *corresponding author: use, but commercially trademark protected. Non-use of the Prof. Dr. H.P. Jennissen, Institut für Physiologische Chemie, Universität symbols  und  does not imply a waiver of trademark rights. Duisburg-Essen, Universitätsklinikum Essen, Hufelandstr. 55, D-45122 Essen, Germany; Email: hp.jennissen@uni-due.de Open Access. © 2019 Herbert P. Jennissen et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 License. H. P. Jennissen et al., Implantomics: A Paradigm Shift in Implantology — 132 3 Results and Discussion similar to fracture healing [21] and leading to implant integration [22]. Based exclusively on in vitro data this has 3.1 Protein Adsorption been the doctrine to the present day [23,24]. In their paper The basic mechanisms of protein adsorption were from 1987 on the Big Twelve proteins Andrade and Hlady elucidated in single protein adsorption systems. From [13] demonstrated 150 electrophoretic bands from plasma equilibrium and kinetic studies it has been concluded that and estimated that a cell contains ca. 5,000 proteins. Today's homogenous pure proteins are adsorbed to non-biological estimates with splice variants lie at ca. 70,000 human surfaces with high affinity by multivalent [6], cooperative [7] proteins. Current doctrine teaches that adsorption from blood non-covalent [8] interactions involving thermodynamically plasma leads to preferential adsorbates such as albumin, irreversible adsorption hysteresis [9,10] with binding fibrinogen and IgG [25], followed by blood coagulation, the 12 -1 affinities of K' ~ 10 M and higher (Fig. 1). In physically formation of a fibrin network as guide rail for oncoming cells controlled kinetic studies the adsorption of proteins is an e.g. Macrophages [26,27] and stem cells. exponential function beginning in milliseconds and reaching Implant surface properties also play a decisive role [28]. equilibrium in less than one minute [11]. Such surfaces are now available and classified either as ultra- /superhydrophilic displaying contact angles < 10° [29] or as Native Unfolded Denatured Protein hyperhydrophilic with complex and/or imaginary contact angles [30]. The rapid, spontaneous reversal from Free hydrophilicity to hydrophobicity can be prohibited for years in a dry state by an exsiccation layer of salt [31]. Bound 3.2 The Hip Implantome t = 0 t = t t =  3.2.1 The Origin of the Hip Implantome Nucleation complex | Conformation 2 | Conformation 3 (Conformation 1) 6 9 12 -1 K' 10 10 10 [M ] As detailed above, the adsorbed protein layer is a result Fig. 1. Multiple metastable protein states in hysteretic Model of the periimplant protein pool and the implant surface. The of protein adsorption [9,12,13]. The steps k , k , k are +2 +3 -5 primary implantome originates not only from the blood I-III slow and the steps k , k are fast. t = time, P = adsorbed -6 -7 plasma proteome [23,24], but from a mixture of blood plasma protein in 3 conformational states of increasing affinity (K ) with the periimplant operational humor (Humor operationis or operationl protein fluid), the composition of which is still In early attempts for understanding more complex systems. unknown. The operational humor depends on the operator Horbett et al. [14] and Cooper's group [15] analyzed pure and the operational and patient situation. Thus tissue injuries binary-protein adsorption systems, demonstrating compe- result, which release large amounts of intracellular proteins tition and heterologous protein-protein displacement on but are operationally mandatory. To a certain extent the polymers. On defined alkyl-residue lattices protein-protein situation cannot be planned. On the other hand in the future displacement was shown to be a form of negative coopera- an optimal implantome might be rationally prefabricated. tivity [16]. A novel observation in binary systems is "protein interference" [17]. Much earlier Vroman had studied more complex multi-protein adsorption systems of fibrinogen from 3.2.2 The Primary Implantome and the Early Bone- blood plasma [18] (Vroman effect). Because of the immense Implant Interface analytical problems this work had been largely discontinued. This has now changed. Today the highest imaginable scale of The bone-implant interface in general, and for titanium complexity in the form of proteomic-scale or myriad-protein implants specifically, possesses biochemical, biological and adsorption on surfaces has become a reality [4], allowing mechanical aspects which must be experimentally assessed. thousands of individual proteins to be monitored In literature searches very little can be found on the early simultaneously during adsorption and desorption. interface healing stages and biochemistry appears to be an At the advent of the field of biomaterials in the 1970ies implantomic and interfacial orphan. and 1980ties it was without question that the first proteins The initial protein layer on an implant, i.e. the primary adsorbed on an implantable device originate from blood implantome, is the crucial host response and an integral part plasma [13,19,20] initiating periimplant endosseous healing of the initial bone-implant interface during the earliest H. P. Jennissen et al., Implantomics: A Paradigm Shift in Implantology — 133 healing stage. An early paper on the composition of the bone- implatome [4] was the zinc-finger protein family with over metal interface was described nine days after implantation in 120 different protein entities containing the three high a rat model by Donath et al. [26] and appears to be 7-15 µm abundance proteins ZNF35, ZNF470 and ZNF 850. wide. The earliest cells to appear after 3 days are histiocytes 3.2.3 Evolution from the Primary Implantome to and multinucleate giant cells, probably related to M2 type the Integratome in the Bone-Implant Interface macrophages in agreement with a macrophage model of osseointegration (see ref. [27,32,33]). A first step has now been made in the pilot determination In the current paper by Jäger et al. [4] it was found that of the primary implantome on a hip implant after 2 min in the primary human hip implantome, at a post-insertion time situ [4]. This protein layer is a principle component of the of 2 min, consisted of 2802 (#peptide number ≥ 2) unique first "cell-containing bone-implant interface" What do we know about the last stage, the bone-implant interface of Table 2 integration (i.e. integratome). Usually it is equated with the [1] Profile of Plasma Proteome in the hip Implantome bone-implant contact, i.e. BIC. The residual interface gap in Abundance [ppm] the BIC region is so small, that cells can no longer enter, Name Symbol Nr mean SD which results in a "cell-free bone-implant interface". This by 1 Serum albumin ALB 48927 17300 no means indicates that an implant proteome is absent from 2 Alpha-1-antichymotrypsin SERPINA3 2342 1174 this interface! What is also often forgotten is, that the BIC 3 Titin TTN 1961 98 4 Carbonic anhydrase 1 CA1 1888 21 generally only covers 50-60% of the integrated bone-implant 5 Collagen alpha-1(V) chain COL5A1 1820 1486 interface [34]. What about the other 40-50% of the non-BIC 6 Catalase CAT 1678 947 regions in this area? Are they part of implant integration and 7 Keratin, type II cytoskeletal 1 KRT1 1540 2002 the integratome? Recently a comprehensive review of the 8 Fibrinogen beta chain FGB 1518 529 SH3 domain-binding glutamic largely submicroscopic evidence obtained for the bone- 9 SH3BGRL2 1411 1946 acid-rich-like protein 2 implant-interface in the BIC region of integration has 10 Keratin, type I cytoskeletal 10 KRT10 1359 811 appeared [3]. It is concluded that the bone-implant interface 11 Serotransferrin TF 1312 256 12 Fibrinogen gamma chain FGG 1161 248 zone at this stage is primarily fibrillar with an electron dense [1] Data from supplementary Table 1 ref. [4]. Hemoglobin, the 20-50 nm thick layer of collagen fibrils and an additional most abundant protein, is not shown, because it is an finely fibrillar mineralized matrix of ca. 200 nm thickness, intrinsically intracellular protein. i.e. in sum 250 nm in width (i.e. enough room for an implantome). Generally only four proteins are repeatedly proteins of which numerically 77% were of intracellular named in the bone-implant-interface of an integrated implant: origin and only 9% (i.e. 247 proteins) from blood plasma [4] collagen, bone silaoprotein, osteopontin and osteocalcin. (see Table 2). The intracellular proteins of the implantome Three major biological phenomena thus appear notable originated from the bone, the bone marrow, the blood cell in periimplant healing: (i) a large, dynamic cell containing and the plasma proteomes respectively. Surprisingly the most bone-implant interface measuring 20-100 µm in width scales abundant implant protein in the implantome was hemoglobin down 100-400-fold (= compaction) during integration to a (10%) and not serum albumin (5%). Fibrinogen and IgG (ii) long-term constant, cell-free bone-implant interface with were absent from the most abundant first 36 proteins (see a width of ~200-500 nm (BIC) [3], characterized probably by also [14]). Thus the in vitro evidence for the doctrine plasma a steady-state turnover of implantome proteins and (iii) to proteins forming the initial protein layer [25], did not non-BIC marrow-type (?) spaces. From this large interface "translate" into the human in vivo proteome. Thus an old compaction it can speculated, that there may be a parallel paradigm failed, at least in hip implantology. Nevertheless, reduction in the number of proteins but not to a complete the sequence of events strongly suggests, that the implantome disappearance of the implantome per se. per se mediates fundamental cell-biomaterial interactions. Interestingly the importance of proteins in Screening through the supplementary Table 1, deposited biocompatibility, and with that in implantology as a whole, is with the paper in ref. [4], some other additional details strongly disputed: "clinically, protein adsorption is of minor emerged. The only proteins related to bone healing were importance in biocompatibility pathways" [2], and the BMP-1, a protease, and inhibitors of BMP-2 i.e. Chordin, and "adsorption of macromolecules has only minimal effects in Gremlins 1 & 2. The mediators BMP-2, VEGF, TGF- and biocompatibility" [2]. In addition it is stated in the same TNF- were not present in the implant proteome at all. The paper that surface modification technologies in implantology largest single family of proteins contained in the hip – with the possible exception of nanostructures – also only H. P. Jennissen et al., Implantomics: A Paradigm Shift in Implantology — 134 [5] Wasinger, V. C., Cordwell, S. J., Cerpa-Poljak, A., Yan, J. play a minor role [2]. This reasoning insinuates that proteins X., Gooley, A. A., Wilkins, M. R., Duncan, M. W., Harris, neither play a significant role in biocompatibility nor in R., Williams, K. L., & Humphery-Smith, I. (1995) periimplant healing and osseointegration. At present this Electrophoresis, 16, 1090-1094. cannot be disproven, but it is at odds with reason and many [6] Jennissen, H. P. (1976) Biol. Chem. (formerly: Hoppe- Seyler's Z. Physiol. Chem. ), 357, 1201-1203. literature findings. One important point appears to have been [7] Jennissen, H. P. (1976) Biochemistry, 15, 5683-5692. overlooked, namely, that not only the fact alone of obtaining [8] Jennissen, H. P. (2005) Methods Mol. Biol., 305, 81-99. implant biocompatibility and/or integration, but also the [9] Jennissen, H. P. & Botzet, G. (1979) Int J Biol Macromol, 1, time-frame i.e. the rates of healing and osseointegration are 171-179. [10] Jennissen,H.P. (1985) Protein Adsorption Hysteresis. In of utmost importance, especially in connection with hospital "Surface and Interfacial Aspects of Biomedical Polymers" costs. That is where the implantome protein composition may Vol.2, Protein Adsorption (Andrade,J.D., ed), pp. 295-320. very effectively come in. Plenum Press, New York. [11] Jennissen, H. P. & Zumbrink, T. (2004) Biosens. Bioelectron., 19, 987-997. Conclusions [12] Jennissen, H. P. (1988) Makromol Chem, Macromol Symp, It is the aim and mission of implantomics to gain 17, 111-134. fundamental insights into proteome function by clarifying [13] Andrade, J. D. & Hlady, V. (1987) Ann. N. Y. Acad. Sci., 516, 158-172. new pathways of implantome maturation from the primordial [14] Horbett, T. A., Weathersby, P. K., & Hoffman, A. S. (1977) implantome over intermediary implantomes to the final J Bioengineering, 1, 61-78. integratome. It is essential to elucidate the role of implant- [15] Pitt, W. G., Young, B. R., Park, K., & Cooper, S. L. (1988) specific implantomes in biocompatibilty and osseointegration Makromol. Chem. , Macromol. Symp., 17, 453-465. [16] Jennissen, H. P. (1986) J colloid Interface Sci, 111, 570-586. as well as to employ this information for future artificial [17] Jennissen, H. P. (2019) Curr. Dir. Biomed. Engineer., 5, in implantome designs. A high priority should also be given to print. diagnostics, for an understanding of the mechanisms of [18] Vroman, L. & Adams, A. L. (1969) J Biomed. Mater Res, 3, premature implant loosenings and failures by generalized, 43-67. [19] Brash,J.L. & Lyman,D.J. (1971) Adsorption of Proteins and systematic explantome research and analyses. Lipids to Nonbiological Surfaces. In The Chemistry of Biosurfaces (Hair,M.L., ed), pp. 177-229. Marcel Dekker, Acknowledgements New York. The support of the German Research Foundation (Deutsche [20] Andrade,J.D. (1985) Principles of Protein Adsorption. In "Surface and Interfacial Aspects of Biomedical Polymers" Forschungsgemeinschaft; DFG Reference No. Je84/15-3) is Vol.2, Protein Adsorption (Andrade,J.D., ed), pp. 1-80. gratefully acknowledged. (https://orcid.org/0000-0002-2615-0184) Plenum Press, New York. [21] Davies, J. E. (2003) J. Dent. Educ., 67, 932-949. Abbreviations: [22] Branemark, P. I., Hansson, B. O., Adell, R., Breine, U., Lindstrom, J., Hallen, O., & Ohman, A. (1977) Scand. J. BIC, bone implant contact; BMP-2, bone morphogenetic Plast. Reconstr. Surg. Suppl, 16, 1-132. protein 2; LC-MS/MS, LC (Liquid Chromatography), MS [23] Kuzyk, P. R. & Schemitsch, E. H. (2011) Indian J. Orthop., (Mass Spectrometry), MS/MS tandem mass spectrometry ; 45, 108-115. VEGF, vascular endothelial growth factor; SEM, scanning [24] Tengvall, P. (2011) Compr. Biomater., 4, 63-73. [25] Kanagaraja, S., Lundstrom, I., Nygren, H., & Tengvall, P. electron microscopy; TEM, transmission electron (1996) Biomaterials, 17, 2225-2232. microscopy; TPS, titanium plasma sprayed. [26] Donath, K., Kirsch, A., & Osborn, J. F. (1984) Fortschr. Zahnärztl. Implantol., 1, 55-58. Author Statement [27] Jennissen, H. P. (2016) Curr. Dir. Biomed. Engineer., 2, 53-56. The Author states no conflict of interest. [28] Schwarz, F., Wieland, M., Schwartz, Z., Zhao, G., Rupp, F., Geis-Gerstorfer, J., Schedle, A., Broggini, N., Bornstein, M. M., Buser, D., Ferguson, S. J., Becker, J., Boyan, B. D., & 4 References Cochran, D. L. (2009) J. Biomed. Mater. Res. B Appl. Biomater., 88, 544-557. [1] Kuhn,T.S. (1962) The Structure of Scientific Revolutions. In [29] Jennissen, H. P. (2001) Bionanomaterials (formerly: Third Edition 1996, pp. 1-212. University of Chicago Press, Biomaterialien), 2, 45-53. Chicago, London. [30] Jennissen, H. P. (2012) Materialwiss. Werkstofftech. [2] Williams, D. F. (2016) ACS Biomater. Sci. Eng., 3, 2-35. (Mater. Sci. Eng. Technol), 43, 743-750. [3] Shah, F. A., Thomsen, P., & Palmquist, A. (2019) Acta [31] Jennissen, H. P. (2010) Materialwiss. Werkstofftech. Biomater., 84, 1-15. (Mater. Sci. Eng. Technol. ), 41, 1035-1039. [4] Jäger, M., Jennissen, H. P., Haversath, M., Busch, A., [32] Trindade, R., Albrektsson, T., Galli, S., Prgomet, Z., Grupp, T., Sowislok, A., & Herten, M. (2019) Proteomics. Tengvall, P., & Wennerberg, A. (2018) Clin. Implant. Dent. Clin. Appl., e1800168. Relat Res., 20, 82-91. H. P. Jennissen et al., Implantomics: A Paradigm Shift in Implantology — 13 5 [33] Schlundt, C., El, K. T., Serra, A., Dienelt, A., Wendler, S., Schell, H., Van, R. N., Radbruch, A., Lucius, R., Hartmann, S., Duda, G. N., & Schmidt-Bleek, K. (2018) Bone, 106, 78-89. [34] Romanos, G. E., Toh, C. G., Siar, C. H., Wicht, H., Yacoob, H., & Nentwig, G. H. (2003) J Periodontol., 74, 1483-1490. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Current Directions in Biomedical Engineering de Gruyter

Implantomics: A Paradigm Shift in Implantology

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Abstract

Current Directions in Biomedical Engineering 2019;5(1):13 3 1-13 6 Herbert P. Jennissen* Abstract: Implantomics is the science of the implantome. The adsorption is the first fundamental interaction between the implantome is a blend of the two terms implant and human body and the surface of a prosthesis on implantation. proteome. The proteome is defined as the protein Every implant poses a non-specific molecular recognition complement of the genome. The term proteome also implies surface in the body with high affinity for the spontaneous the mass screening of proteins for the determination of all adsorption of human proteins. The adsorbed protein layer is proteins – and indirectly of all genes – involved in a certain thus a result of the protein composition in the environment tissue or organ response. In this sense the term proteome is (periimplant protein pool) and the respective surface with employed here in a new way to specify the totality of proteins affinity for proteins. As will be shown the proteomic associated with a foreign body inserted into the human body. approach (for definition see [5]) necessitates a reevaluation of It will be addressed, why the determination of the implant- present models in implant science. In a sandwich model of tome is important and which role the implantome may play in the bone-implant interface the implant proteome can be the bone-implant interface. viewed as the sandwich-spread in-between. It's understanding may be the key to master biocompatibility and implant integration. Keywords: implant proteome, bone-implant interface, biocompatibility, osseointegration, peri-implant bone healing, 2 Materials and Methods https://doi.org/10.1515/cdbme-2019-003 4 All methods related to the hip implant proteome are described in ref. [4], together with the primary proteome data, available as supplementary Table 1 for download [4] 1 Introduction allowing third party evaluations. Briefly [4] the in situ femoral stems were retrieved 2 min after implantation, The term "paradigm Shift" was coined by Kuhn in 1962 washed with saline, quick frozen in liquid nitrogen and stored at -80 °C. Proteins were by solubilized by SDS and reducing [1]. Three steps are involved: "crisis", "reform" and agents at room temperature and analyzed by tandem LC- emergence of a "new paradigm". The crisis appears to be, MS/MS with the Proteome Discoverer software (Thermo that after ca. 35 years of research biocompatibility and Scientific) by Mosaique GmbH (D-30659 Hannover) [4]. osseointegration are still only poorly understood (see Biocontact hip implants (Braun Aesculap, D-78532 comprehensive reviews [2,3]). Published conclusions of Tuttlingen) with a rough plasmapore TPS surface and a protein adsorption being only of minor importance in smoother glass pearl blasted surface were employed. implantology are up for revision. A radical reform of this thinking has been triggered by a new technology, i.e. LC- Table 1 MS/MS, allowing the complete, simultaneous identification Suggested Nomenclature for implant proteome research* of all initial proteins layered on a human implant [4]. A new Blended Connotation paradigm is suggested as the emergence of a proteomic scale Terms new understanding of protein function in implantology. Implantomics Science that deals with the proteome on implants in general Biocompatibility is primarily a function of the implant or Explantomics Science that deals with the explant proteome biomaterial surface. Corresponding research is Genome- and host-based in situ protein layer on implants Implantome multidisciplinary involving e.g. surface physics, surface during physiological peri-implant healing chemistry and surface biochemistry, terms which could be Genome- and host-based in situ protein layer on explanted Explantome implants, due to pathological peri-implant events. condensed to one word, epiphanostics (from greek Genome- and host-based physiological steady-state "epiphaneia" = surface). On the biochemical level protein Integratome protein layer on a tissue-integrated implant in situ *implantome genesis is the dynamic product of a foreign-body ______ reaction of the host. The blended terms are fully free for scientific *corresponding author: use, but commercially trademark protected. Non-use of the Prof. Dr. H.P. Jennissen, Institut für Physiologische Chemie, Universität symbols  und  does not imply a waiver of trademark rights. Duisburg-Essen, Universitätsklinikum Essen, Hufelandstr. 55, D-45122 Essen, Germany; Email: hp.jennissen@uni-due.de Open Access. © 2019 Herbert P. Jennissen et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 License. H. P. Jennissen et al., Implantomics: A Paradigm Shift in Implantology — 132 3 Results and Discussion similar to fracture healing [21] and leading to implant integration [22]. Based exclusively on in vitro data this has 3.1 Protein Adsorption been the doctrine to the present day [23,24]. In their paper The basic mechanisms of protein adsorption were from 1987 on the Big Twelve proteins Andrade and Hlady elucidated in single protein adsorption systems. From [13] demonstrated 150 electrophoretic bands from plasma equilibrium and kinetic studies it has been concluded that and estimated that a cell contains ca. 5,000 proteins. Today's homogenous pure proteins are adsorbed to non-biological estimates with splice variants lie at ca. 70,000 human surfaces with high affinity by multivalent [6], cooperative [7] proteins. Current doctrine teaches that adsorption from blood non-covalent [8] interactions involving thermodynamically plasma leads to preferential adsorbates such as albumin, irreversible adsorption hysteresis [9,10] with binding fibrinogen and IgG [25], followed by blood coagulation, the 12 -1 affinities of K' ~ 10 M and higher (Fig. 1). In physically formation of a fibrin network as guide rail for oncoming cells controlled kinetic studies the adsorption of proteins is an e.g. Macrophages [26,27] and stem cells. exponential function beginning in milliseconds and reaching Implant surface properties also play a decisive role [28]. equilibrium in less than one minute [11]. Such surfaces are now available and classified either as ultra- /superhydrophilic displaying contact angles < 10° [29] or as Native Unfolded Denatured Protein hyperhydrophilic with complex and/or imaginary contact angles [30]. The rapid, spontaneous reversal from Free hydrophilicity to hydrophobicity can be prohibited for years in a dry state by an exsiccation layer of salt [31]. Bound 3.2 The Hip Implantome t = 0 t = t t =  3.2.1 The Origin of the Hip Implantome Nucleation complex | Conformation 2 | Conformation 3 (Conformation 1) 6 9 12 -1 K' 10 10 10 [M ] As detailed above, the adsorbed protein layer is a result Fig. 1. Multiple metastable protein states in hysteretic Model of the periimplant protein pool and the implant surface. The of protein adsorption [9,12,13]. The steps k , k , k are +2 +3 -5 primary implantome originates not only from the blood I-III slow and the steps k , k are fast. t = time, P = adsorbed -6 -7 plasma proteome [23,24], but from a mixture of blood plasma protein in 3 conformational states of increasing affinity (K ) with the periimplant operational humor (Humor operationis or operationl protein fluid), the composition of which is still In early attempts for understanding more complex systems. unknown. The operational humor depends on the operator Horbett et al. [14] and Cooper's group [15] analyzed pure and the operational and patient situation. Thus tissue injuries binary-protein adsorption systems, demonstrating compe- result, which release large amounts of intracellular proteins tition and heterologous protein-protein displacement on but are operationally mandatory. To a certain extent the polymers. On defined alkyl-residue lattices protein-protein situation cannot be planned. On the other hand in the future displacement was shown to be a form of negative coopera- an optimal implantome might be rationally prefabricated. tivity [16]. A novel observation in binary systems is "protein interference" [17]. Much earlier Vroman had studied more complex multi-protein adsorption systems of fibrinogen from 3.2.2 The Primary Implantome and the Early Bone- blood plasma [18] (Vroman effect). Because of the immense Implant Interface analytical problems this work had been largely discontinued. This has now changed. Today the highest imaginable scale of The bone-implant interface in general, and for titanium complexity in the form of proteomic-scale or myriad-protein implants specifically, possesses biochemical, biological and adsorption on surfaces has become a reality [4], allowing mechanical aspects which must be experimentally assessed. thousands of individual proteins to be monitored In literature searches very little can be found on the early simultaneously during adsorption and desorption. interface healing stages and biochemistry appears to be an At the advent of the field of biomaterials in the 1970ies implantomic and interfacial orphan. and 1980ties it was without question that the first proteins The initial protein layer on an implant, i.e. the primary adsorbed on an implantable device originate from blood implantome, is the crucial host response and an integral part plasma [13,19,20] initiating periimplant endosseous healing of the initial bone-implant interface during the earliest H. P. Jennissen et al., Implantomics: A Paradigm Shift in Implantology — 133 healing stage. An early paper on the composition of the bone- implatome [4] was the zinc-finger protein family with over metal interface was described nine days after implantation in 120 different protein entities containing the three high a rat model by Donath et al. [26] and appears to be 7-15 µm abundance proteins ZNF35, ZNF470 and ZNF 850. wide. The earliest cells to appear after 3 days are histiocytes 3.2.3 Evolution from the Primary Implantome to and multinucleate giant cells, probably related to M2 type the Integratome in the Bone-Implant Interface macrophages in agreement with a macrophage model of osseointegration (see ref. [27,32,33]). A first step has now been made in the pilot determination In the current paper by Jäger et al. [4] it was found that of the primary implantome on a hip implant after 2 min in the primary human hip implantome, at a post-insertion time situ [4]. This protein layer is a principle component of the of 2 min, consisted of 2802 (#peptide number ≥ 2) unique first "cell-containing bone-implant interface" What do we know about the last stage, the bone-implant interface of Table 2 integration (i.e. integratome). Usually it is equated with the [1] Profile of Plasma Proteome in the hip Implantome bone-implant contact, i.e. BIC. The residual interface gap in Abundance [ppm] the BIC region is so small, that cells can no longer enter, Name Symbol Nr mean SD which results in a "cell-free bone-implant interface". This by 1 Serum albumin ALB 48927 17300 no means indicates that an implant proteome is absent from 2 Alpha-1-antichymotrypsin SERPINA3 2342 1174 this interface! What is also often forgotten is, that the BIC 3 Titin TTN 1961 98 4 Carbonic anhydrase 1 CA1 1888 21 generally only covers 50-60% of the integrated bone-implant 5 Collagen alpha-1(V) chain COL5A1 1820 1486 interface [34]. What about the other 40-50% of the non-BIC 6 Catalase CAT 1678 947 regions in this area? Are they part of implant integration and 7 Keratin, type II cytoskeletal 1 KRT1 1540 2002 the integratome? Recently a comprehensive review of the 8 Fibrinogen beta chain FGB 1518 529 SH3 domain-binding glutamic largely submicroscopic evidence obtained for the bone- 9 SH3BGRL2 1411 1946 acid-rich-like protein 2 implant-interface in the BIC region of integration has 10 Keratin, type I cytoskeletal 10 KRT10 1359 811 appeared [3]. It is concluded that the bone-implant interface 11 Serotransferrin TF 1312 256 12 Fibrinogen gamma chain FGG 1161 248 zone at this stage is primarily fibrillar with an electron dense [1] Data from supplementary Table 1 ref. [4]. Hemoglobin, the 20-50 nm thick layer of collagen fibrils and an additional most abundant protein, is not shown, because it is an finely fibrillar mineralized matrix of ca. 200 nm thickness, intrinsically intracellular protein. i.e. in sum 250 nm in width (i.e. enough room for an implantome). Generally only four proteins are repeatedly proteins of which numerically 77% were of intracellular named in the bone-implant-interface of an integrated implant: origin and only 9% (i.e. 247 proteins) from blood plasma [4] collagen, bone silaoprotein, osteopontin and osteocalcin. (see Table 2). The intracellular proteins of the implantome Three major biological phenomena thus appear notable originated from the bone, the bone marrow, the blood cell in periimplant healing: (i) a large, dynamic cell containing and the plasma proteomes respectively. Surprisingly the most bone-implant interface measuring 20-100 µm in width scales abundant implant protein in the implantome was hemoglobin down 100-400-fold (= compaction) during integration to a (10%) and not serum albumin (5%). Fibrinogen and IgG (ii) long-term constant, cell-free bone-implant interface with were absent from the most abundant first 36 proteins (see a width of ~200-500 nm (BIC) [3], characterized probably by also [14]). Thus the in vitro evidence for the doctrine plasma a steady-state turnover of implantome proteins and (iii) to proteins forming the initial protein layer [25], did not non-BIC marrow-type (?) spaces. From this large interface "translate" into the human in vivo proteome. Thus an old compaction it can speculated, that there may be a parallel paradigm failed, at least in hip implantology. Nevertheless, reduction in the number of proteins but not to a complete the sequence of events strongly suggests, that the implantome disappearance of the implantome per se. per se mediates fundamental cell-biomaterial interactions. Interestingly the importance of proteins in Screening through the supplementary Table 1, deposited biocompatibility, and with that in implantology as a whole, is with the paper in ref. [4], some other additional details strongly disputed: "clinically, protein adsorption is of minor emerged. The only proteins related to bone healing were importance in biocompatibility pathways" [2], and the BMP-1, a protease, and inhibitors of BMP-2 i.e. Chordin, and "adsorption of macromolecules has only minimal effects in Gremlins 1 & 2. The mediators BMP-2, VEGF, TGF- and biocompatibility" [2]. In addition it is stated in the same TNF- were not present in the implant proteome at all. The paper that surface modification technologies in implantology largest single family of proteins contained in the hip – with the possible exception of nanostructures – also only H. P. Jennissen et al., Implantomics: A Paradigm Shift in Implantology — 134 [5] Wasinger, V. C., Cordwell, S. J., Cerpa-Poljak, A., Yan, J. play a minor role [2]. This reasoning insinuates that proteins X., Gooley, A. A., Wilkins, M. R., Duncan, M. W., Harris, neither play a significant role in biocompatibility nor in R., Williams, K. L., & Humphery-Smith, I. (1995) periimplant healing and osseointegration. At present this Electrophoresis, 16, 1090-1094. cannot be disproven, but it is at odds with reason and many [6] Jennissen, H. P. (1976) Biol. Chem. (formerly: Hoppe- Seyler's Z. Physiol. Chem. ), 357, 1201-1203. literature findings. One important point appears to have been [7] Jennissen, H. P. (1976) Biochemistry, 15, 5683-5692. overlooked, namely, that not only the fact alone of obtaining [8] Jennissen, H. P. (2005) Methods Mol. Biol., 305, 81-99. implant biocompatibility and/or integration, but also the [9] Jennissen, H. P. & Botzet, G. (1979) Int J Biol Macromol, 1, time-frame i.e. the rates of healing and osseointegration are 171-179. [10] Jennissen,H.P. (1985) Protein Adsorption Hysteresis. In of utmost importance, especially in connection with hospital "Surface and Interfacial Aspects of Biomedical Polymers" costs. That is where the implantome protein composition may Vol.2, Protein Adsorption (Andrade,J.D., ed), pp. 295-320. very effectively come in. Plenum Press, New York. [11] Jennissen, H. P. & Zumbrink, T. (2004) Biosens. Bioelectron., 19, 987-997. Conclusions [12] Jennissen, H. P. (1988) Makromol Chem, Macromol Symp, It is the aim and mission of implantomics to gain 17, 111-134. fundamental insights into proteome function by clarifying [13] Andrade, J. D. & Hlady, V. (1987) Ann. N. Y. Acad. Sci., 516, 158-172. new pathways of implantome maturation from the primordial [14] Horbett, T. A., Weathersby, P. K., & Hoffman, A. S. (1977) implantome over intermediary implantomes to the final J Bioengineering, 1, 61-78. integratome. It is essential to elucidate the role of implant- [15] Pitt, W. G., Young, B. R., Park, K., & Cooper, S. L. (1988) specific implantomes in biocompatibilty and osseointegration Makromol. Chem. , Macromol. Symp., 17, 453-465. [16] Jennissen, H. P. (1986) J colloid Interface Sci, 111, 570-586. as well as to employ this information for future artificial [17] Jennissen, H. P. (2019) Curr. Dir. Biomed. Engineer., 5, in implantome designs. A high priority should also be given to print. diagnostics, for an understanding of the mechanisms of [18] Vroman, L. & Adams, A. L. (1969) J Biomed. Mater Res, 3, premature implant loosenings and failures by generalized, 43-67. [19] Brash,J.L. & Lyman,D.J. (1971) Adsorption of Proteins and systematic explantome research and analyses. Lipids to Nonbiological Surfaces. In The Chemistry of Biosurfaces (Hair,M.L., ed), pp. 177-229. Marcel Dekker, Acknowledgements New York. The support of the German Research Foundation (Deutsche [20] Andrade,J.D. (1985) Principles of Protein Adsorption. In "Surface and Interfacial Aspects of Biomedical Polymers" Forschungsgemeinschaft; DFG Reference No. Je84/15-3) is Vol.2, Protein Adsorption (Andrade,J.D., ed), pp. 1-80. gratefully acknowledged. (https://orcid.org/0000-0002-2615-0184) Plenum Press, New York. [21] Davies, J. E. (2003) J. Dent. Educ., 67, 932-949. Abbreviations: [22] Branemark, P. I., Hansson, B. O., Adell, R., Breine, U., Lindstrom, J., Hallen, O., & Ohman, A. (1977) Scand. J. BIC, bone implant contact; BMP-2, bone morphogenetic Plast. Reconstr. Surg. Suppl, 16, 1-132. protein 2; LC-MS/MS, LC (Liquid Chromatography), MS [23] Kuzyk, P. R. & Schemitsch, E. H. (2011) Indian J. Orthop., (Mass Spectrometry), MS/MS tandem mass spectrometry ; 45, 108-115. VEGF, vascular endothelial growth factor; SEM, scanning [24] Tengvall, P. (2011) Compr. Biomater., 4, 63-73. [25] Kanagaraja, S., Lundstrom, I., Nygren, H., & Tengvall, P. electron microscopy; TEM, transmission electron (1996) Biomaterials, 17, 2225-2232. microscopy; TPS, titanium plasma sprayed. [26] Donath, K., Kirsch, A., & Osborn, J. F. (1984) Fortschr. Zahnärztl. Implantol., 1, 55-58. Author Statement [27] Jennissen, H. P. (2016) Curr. Dir. Biomed. Engineer., 2, 53-56. The Author states no conflict of interest. [28] Schwarz, F., Wieland, M., Schwartz, Z., Zhao, G., Rupp, F., Geis-Gerstorfer, J., Schedle, A., Broggini, N., Bornstein, M. M., Buser, D., Ferguson, S. J., Becker, J., Boyan, B. D., & 4 References Cochran, D. L. (2009) J. Biomed. Mater. Res. B Appl. Biomater., 88, 544-557. [1] Kuhn,T.S. (1962) The Structure of Scientific Revolutions. In [29] Jennissen, H. P. (2001) Bionanomaterials (formerly: Third Edition 1996, pp. 1-212. University of Chicago Press, Biomaterialien), 2, 45-53. Chicago, London. [30] Jennissen, H. P. (2012) Materialwiss. Werkstofftech. [2] Williams, D. F. (2016) ACS Biomater. Sci. Eng., 3, 2-35. (Mater. Sci. Eng. Technol), 43, 743-750. [3] Shah, F. A., Thomsen, P., & Palmquist, A. (2019) Acta [31] Jennissen, H. P. (2010) Materialwiss. Werkstofftech. Biomater., 84, 1-15. (Mater. Sci. Eng. Technol. ), 41, 1035-1039. [4] Jäger, M., Jennissen, H. P., Haversath, M., Busch, A., [32] Trindade, R., Albrektsson, T., Galli, S., Prgomet, Z., Grupp, T., Sowislok, A., & Herten, M. (2019) Proteomics. Tengvall, P., & Wennerberg, A. (2018) Clin. Implant. Dent. Clin. Appl., e1800168. Relat Res., 20, 82-91. H. P. Jennissen et al., Implantomics: A Paradigm Shift in Implantology — 13 5 [33] Schlundt, C., El, K. T., Serra, A., Dienelt, A., Wendler, S., Schell, H., Van, R. N., Radbruch, A., Lucius, R., Hartmann, S., Duda, G. N., & Schmidt-Bleek, K. (2018) Bone, 106, 78-89. [34] Romanos, G. E., Toh, C. G., Siar, C. H., Wicht, H., Yacoob, H., & Nentwig, G. H. (2003) J Periodontol., 74, 1483-1490.

Journal

Current Directions in Biomedical Engineeringde Gruyter

Published: Sep 1, 2019

Keywords: implant proteome; bone-implant interface; biocompatibility; osseointegration; peri-implant bone healing

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