Shanbhag et al.

International Journal of Implant Dentistry (2015) 1:5

DOI 10.1186/s40729-015-0006-2

REVIEW Open Access

Genomic analyses of early peri-implant bone

healing in humans: a systematic review
Siddharth Shanbhag1,2, Vivek Shanbhag2 and Andreas Stavropoulos1*

Abstract
Objective: The objective of the study was to systematically review the literature for studies reporting gene expression
analyses (GEA) of the biological processes involved in early human peri-implant bone healing.
Methods: Electronic databases (MEDLINE, EMBASE) were searched in duplicate. Controlled and uncontrolled studies
reporting GEA of human peri-implant tissues - including ≥5 patients and ≥2 time points - during the first 4 weeks of
healing were eligible for inclusion. Methodological quality and risk of bias were also assessed.
Results: Four exploratory studies were included in reporting GEA of either tissues attached to SLA or SLActive implants
after 4 to 14 days or cells attached to TiOBlast or Osseospeed implants after 3 to 7 days. A total of 111 implants from 43
patients were analyzed using validated array methods; however, considerable heterogeneity and risk of bias were
detected. A consistent overall pattern of gene expression was observed; genes representing an immuno-inflammatory
response were overexpressed at days 3 to 4, followed by genes representing osteogenic processes at day 7. Genes
representing bone remodeling, angiogenesis, and neurogenesis were expressed concomitantly with osteogenesis.
Several regulators of these processes, such as cytokines, growth factors, transcription factors, and signaling pathways,
were identified. Implant surface properties seemed to influence the healing processes at various stages via differential
gene expression.
Conclusion: Limited evidence from gene expression studies in humans indicates that osteogenic processes
commence within the first post-operative week and they appear influenced at various stages by implant
surface properties.
Keywords: Dental implant; Osseointegration; Gene expression; Molecular assessment

Review microenvironment (i.e., cells, signaling molecules, and

Introduction matrix) into which the implant is placed and have many
Osseointegrated oral implants are an integral part of similarities with general wound healing mechanisms [7].
modern reconstructive dentistry and are associated with Implant surgery induces trauma, resulting in bleeding
favorable long-term therapeutic outcomes [1]. Osseoin- and fibrin clot formation and an inflammatory reaction
tegration was originally defined as the direct contact be- that dominate the events of the first post-operative week.
tween vital bone and a load-bearing implant observed at The deposition of vital new bone on the implant surface
the light microscopic, i.e., histological, level [2]. Morpho- by osteoblasts (osteogenesis), a fundamental requirement
genesis of implant osseointegration has been assessed in for osseointegration, occurs via secretion of a complex
several preclinical in vivo and clinical histological studies extracellular matrix (ECM) of proteins, which subsequently
[3-6], providing the basis for understanding the bio- undergoes mineralization to form bone [8,9]. Primary
logical process. (woven) bone lined by osteoblasts can indeed be observed
The biological events during the early phase of on the implant surface already after 1 week [3,5]. In
osseointegration are directly influenced by the osseous parallel, removal of the created bone debris and remod-
eling of necrotized bone (due to the pressure exerted
* Correspondence: [email protected] by the implant) is underway. Replacement of woven
1
Department of Periodontology, Faculty of Odontology, Malmö University,
Carl Gustafs väg 34, 214 21 Malmö, Sweden bone by organized and mechanically superior lamellar
Full list of author information is available at the end of the article

© 2015 Shanbhag et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly credited.
Shanbhag et al. International Journal of Implant Dentistry (2015) 1:5 Page 2 of 12

bone can be observed from the second to fourth week available literature on gene expression analyses of the
(depending on the species) and progressively increases biological processes involved in early human peri-implant
until woven bone is almost entirely replaced (8 to 12 weeks). bone healing.
These events, including the nutrition of the newly formed
tissue, are sustained through concomitantly occurring Methods
angiogenesis, i.e., formation of new blood vessels from Study design
existing ones [10,11]. Thus, osseointegration is a dynamic A study protocol for a systematic qualitative literature
process whereby bone formation and remodeling occur in review was developed based on recommended methods
parallel around the implant [4,6]. [29]. The focused question was ‘what biological processes
Morphogenesis of osseointegration and assessment of are reflected by gene expression analyses in peri-implant
the degree of bone-to-implant contact is usually performed tissues of humans during the early stages (up to 4 weeks)
by means of histological evaluation [12], while the under- of healing?’
lying molecular processes may be more precisely evaluated
at genetic level [13,14]. Data from gene expression analyses Inclusion and exclusion criteria
of fracture healing provide the basis for understanding All studies, controlled (using different implants) or un-
these processes [15]. These studies have identified the cells, controlled, reporting gene expression analyses of peri-
signals, and interactions governing the key processes of implant tissues harvested from ≥5 human patients at ≥2
bone regeneration. Bone-forming osteoblasts are primarily time points during the first 4 weeks of healing, were
derived from marrow-resident multipotent progenitor cells eligible for inclusion. Studies reporting the use of either
(mesenchymal stem cells (MSCs)), which are recruited to ‘experimental’ (micro) or standard implants with clear
the regeneration site. This process of MSC recruitment description of implant surface properties, placed in the
and differentiation along the osteogenic lineage is termed maxilla or mandible and retrieved at a later time point,
as osteoinduction and is controlled primarily by various were eligible for inclusion. Studies reporting (1) analyses
pro/anti-inflammatory cytokines (CKs) and by growth of peri-implant mucosa or sulcular fluid or peri-implant
factors (GFs) secreted by inflammatory cells and/or os- tissues of failing or infected implants (peri-implantitis), (2)
teoblasts or by GF resident within the extracellular matrix only histological or immunohistochemical analyses with-
(e.g., bone morphogenetic proteins (BMPs)) in response to out gene expression of harvested tissues, and (3) in vitro
injury [16-18]. Moreover, CKs and GFs act as signaling and preclinical in vivo studies were excluded. Primary out-
molecules via specific signaling pathways and guide the come of interest was the biological process (or processes)
process of cell differentiation in the proper temporal se- reflected by gene expression at a particular time point of
quence [19,20]. Intermediaries in this process are various peri-implant tissue healing.
bone-specific transcription factors (TFs), which act as
‘molecular switches’ during cell differentiation and are Search strategy
targets of CKs and GFs [21]. TFs facilitate bone-specific Electronic databases of MEDLINE (via PubMed) and
gene transcription and ultimately gene expression by EMBASE were searched by one author (SS) for rele-
which MSCs undergo differentiation and acquire the vant English-language literature up to and including
osteoblastic phenotype [22]. While GFs regulate mainly June 2014. The search strategy used for MEDLINE was
osteoinduction and osteogenesis, pro-inflammatory CKs ((((("gene expression" OR transcriptome OR transcriptional
regulate the antagonist process of bone resorption by OR molecular OR microarray))) AND ((osseointegration
inducing the differentiation of hematopoietic stem cells OR healing OR "peri implant"))) AND implants) AND
(HSCs) into osteoclasts and macrophages [23], contrib- ((human OR humans OR patients OR subjects)). Unpub-
uting to the dynamic nature of bone regeneration and lished literature was searched via the Google and Google
remodeling. Scholar search engines. Additionally, the bibliographies of
Recent in vitro [24] and preclinical in vivo [25] studies all relevant studies and review articles were searched.
have focused on the early molecular biological responses
to various titanium implant surfaces. Understanding these Study selection
early responses is essential for efforts aiming to accelerate Titles and abstracts of the search identified studies were
and enhance the process of osseointegration [26]. Upregu- screened by two authors (SS and VS) based on the inclu-
lation or downregulation of specific genes in peri-implant sion criteria, and full texts of all eligible studies were
tissues identified by analyses of genetic material (DNA, obtained. Differences in assessment of eligibility were
RNA) reflects the nature and timing of the various healing resolved by discussion with the third author (AS). Full
processes, which in turn could be potential ‘molecular texts were independently reviewed by both reviewers,
targets’ for enhancing osseointegration [27,28]. The aim and final inclusion was based on the aforementioned
of the present study was to systematically review the inclusion criteria.
Shanbhag et al. International Journal of Implant Dentistry (2015) 1:5 Page 3 of 12

Data extraction guidance [33] regarding the qualitative and quantitative

Both reviewers independently extracted data from the syntheses of results from genetic association studies was
full texts of included articles using specially designed forms. consulted, and heterogeneity across the included studies
Data on author(s), study design, implant type/surface, was assessed to explore the possibility of a meta-analysis.
any additional procedures performed, number of patients
(in each group), presence of a control group, procedure Results and discussion
and time of implant retrieval, methods of gene expression The included studies basically report on commercially
analysis, and main results, were extracted. Descriptive available implants from two major manufacturers and in-
summaries of the studies were entered into tables, and volve comparisons of different implant surface technologies
a qualitative synthesis of evidence was planned. Any in regard with topography and/or chemistry modifications
disagreement between the reviewers regarding data ex- within each implant system. Various analyses were per-
traction was resolved by discussion. formed in the included studies; however, an attempt has
been made to synthesize the various findings and discuss
Assessment of methodology and risk of bias them herein irrespective of the specific implant systems,
Assessment of the methodological validity of the included based on the assumption that basic biological mechanisms
studies was performed using criteria adapted from previ- of peri-implant bone wound healing are largely implant
ous reports [30,31]. Aspects of study design, genotyping system independent.
methods, and data analyses were considered using nine
criteria (Table 1). Search results and study characteristics
The risk of bias in the included studies was assessed Of the 242 search identified studies, only four studies
using an adaptation of published guidelines for reporting were finally included in the review, all focusing on the
systematic reviews of periodontal genetic association stud- impact of implant surface on early human peri-implant
ies [32]. Mainly, aspects of study design and methodo- bone healing (Figure 1; Table 3). Genetic analyses of total
logical validity were assessed using 15 criteria and scored RNA isolated from either newly formed peri-implant bone
as ‘yes,’ ‘no,’ or ‘unclear’ based on the information provided harvested by trephination [34,35] or from cells adherent
in the study manuscript (Table 2). Moreover, published to implants retrieved by reverse threading [36,37] were
Table 1 Assessment of the genotyping methodology in the included studies
Methodology Ivanovski et al. [34] Donos et al. [35] Bryington et al. [36] Thalji et al. [37]
Tissue harvesting Tissue attached to implant Tissue attached to implant Implants removed by reverse Implants removed by reverse
carefully removed with a carefully removed with a threading and homogenized; threading and homogenized;
curette, preexisting hard curette and homogenized cell lysates isolated cell lysates isolated
tissue discarded
Sample preparation Total RNA isolation, Total RNA isolation, Total RNA isolation, Total RNA isolation, purification,
purification, quantity/ purification, quantity/ quantification quantity/quality analysis and
quality analysis and quality analysis and biotin-labeling
biotin-labeling biotin-labeling
Array technique Microarray hybridization Microarray hybridization RT-PCR (custom RT-PCR array Microarray hybridization (Affymetrix
(Human WG-6 V3) (Human WG-6 V3) for osteogenesis genes; human Human Gene 1.1 ST)
inflammatory cytokines and
receptors PCR array)
Scanning, data Bead Station 500/Bead Bead Station 500/ Bead RT2 SYBR Green qPCR Master Affymetrix Gene Chip Scanner
preparation Studio v3 software, raw Studio v3 software, raw Mix/7500 Real-Time PCR system
probe expression values probe expression values
extracted extracted
Processing Noisy data discarded Noisy data discarded Normalization of osteogenesis Unclear
and cytokine array
Clustering GO categories (DAVID GO categories (DAVID tool) Osteogenesis genes; cytokine- GO categories (Gene Spring)
tool) related genes
Statistical analysis Gene Spring software Gene Spring software RT2 Profiler software Gene Spring software
Comparisons Pair-wise comparisons Pair-wise comparisons at T-test to evaluate differences Two-way ANOVAs to determine
between three time each time point (4, 7, and between each implant surface differences between implant
points (4 vs. 7 days, 7 14 days) between SLA and per time point surface type and time points;
vs. 14 days, and 4 vs. SLActive surfaces pair-wise comparisons of each
14 days) implant surface independently
at different time points (day 7
vs. day 3)
GO, gene ontology; DAVID, Database for Annotation, Visualization and Integrated Discovery.
Shanbhag et al. International Journal of Implant Dentistry (2015) 1:5 Page 4 of 12

Table 2 Assessment of risk of bias and heterogeneity within and across the included studies
Category Ivanovski et al. [34] Donos et al. 2011 [35] Bryington et al. [36] Thalji et al. [37]
Study design
Comparison None (only SLActive) SLA vs. SLActive TiOBlast vs. Osseospeed TiOBlast vs. Osseospeed
Setting University University University University
Population, inclusion 9 healthy volunteers with 9 healthy volunteers with 6 women, 4 men; implant 9 women, 2 men; implant
criteria no mandibular third molars, no mandibular third patients, systemically healthy patients, systemically healthy;
no contraindications for oral molars; age 21 to 48, (no HTN, diabetes, CVD); age age 47 to 69, mean 60.2 years
surgery; age 21 to 48, median 29 years 25 to 58, mean 36.2 years
median 29 years
Exclusion criteria Smokers Smokers Smokers, pregnancy, Smokers, uncontrolled
periodontal/periapical diabetes, history of
disease, subjects taking head/neck radiotherapy,
bisphosphonates, taking corticosteroids,
hormone replacement bisphosphonates
therapy, corticosteroids
Comparability of groups Unclear Unclear Unclear Unclear
Potential confounders, Unclear Unclear Unclear Unclear
e.g., post-op medication
Power calculation No No No No
Statistical correction For multiple sampling For multiple sampling Unclear For multiple sampling
Methods
Tissue analyzed Peri-implant tissue Peri-implant tissue Implant-adherent cells Implant-adherent cells
Genetic material Total RNA Total RNA Total RNA Total RNA
analyzed
Success rate Unclear 16/18 samples (88.8%) 7/10 subject samples (70%) Unclear
Genotyping method Microarray Microarray RT-PCR Whole-genome microarray
Genotype counts Yes Yes Yes Yes
Blinding Unclear Unclear Yes Unclear
Reproducibility, validated No No No No
genotyping accuracy
All studies were judged to be at a high risk of bias with substantial heterogeneity across studies.

performed. In total, 111 implants from 43 patients were hydrophilic (SLActive®) versus a hydrophobic unmodi-
analyzed. All four studies reported the use of commercially fied SLA® (Institute Straumann AG, Basel, Switzerland)
existing implant surfaces, i.e., either a chemically modified, surface; or a micro-topographic titanium-oxide grit-
hydrophilic, sand-blasted, acid-etched surface (SLActive®, blasted surface (TiOBlast®, AstraTech, Molndal, Sweden)
Institute Straumann AG, Basel, Switzerland); or a versus a chemically modified nano-topographic grit-blasted

Figure 1 Flowchart for study selection (n = number of studies).

Shanbhag et al. International Journal of Implant Dentistry (2015) 1:5 Page 5 of 12

Table 3 Summary of findings from the included studies (n = 4)

Study Ivanovski et al. [34] Donos et al. [35] Bryington et al. [36] Thalji et al. [37]
Design 9 patients; 9 implants placed 18 patients; 18 implants 10 patients; 60 implants 11 patients; 44 implants placed
placed placed
Total RNA extracted from 16 samples analyzed 42 samples analyzed Total RNA extracted from implant
peri-implant tissue adherent cells (reverse thread)
(trephine)
Total RNA extracted from Total RNA extracted from
peri-implant tissue (trephine) implant adherent cells
(reverse thread)
Surface SLActive SLA vs. SLActive TiOBlast vs. Osseospeed TiOBlast vs. Osseospeed
GE day 3/4 Upregulated Upregulated on SLA Upregulated on both surfaces No significant differences between
surfaces at any time point (P > 0.05)
CKs (TNF-a, IL-6, IL-2) Neurogenesis Osteogenesis (Runx2, Osx, Results presented as GE at day 7 vs.
BMP6, OPN) day 3 for each surface
Immune-inflammatory cells Collagen organization Inflammatory CKs (IL-1A,B,
(LC, MP) TNF)
Inflammatory NF-kB p/w Upregulated on SLActive MP activity
Ras protein p/w Upregulated on Osseospeed
Collagen organization Chemotaxis (CCL18, CXCL10,
CXCL14)
CK response Anti-inflammatory CKs
(TOLLIP, IL9, IL22)
GE day 7 Upregulated Upregulated on both surfaces Upregulated on both surfaces Upregulated on both surfaces
MSC genes (HOX, Sp3) Inflammatory CKs (IL1, IL2, Osteogenesis (Runx2, Osx*, ECM (Coll, GPs, PGs)
IL6, TNFS) OCN*, OPN, BMP6, BSP)
GF (TGF-B receptor) Neurogenesis [* Osseo > TiOB; P < 0.05] Collagen organization (PLODs, LOX,
PCOLCE)
VEGF sig. (vs. day 14) Upregulated on SLActive Angiogenesis/VEGF sig. (ANXA,
EPAS1)
Wnt p/w Neurogenesis (BDNF, Ossification
NTF3)
Downregulated ECM (OPN) Remodeling (MMPs, TIMPs)
Inflammatory NF-kB p/w BMP p/w (BMP4, BMP2K) Osteoclastic (CTSK, ACP5)
(vs. day 4)
MAPK sig. Chemotaxis (CKs, MP activity)
Mineralization Anti-inflammatory CKs (CCL22,
CCL18)
Focal adhesion (integrins) Downregulated on both surfaces
Angiogenesis (VEGF sig., Inflammatory CKs (IL1A, IL1B)
P13-Akt p/w)
Downregulated on SLActive
Inflammatory cells (LC)
GE day 14 Upregulated Upregulated on both -
surfaces
ECM (Coll, OC, ON, ALP) BMP p/w (BMP4, BMP2K)
TFs (Osx, Dlx5, Twist1, Smad6) Downregulated on both
surfaces
Remodeling (MMP, CTSK) Inflammatory cells (LC)
GFs (BMP, GDF)
Angiogenesis (VEGF sig.)
Neurogenesis
Shanbhag et al. International Journal of Implant Dentistry (2015) 1:5 Page 6 of 12

Table 3 Summary of findings from the included studies (n = 4) (Continued)

TGF-b/BMP, Notch p/w
Ras protein p/w
Wnt-receptor genes
Notch genes (up/down)
Downregulated
Inflammatory response
(vs. day 7)
GE, gene expression; CKs, cytokines; p/w, pathway; MSC, mesenchymal stem cells; GF, growth factors; sig., signaling; ECM, extracellular matrix; TFs, transcription
factors; MP, macrophage; LC, lymphocytes; GPs, glycoproteins; PGs, proteoglycans.

surface (Osseospeed®, AstraTech, Molndal, Sweden). Biological processes identified through gene expression
Implant retrieval times were at 3 or 4 days and in peri-implant tissues
7 days in all studies and additionally at 14 days in Conventional implant surgery involves osteotomy prep-
two studies [34,35]. aration and insertion of the implant into the alveolar bone.
The immediate local effects of this procedure, functionally
Assessment of methodology and risk of bias relevant to subsequent healing processes, are (1) bone
All studies used validated methods for gene expression trauma, (2) formation of bone debris, (3) hemostasis and
analysis; genetic data was analyzed using microarray (three clot formation, and (4) hypoxia. These effects involve
studies) or real-time PCR (RT-PCR) (one study) methods the release of specific CKs and GFs within the local en-
(Table 1). Total RNA was isolated from lysates of either vironment [7], resulting in recruitment of two primary
trephined peri-implant tissues or implant-adherent cells, cell types to the site, inflammatory cells and progenitor
and subjected to microarray processing or RT-PCR. cells (MSCs and HSCs) [19], which in turn regulate the
Although moderate-to-good agreement has been re- subsequent healing processes. A summary of differen-
ported between the two methods, validation of DNA tially regulated genes relating to the involved biological
microarray results by the more sensitive PCR array is processes is presented in Table 4, while Figure 2 repre-
generally recommended [38]. None of the microarray sents an evidence-based illustrative model summarizing
studies identified have validated their results using these processes.
RT-PCR. Genotyping data (gene lists) were imported
and analyzed using computer software and further Inflammation
condensed into functionally and biologically relevant All studies reported a significant upregulation of genes
categories. Nevertheless, differential gene expression associated with inflammation during the first time point
in relation to a particular cell type or region of tissue of observation (day 3 or 4) regardless of the implant sur-
analyzed was not performed [35]. Gene ‘upregulation’ face. Specifically, upregulation regarded pro-inflammatory
was reported when genes were expressed at a higher cytokines of the interleukin (IL), tumor necrosis factor
level on one implant surface in comparison to another; in (TNF), and interferon (IFN) families, as well as genes asso-
context, differentiation between gene expression and over- ciated with proliferation of lymphocytes and macrophages
expression may be difficult to define. Statistical methods (MPs). Previous in vitro [40,41] and animal [42] studies
were used to compare differences in gene expression have reported the significance of MPs at the bone-implant
between different time points and/or implant surfaces interface and identified favorable MP activity in relation to
(P < 0.05 significance level), while correcting for possible modified rough surfaces as demonstrated by in vitro gene
errors, i.e., false gene discovery rate due to multiple sam- expression that was associated with increased in vivo bone
pling [39]. There was considerable heterogeneity across formation. Also, the nuclear factor-kB (NF-kB) inflamma-
the included studies in terms of study design, population, tory pathway was upregulated at day 4 [34], while macro-
implant surface technology, genotyping methods, and data phage activity and chemokines of the CCL and CXL
analyses (Table 2). Therefore, no meta-analysis of associ- families in the peri-implant tissues continued to remain
ation between gene expression and implant surface prop- prominent at day 7.
erties was relevant. However, this inflammatory response was generally down-
Thus, high risk of bias should be considered when regulated at later time points (day 7 or 14). For example, in
interpreting the results, due to the above methodological one study, genes associated with pro-inflammatory cytokines
limitations and the overall limited information (four (IL-1B, IL-1A, IL-1R2) and chemokines (CCL22, CCL18)
studies) available. were downregulated and upregulated, respectively, on day 7,
Shanbhag et al. International Journal of Implant Dentistry (2015) 1:5 Page 7 of 12

Table 4 Summary of biological processes and associated Table 4 Summary of biological processes and associated
genes reported in the included studies genes reported in the included studies (Continued)
Process Upregulated genes Osteoclast activity/ Cathepsin K (CTSK, CTSK-receptor)
Category (gene code) remodeling
Tartarate-resistant acid phosphatase (TRAP/ACP5)
Inflammation/immune response
Matrix metallopeptidase (MMP2, MMP12, MMP9,
Pro-inflammatory Tumor necrosis factor (TNF-a, TNFSF9) MMP7, MMP13)
cytokines
Interleukin (IL-6, IL-2, IL-1 F9, IL-23A, IL-6ST) Tissue inhibitor metallopeptidase (TIMP2, TIMP3)
Interferon (IFNA2) Angiogenesis Vascular endothelial GF-signaling (EPAS1, ANXA2,
EGR1-binding protein)
Nuclear factor-kB (I-kB kinase/NF-kB)
Phosphatidyl-inositol 3-kinase (PI3K)-Akt signaling
Anti-inflammatory Interleukin (IL-22, IL-9)
cytokines Neurogenesis Brain-derived neurotrophic factor (BDNF)
Toll interacting protein (TOLLIP)
Neurotrophin 3 (NTF3)
Cells Lymphocyte, macrophage negative proliferation
(BTLA, LST1) NK2 homeobox 2(NKX2-2)
Macrophage scavenger receptor (MSR1) Tubby-like protein 3 (TULP3)
Chemotaxis Chemokines (CCR8, CCL18, CCL22, CXCL10,
CXCL14)
Osteoinduction/osteogenesis
at both implant surface technologies examined (Osseospeed
and TiOBlast) [37]. Moreover, the anti-inflammatory re-
Growth factors (GF)/ Insulin-like GF (IGF1)
signaling pathways sponse seemed to be modulated by surface properties. In
Transforming GF (TGF-b, TGF-b receptor 1, 2 one study, genes related to anti-inflammatory cytokines such
and 3, TGF-a)
as IL-9, IL-22, toll-like receptor inhibitor protein (TOLLIP),
Platelet-derived GF (PDGF receptor)
and several key chemokines (CCL18, CXCL10, CXCL18)
Bone morphogenetic proteins (BMP4, BMP6, were significantly upregulated on Osseospeed surfaces but
BMP receptor 1A, BMP2-K)
not TiOBlast, at day 3 [36]. In another study, genes associ-
Growth and differentiation factor (GDF10) ated with inflammatory cell proliferation were significantly
Wnt frizzled receptor (FZD3, FZD8, FRZB) downregulated earlier on SLActive surfaces compared to the
Notch (NOTCH2) SLA, i.e., at day 7 instead of day 14 [35]. Therefore, the
Ras-protein signal transduction (RAP1B, RAP1A, initial inflammatory response seems to be important for
RASGRP4) the recruitment of cells that govern subsequent healing
Mitogen activated protein kinase (MAP3K7IP2, processes and is regulated by a natural biological immune
MAPK9, MAP2K3, MAP3K2) response which may be further modified by implant
Transcription ‘Master switches’ [RUNX2, SP7 (OSX)] surface properties.
factors
Homeobox (DLX1, DLX5, HOXD12, MSX1,
HOXA5, HOXB1, HOXB6, HOXC6) Osteogenic differentiation
SP [SP1, SP3, SP7 (Osx)] Cells along the osteogenic differentiation pathway may
Twist (TWIST 1-receptor) be artificially categorized as (1) undifferentiated MSCs,
(2) osteo-chondro-progenitor cells, (3) pre-osteoblasts,
ECM deposition/ Collagen (Col1A1, Col12A1, Col6A3, Col3A1,
organization Col6A1, Col11A1, Col11A2, Col13A1, Col5A2) and (4) osteoblasts; although in reality, a developmental
Non-collagen proteins [BGLAP (OC), SPARC (ON),
continuum without distinct boundaries may exist [43].
SPP1 (OP), BSP, IBSP, POSTN, ECM1] While pre-differentiated osteoblasts in the marrow com-
Small leucine-rich proteoglycans (SLRP) (DCN, partment only play a minor role in bone wound healing,
BGN, LUM) a more prominent role is that of undifferentiated MSCs
Heat-shock protein 47 (HSP47) which are recruited to the regeneration site where they
Alkaline phosphatase (ALPL)
differentiate into osteoblasts [16]. The recruitment and
differentiation of MSCs is regulated by CKs and GFs
Cadherin (CDH11)
[17,19]. The GFs most commonly implicated in bone
Integrin (ITGB4, ITGB5) wound healing are BMPs, members of the TGF-β family,
Laminin (LAMA2, LAMA3) PDGF, and IGF-1 [19,20]. Moreover, the bone debris cre-
Pro-collagen lysyl-hydroxylase (PLOD1, PLOD2, ated during implant surgery, the peri-implant blood clot
PLOD3) (i.e., platelets) and the differentiating MSCs themselves
Pro-collagen C-endopeptidase enhancer further contribute to release of GFs at the site [44,45].
(PCOLCE) All studies reported some evidence of osteogenic dif-
Lysyl-oxidase (LOX) ferentiation at an early time point (day 3 or 4) via
Shanbhag et al. International Journal of Implant Dentistry (2015) 1:5 Page 8 of 12

Figure 2 Summary of biological processes identified via gene expression during early peri-implant bone healing. CKs, cytokines;
GFs, growth factors; EPC, endothelial progenitor cells; EC, endothelial cells; MSC, mesenchymal stem cells; OB, osteoblasts; ECM, extracellular
matrix; HSC, haematopoietic stem cells; MP, macrophages; OC, osteoclasts.

expression of genes associated with key growth factors The key signaling pathways, via which GFs guide
(bone morphogenetic proteins (BMP4, BMP6, BMP2- osteogenic cell differentiation, are the TGF-β/BMP- and
kinase), growth and differentiation factor-10 (GDF10), Wnt-mediated pathways [19,47]. While the BMP pathway
transforming growth factors (TGF-α, TGF-β), platelet- ensures differentiation of MSCs into osteo-chondro-
derived growth factor (PDGF), and insulin-like growth progenitors (OCPs), the Wnt pathway is essential for
factor-1 (IGF1)), transcription factors (Runx2, Osx, Dlx3, subsequent osteoblastic commitment, i.e., Wnt acts
Dlx5, Msx1, HOX genes, Sp1, Sp3), and/or osteogenic ‘downstream’ of BMP to ensure that OCPs differentiate
signaling pathways (TGF-β/BMP signaling, Wnt-receptors, into osteoblasts and not chondroblasts [47]. Genes as-
Ras-protein/mitogen-activated protein kinase (Ras/MAPK) sociated with both TGF-β/BMP and Wnt pathway (Wnt
signal transduction). In all studies, these genes were further receptors) were upregulated at day 7 [34,35] and day 14
upregulated at day 7. Upregulation of osteogenic factors [34] on SLA and SLActive surfaces, suggesting the oc-
seemed regulated by implant surface. The key transcription currence of osteogenic differentiation at these time points.
factor osterix (Osx) was upregulated on the Osseospeed GF-regulated signaling pathways exert their effects on
surface, but not TiOBlast at day 7 [36], while tissues adja- differentiating cells via activation of TFs. The TFs Runx2
cent to SLActive surfaces demonstrated comparatively and Osx are considered as ‘master switches’ and absolute
greater BMP and Ras/MAPK expression compared to SLA requirements for osteoblast differentiation [21] - while
surfaces at day 7 [35]. Previous in vivo animal studies have Runx2 is essential for MSC differentiation, Osx acting
reported correlations between upregulated osteogenic gene ‘downstream’ of Runx2 controls osteoblastic fate deter-
expression in peri-implant tissues and enhanced histo- mination [48,49]. An upregulation of these genes was
logical and biomechanical measures of osseointegration observed in relation to the TiOBlast, Osseospeed, and
during early (1- to 4-week) healing times [27,46]; neverthe- SLActive surfaces in the present review. However, at
less, it is unclear whether upregulation and/or overexpres- day 7, expression of Osx was significantly greater on
sion of genes at a specific time point directly correlates to Osseospeed than TiOBlast surfaces. This finding is con-
increased protein production in vivo. sistent with previous animal [50,51] and human studies
Shanbhag et al. International Journal of Implant Dentistry (2015) 1:5 Page 9 of 12

[52] where superior in vivo osseointegration (i.e., larger Osteoclastic activity and remodeling
amount of bone-to-implant contact occurring earlier) While GFs regulate osteogenesis, pro-inflammatory CKs
of Osseospeed versus TiOBlast implants was reported. (e.g., IL-1, IL-6, TNF-α) simultaneously regulate the an-
Thus, it appears that implant surface topography and/ tagonist process of bone resorption via osteoclasts [23].
or chemistry influence peri-implant bone healing in Moreover, osteoblasts themselves stimulate osteoclasto-
humans both at the signaling pathway and transcrip- genesis via macrophage colony stimulating factor (M-CSF)
tion factor level. and receptor activator of NF-kB ligand (RANKL) genes
but also closely regulate this process via osteoprotegerin
(OPG), an inhibitor of RANKL [59].
ECM production Two studies reported expression of genes associated with
Deposition of new bone on the implant surface involves osteoclastic activity and ECM degradation (cathepsin-K
the secretion of a complex ECM (scaffold) of proteins by (CTSK), tartarate-resistant acid phosphatase (ACP5), and/
osteoblasts, which subsequently undergoes mineralization or matrix metalloproteinases (MMPs)), on Osseospeed
[9]. Expression of ECM proteins is a reliable indicator of and TiOBlast surfaces at day 7 [37], and SLActive sur-
early osteogenic activity [19] and was identified in all four faces at day 14. However, upregulation of MMP inhibi-
studies at days 7 and 14. All studies reported some tors (TIMP-2, -3) was also reported on TiOBlast and
evidence of ECM production and/or organization at Osseospeed surfaces suggesting a control of the resorp-
days 7 and 14. Upregulated genes associated with ECM tion process. Although no studies reported differential
deposition included various collagens (Col 1 to 11), RANKL/OPG expression, a previous in vitro study [60]
non-collagen proteins (osteopontin (OPN), osteonec- reported significant downregulation of osteoclastogenic
tin (ON), osteocalcin (OCN), bone sialoprotein (IBSP), genes on SLActive surfaces. Collectively, these data re-
periostin (POSTN), and ECM protein-1), alkaline phosphat- affirm the dynamic nature of bone formation and resorp-
ase (ALP), and bone-specific adhesion proteins (integrins tion at the implant-bone interface, even in early healing
(ITGB4, ITGB5), laminins (LAMA2, LAMA3), and cadher- stages, and suggest the possibility for implant surface tech-
ins (CDH11)). Osteocalcin, the most bone-specific ECM nology modulation of bone remodeling.
protein and a late marker of osteogenic differentiation [19],
was significantly upregulated on Osseospeed (versus Angiogenesis
TiOBlast) surfaces at day 7 [36]. Osteopontin, an ECM Angiogenesis is closely related to osteogenesis and occurs
protein essential for mineralization [53], was significantly simultaneously during bone regeneration [11]. Physiological
upregulated on SLActive comparing to SLA surfaces at oxygen tensions in bone are about 12.5% O2 but fall to 1%
day 7 [35]. The possibility that implant surface features O2 in regeneration sites due to disruption of the local
enhance osteogenic differentiation of MSCs via upregula- vasculature as a result of injury and/or surgery [61,62]. A
tion of specific genes (e.g., SLActive versus SLA in regard key event that stimulates angiogenesis (and osteogenesis)
with BMP and Wnt signaling) has been demonstrated at regeneration sites is hypoxia, via the hypoxia inducible
in vitro [54]. (transcription) factor-1 (HIF-1) that regulates expression
Furthermore, genes associated with collagen fibril for- of angiogenic genes [63]. The key cells involved in angio-
mation/organization (heat-shock protein-47 (HSP-47), genesis are macrophages, which in response to hypoxia
pro-collagen C-endopeptidase enhancer (PCOLCE), small and inflammation release chemotactic and angiogenic
leucine-rich proteoglycans (SLRP)) and post-translational growth factors (e.g., VEGF) [40,64], and endothelial pro-
modification (pro-collagen lysyl-hydroxylases (PLOD1, genitor cells (EPCs) which differentiate into endothelial
PLOD2, PLOD3) and lysyl-oxidase (LOX)) were upregu- cell lining blood vessels [65]. VEGF is the single most
lated on Osseospeed and TiOBlast surfaces [37]. Collagen important regulator of EPC differentiation and vessel
comprises approximately 90% of the ECM and collagen formation [66]. Moreover, a role for VEGF in osteogenic
fibrillogenesis and organization directly determine the differentiation has also been suggested mainly via inter-
biomechanical properties of bone [55,56]. Genes associ- action with the BMP signaling pathway [67].
ated with collagen fibril formation, maturation, and post- In the present review, a significant simultaneous upregu-
translational modification expressed by osteoblasts [57,58] lation of several angiogenesis-related genes was identified
were upregulated on TiOBlast and Osseospeed implants, at day 7 in all included studies. Pro-angiogenic factors
representing early ECM organization at the bone-implant (ANXA2, EPAS-1) were upregulated at TiOBlast and
interface. These modifications determine the pattern of Osseospeed surfaces at day 7 [37]. Genes associated
collagen cross-linking which in turn influences tissue with VEGF and P13K-AKT signaling pathways were up-
organization, mineralization, and ultimately mechanical regulated at SLActive (but not SLA) surfaces on day 7
bone strength [56], and in the case of osseointegration, and continued to be upregulated on day 14 [35]. The
the integrity of the bone-implant interface [37]. P13K-AKT pathway is reported to be important for
Shanbhag et al. International Journal of Implant Dentistry (2015) 1:5 Page 10 of 12

endothelial cell survival, migration, and vessel formation, osteogenesis (TGF, BMP4, BMP7, OCN and ALP), and
in addition to aiding VEGF-mediated angiogenesis [68]. angiogenesis (VEGF) were upregulated, continuing until
Previous in vitro studies have reported the pro-angiogenic day 14, suggesting that the basic biological processes
effects of SLActive surfaces by promoting VEGF expres- governing alveolar wound healing and osseointegration
sion in EPCs and osteoblasts [65,69], while enhanced are the same.
histological osseointegration of SLActive implants has
been directly correlated with increased angiogenesis in
Conclusions
a dog model [70,71]. Thus, implant surface technology
Based on limited evidence of gene expression data from
appears to have the possibility to also influence angio-
four studies involving 43 patients, the following remarks
genesis at early stages of wound healing.
can be made:
Neurogenesis
1. Early peri-implant healing (2 weeks) involves a
Bone innervation includes both myelinated and unmyelin-
sequence of biological events which are similar to
ated nerve fibers located in the periosteum, bone cortex,
those observed in other bone wound healing
Haversian systems, Volkmann’s canals, and the marrow
scenarios (fractures, extraction-sockets).
spaces [72]. An interesting finding in the present review
2. Osseointegration depends on osteogenesis at the
was the significant upregulation of genes associated with
implant interface, but other simultaneously
neurogenesis, more than any other biological process, on
occurring processes such as inflammation, bone
SLActive and SLA surfaces at all time points [34,35]. Spe-
resorption, angiogenesis and neurogenesis also play
cific processes represented were axon formation, growth
an important role, as evidenced by consistent and
and differentiation, and the neural signaling pathway. This
concomitant gene expression.
is consistent with previous in vivo reports of murine frac-
3. Several genes associated with key regulators of
ture healing [73] and calvarial defect regeneration in rela-
biological processes, such as cells, cytokines, growth
tion to SLA surfaces [74,75]. Key neurotrophic factors
factors, transcription factors, signaling pathways,
(brain-derived neurotrophic factor (BDNF) and neurotro-
and secretory products, were shown to be
phin 3 (NTF3)), essential for neuronal survival and dif-
differentially regulated during peri-implant healing
ferentiation during development [76], were significantly
in a manner that was largely consistent - in terms of
upregulated on SLActive versus SLA surfaces at day 7
nature and timing - with previous in vitro and
suggesting an effect of surface modulation. The P13K-
preclinical in vivo histological studies of
AKT pathway, upregulated on SLActive surfaces (in
osseointegration.
relation to angiogenesis), has also been implicated in
4. Implant surface technology can influence
neuronal survival and subsequent neural development
osseointegration, at every step of the early wound
[77,78] and could have contributed to upregulation of
healing process, i.e., anti-inflammatory response,
neurogenic genes at these surfaces. Indeed, previous
progenitor cell recruitment, osteoinduction, growth
histologic reports have described changes in bone in-
factor/transcription factor expression, signaling
nervation after implant placement (and loading) and the
pathway regulation, and extracellular matrix
presence of nerve fibers within the peri-implant bone, in
production. However, the relevance of those
animals and humans [79-81].
observations is questionable; no distinct differences
It can be hypothesized that peri-implant neurogenesis
have been demonstrated in terms of histological
is one of the underlying mechanisms governing the
outcomes at later time points or short- and long-term
phenomenon of osseoperception, defined as the tactile
clinical performance among the various implant
sensibility of osseointegrated implants to occlusal forces
surface technologies discussed herein.
induced via activation of nerve endings and/or receptors
in the peri-implant environment [82,83]. Moreover, re-
Competing interests
cent evidence suggests that implant surface properties The authors declare that they have no competing interests.
may influence the degree of osseoperception in humans
[84], which can be correlated with the genomic evidence Authors’ contributions
for implant surface modulation of neurogenesis during SS conceived and carried out the study and drafted the manuscript. VS and
osseointegration. AS participated in carrying out the study and drafting the manuscript. All
authors read and approved the final manuscript.
Finally, the present review findings are consistent with
a recent gene expression study of healing extraction sockets Author details
1
in humans [85]. This study reported an initial upregulation Department of Periodontology, Faculty of Odontology, Malmö University,
Carl Gustafs väg 34, 214 21 Malmö, Sweden. 2Centre for Oral Rehabilitation &
of pro-inflammatory cytokines (IL-1, IL-6) at day 1, but Implant Dentistry, 1 Laxmi Niwas, 87 Bajaj Road, Vile Parle West, Mumbai
by day 7, genes suggestive of immune response (IL-10), 400056, India.
Shanbhag et al. International Journal of Implant Dentistry (2015) 1:5 Page 11 of 12

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