Journal of Dental Research

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Molecular Analysis of Healing at a Bone-Implant Interface

C. Colnot, D.M. Romero, S. Huang, J. Rahman, J.A. Currey, A. Nanci, J.B. Brunski and J.A. Helms
J DENT RES 2007 86: 862
DOI: 10.1177/154405910708600911

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 RESEARCH REPORTS
Biomaterials & Bioengineering
C. Colnot1, D.M. Romero1, S. Huang1,
J. Rahman1, J.A. Currey2, A. Nanci3,
J.B. Brunski2*, and J.A. Helms4
1 Department of Orthopaedic Surgery, University of
California, San Francisco, CA 94110-1342, USA;
2 Department of Biomedical Engineering, Jonsson
Engineering Center, Rensselaer Polytechnic Institute, Troy,
NY 12180-3590, USA; 3Faculty of Dentistry, Université de
Montréal, Canada; and 4 Department of Plastic and
Reconstructive Surgery, Stanford University, 257 Campus
Drive, Room GK207, Stanford, CA 94305, USA;
*corresponding author, brunsj@rpi.edu
J Dent Res 86(9):862-867, 2007
ABSTRACT
While bone healing occurs around implants, the
extent to which this differs from healing at sites
without implants remains unknown. We tested the
hypothesis that an implant surface may affect the
early stages of healing. In a new mouse model, we
made cellular and molecular evaluations of
healing at bone-implant interfaces vs. empty
cortical defects. We assessed healing around Ti-
6Al-4V, poly(L-lactide-co-D,L,-lactide), and 303
stainless steel implants with surface characteristics
comparable with those of commercial implants.
Our qualitative cellular and molecular evaluations
showed that osteoblast differentiation and new
bone deposition began sooner around the implants,
suggesting that the implant surface and
microenvironment around implants favored
osteogenesis. The general stages of healing in this
mouse model resembled those in larger animal
models, and supported the use of this new model
as a test bed for studying cellular and molecular
responses to biomaterial and biomechanical
conditions.
KEY WORDS: bone, healing, implants, osteoblast.
Received October 11, 2006; Last revision April 21, 2007;
Accepted May 4, 2007
A supplemental appendix to this article is published
electronically only at http://www.dentalresearch.org.
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International and American Associations for Dental Research
Molecular Analysis of Healing
at a Bone-Implant Interface
INTRODUCTION
D espite high success rates, implants still fail, due to wear debris (Wang et
al., 2004), excessive micromotion (Soballe et al., 1992; Brunski, 1999),
or excessive loading (Hoshaw et al., 1994; Isidor, 1997; Esposito et al.,
2000). An understanding of the biological mechanisms of implant success
and failure is fundamental to the development of preventive or remedial
strategies for the treatment of loosened implants. Numerous studies
(Brånemark et al., 1977; Lazzara et al., 1999; Schatzker, 2002; Berglundh et
al., 2003; Watzak et al., 2005) have indicated that a typical initial interface
will have some regions with direct bone-implant contact and other regions
with bone-implant gaps. Regions of contact provide initial mechanical
stability, despite surgical damage to bone and bone remodeling, which
transiently increase porosity (Hoshaw et al., 1995; Huja et al., 1999). Gaps
heal through blood clotting, matrix remodeling, angiogenesis, cell
differentiation, woven bone formation, and turnover of woven bone
(Berglundh et al., 2003; Davies and Hosseini, 2000). However, the source of
skeletal progenitor cells, the rate-limiting steps that control their
differentiation into bone, the signaling molecules that direct this process,
and the role of biomechanical and biomaterial factors on cell fate decisions
remain to be elucidated. We have developed a murine model for studying
such topics at cellular and molecular levels. Here, we compared healing
around implants with healing of empty implant sites, to assess whether the
presence of an implant accelerated the initiation of bone healing.
MATERIALS & METHODS
Implants
Ti-6Al-4V implants ("Ti alloy", Whaledent, Inc., Akron, OH, USA), 1.0 mm
diameter by 2.0 mm length, were lightly sand-blasted with 25 ␮m Al2O3 powder
and ultrasonically cleaned for 5 min. Poly(L-lactide-co-D,L,-lactide) implants
were made from "BioPin" implants (Imtec Corp., Ardmore, OK, USA). We
modified pre-sterilized BioPin implants (resembling miniature thumbtacks with
1.0 mm shank diameter and 3.5 mm length) by cutting off the pointed tip,
leaving a blunt tip and shank length 1.5 mm. Stainless steel implants ("303 SS",
Insect Pins, Fine Science Tools, Foster City, CA, USA) were 303 medical-grade
stainless steel, 0.25 mm diameter.
Preparation of Empty Holes and Placement of Implants
All procedures followed protocols approved by the Institutional Committee on
Animal Research. Adult wild-type mice (males, 3-5 mos old) were anesthetized.
For empty holes, Ti alloy, and BioPin implants, a 0.8-mm hole was drilled in the
anterio-proximal tibia and enlarged via a 1.0-mm drill to minimize bone
damage, as previously described (Colnot et al., 2005). Implants were press-fitted
into slightly undersized holes, and wounds were closed. For 303 SS implants,
each pin was transfixed percutaneously in the proximal tibia, leaving only a small
J Dent Res 86(9) 2007 Molecular Analysis at Bone-Implant Interface 863

segment across the leg. Following surgery, mice received

subcutaneous injections of buprenorphine for analgesia and were
allowed to ambulate freely. Mice were killed at days 3, 5, 7, 10, 14,
21, and 28 post-surgery (n = 2 d3, 2 d7, and 3 d10 for 303 SS
implants; n= 3 d3, 2 d5, 2 d7, 2 d10, 1 d14, 2 d21, and 2 d28 for Ti
alloy implants; and n = 3 d3, 4 d5, 5 d7, 5 d10, 2 d14, 4 d21, and 3
d28 for BioPin implants).
Tissue Processing, Histology, and in situ Hybridization
Tibiae were dissected and processed as described (Colnot et al.,
2003). Ti alloy and 303 SS implants were gently removed after
decalcification and prior to dehydration, while BioPin implants
dissolved during processing. Tissue sections were prepared for
histology with Safranin-O Fast green (SO/FG) and Trichrome
(TC), and for histochemistry with tartrate-resistant acid
phosphatase (TRAP) and alkaline phosphatase (AP). Adjacent
sections were subjected to in situ hybridization analyses with
collagen type I (Col1), collagen type (Col2)II, osteocalcin (Oc),
osteopontin (Op), and runx2 (cbfa1) probes as previously
described (Colnot et al., 2003).
Surface Characterization of Implants
Implant surface roughness was measured by optical interferometry
(MicroXam TM ; ADE Phase-Shift, Tucson, AZ, USA) at a
resolution of 0.05 nm (vertical) and 0.3 ␮m (horizontal), with Sa =
arithmetic average value of vertical departures of the profile or Figure 1. Establishing the time-course and type of healing in the implant
surface from the mean line throughout the sampling length or area; bed. A 1.0-mm drill hole was created in the proximal tibiae; samples
St = maximum peak-to-valley height of the entire measurement were then collected at d5, d7, and d28, and stained with Trichrome
(TC) and tartrate-resistant acid phosphatase (TRAP). (A-B) At d5, there
trace; and Sq = root mean square of values of all points of the is no evidence of new bone formation. The injury site is filled with
profile. Implant surfaces were analyzed by ESCA (electron densely packed fibroblasts, (B) while bone debris from the drilling
spectroscopy for chemical analysis) at NESAC/BIO (Univ. of process is engulfed by TRAP-positive osteoclasts. (C-D) By d7, new bone
Washington, Surface Science Instruments S-probe spectrometer), is first detected and undergoes rapid remodeling. (E-F) By d28, woven
bone has been replaced by cortical bone fused with the old cortex.
with x-ray spot size ~ 800 ␮m and sampling depth ~ 50 Å. Scale bar = 100 microns. (See color version of this Fig. in the online
APPENDIX.)
RESULTS
Healing in Empty Implant Sites completely remodeled by d28.
We first determined how an implant site healed in the absence Histological analyses indicated that new bone formed in
of any device, as a baseline for comparing healing around the empty implant site by d7. To pinpoint the actual onset of
various implants. We examined the program of bone healing osteogenesis, rather than its histological manifestation, we
using histological stains to detect cartilage and bone. At all conducted molecular analyses at earlier time-points. We
time-points (post-surgical d3-28), we never detected cartilage examined expression of 3 genes: Collagen type I (Col I),
(data not shown) and found evidence of only new bone (Fig. 1, Osteocalcin (Oc), and Osteopontin (Op). On d3, we
Trichrome panels). Thus, empty implant sites healed identified cells in the injury site that expressed Col I (Fig.
exclusively through intramembranous ossification. 2A, arrow), suggesting initiation of an osteogenic program.
Next, we determined the time-course of reparative The lack of Oc and Op expression in adjacent sections
osteogenesis. On d3, there was minimal Aniline-blue-stained indicated that cells had not yet differentiated into osteoblasts
bony matrix, no Safranin-O-stained cartilaginous matrix, and (Figs. 2B, 2C). The progressive increase in Col1, Oc, and Op
no TRAP staining (data not shown), indicating that bone expression from d5 to d7 indicated that cells initiated
healing had not yet initiated. By d5, some TRAP activity was differentiation into osteoblasts during this window (Figs. 2D-
detectable, but in the absence of new cartilage or bone matrix, 2I). These analyses provided a molecular map illustrating
this activity probably represented osteoclasts resorbing old initiation of osteoblast differentiation and bone formation in
bone debris, or macrophages at the injury site (Figs. 1A, 1B). an empty site.
By d7, abundant amounts of bone matrix were evident, which
was in the process of being remodeled by osteoclasts (Figs. 1C Healing at Ti Alloy and PLA Bone-Implant Interfaces
1D). From d7 to d28, new bone formation was coupled with Next, we ascertained how the presence of an implant in the
bone remodeling, resulting in a patent bone marrow cavity and injury site affected the course of new bone formation and cell
intact cortical plate (Figs. 1E 1F), with only microscopic differentiation. Because of their common use in dental
evidence of the site where newly deposited bone matrix applications, we focused on cellular responses to Ti alloy and
juxtaposed with the old injured cortical bone (arrow, Fig. 1E). BioPin implants. By d3, no sign of new bone deposition was
Thus, a 1.0-mm implant bed showed the first signs of detected by histological analyses (Figs. 3J, 3K). On d5, we
reparative intramembranous ossification at d7, and was noted approximately the same amount of new bone at the
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International and American Associations for Dental Research

864 Colnot et al. J Dent Res 86(9) 2007

differentiation of peri-implant cells

into osteoblasts, and acceleration in
the remodeling of new bone
matrix. We confirmed this using in
situ hybridization, noting that cells
around the implant up-regulated
the osteoblast-related transcription
factors Runx2 and Op at d3 (Figs.
4D, 4E). Lack of Collagen type II
(Col II) expression around the
implant indicated that intra -
membranous ossification was the
mechanism of new bone formation
at implant sites (Fig. 4F).
Surface Characterization
of Implants
Given the importance of a
biomaterial's surface (Albrektsson
and Wennerberg, 2004; Monsees
et al., 2005; Sul et al., 2005), we
measured roughness at 5-10
locations on each implant type:
mean and standard deviations for
S a were: 0.548 ± 0.0336 ␮m for
Figure 2. Molecular analyses of healing in the implant bed. Localization of mRNA for collagen type I
(Col 1), Osteocalcin (Oc), and Osteopontin (Op) by in situ hybridization on sections adjacent to those Ti alloy implants; 0.709 ± 0.217
shown in Fig. 1. (A-C) By d3, low expression levels for Col 1 are found in the marrow cavity (arrow), ␮m for BioPin implants; and
while Oc and Op are not expressed. (D-F) By d5, Col 1 is up-regulated in and around the defect within 0.185 ± 0.08 ␮m for 303 SS
the marrow cavity. Oc mRNA is still not detected. Op-expressing cells are localized near the cortical implants. ESCA revealed that Ti
defect (asterisk). (G-I) By d7, strong expression of all three markers is detected in the defect and in the
marrow space underlying the defect. Scale bar = 200 microns. (See color version of this Fig. in the
alloy implants had surface
online APPENDIX.) contamination represented by C
(53-56 at.%), N (1.5-1.7 at.%), Si
(2-2.8 at.%), Na (2.1-2.5 at.%),
and Ca (0.4-0.6 at.%), besides O
endosteal surface and marrow interfaces in both Ti alloy and and Ti in the oxide on this alloy (Ask et al., 1989). BioPin
BioPin samples (Figs. 3A, 3B). By d7, there was an increase in surfaces showed C (61-62 at.%) and O (37-38 at.%)
new bone trabeculae (Figs. 3D, 3E), which became consistent with its polylactic acid (PLA) content, plus Si (1.3
progressively remodeled. By d28, a thin shell of bone encased at.%) concentrated on the circumferential ridges. The C/O
both Ti alloy and BioPin implants (Figs. 3G, 3H). These ratio at the BioPin surface was 1.65, indicating hydrocarbon
analyses suggested that cells responded to Ti alloy and BioPin contamination, since the C/O ratio for PLA is 1.3. For 303
implant surfaces equivalently. SS pins, analyses revealed polydimethylsiloxane (PDMS),
Although each implant was press-fit into its site, the whose theoretical composition is 50 at.% C, 25 at.% O, and
cortical edges did not always come uniformly into contact with 25 at.% Si. This layer was thick enough to prevent detection
the implant surface; there were regions of bone contact and of the expected Cr2O3 oxide on the 303 SS surface (Sundgren
gaps (~ 0-60 ␮m) between implant and cortex (Fig. 3). We et al., 1985).
found that the time-course of repair was equivalent whether or
not a small gap existed (compare Figs. 3J, 3L [no gap], with
Figs. 3K, 3M [gap]). Larger gaps were occupied by DISCUSSION
extracellular matrix and cells, which gradually differentiated Cortical Bone Defect Healing and Interfacial Healing
into bone (Figs. 3K, 3M), whereas smaller gaps were occupied in the Mouse Model vs. Larger Animal Models
by new osteoblasts (Fig. 3L arrowheads).
This study identified similarities and differences between
Osteoblast Differentiation cortical defect and interfacial healing. Early stages of healing in
is Accelerated in the Presence of an Implant both were characterized by the formation of a hematoma,
Since new bone was detected earlier by histological analyses recruitment of matrix-resorbing cells, and deposition of woven
around implants vs. empty defects, we further analyzed early bone in the empty defect or surrounding the implant. Woven
stages of healing. With implants present, Trichrome staining bone was remodeled into lamellar bone, bridging the two
revealed a faint amount of Aniline-blue-positive matrix at d5 cortical bone ends of the empty defect, or leaving behind a thin
(Fig. 4A, arrowhead). Alkaline phosphatase activity indicated interfacial layer of bone at the implant surface. Our data on
the onset of mineralization (Fig. 4B, arrows), and TRAP cortical defect healing complement previous findings of defect
activity indicated matrix remodeling (Fig. 4C). Thus, relative to healing in rodent and larger animal models (Pritchard, 1964;
an empty site, implant presence resulted in accelerated Schenk and Hunziker, 1994; Chiba et al., 2001; Uusitalo et al.,
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J Dent Res 86(9) 2007 Molecular Analysis at Bone-Implant Interface 865

2001; Campbell et al., 2003), wherein small islands of cartilage

were sometimes also observed on periosteal surfaces.
Interfacial healing in our mouse model shared some key
features with interfacial healing around implants in other
animal models, such as rats (Nanci et al., 1994; Masuda et al.,
1997), rabbits (Schenk and Hunziker, 1994), dogs (Hoshaw et
al., 1994), sheep (Plenk and Zitter, 1996), monkeys (Watzak et
al., 2005), and humans (Lazzara et al., 1999)—with
intramembranous bone healing in empty defects and around
implants.
Differences in Timing of Bone Formation
around Implants and in Empty Holes
Analysis of our cellular and molecular data indicates that: (1)
cells surrounding implants initiated differentiation into
osteoblasts sooner than when no implant was at the site; and (2)
the timing of osteoblast differentiation and new bone matrix
deposition was equivalent among the three implant
biomaterials. One explanation for this is that an implant
provides a surface onto which osteoblasts can adhere and
deposit a matrix that mineralizes, and that this surface was
similar among our implants. The range of roughness values for
our implants was narrow (Sa, 0.185-0.709 ␮m) and at the lower
end of values for "smooth" (Sa, 0 to 0.4 ␮m) and "minimally
rough" (S a , 0.5 to 1.0 ␮m) implants (e.g., S a ~ 0.46 for
machined Brånemark implants). Also, our implants were not as
rough as "moderately rough" (Sa, 0.5 to 1.0 ␮m) or "rough" (Sa,
> 2.0 ␮m) implants, e.g., S a ~ 0.91 ␮m for "OsseoSpeed"
implants; and Sa ~ 1.6 ␮m for SLA implants (Albrektsson and
Wennerberg, 2004; Ellingsen et al., 2004; Sul et al., 2005).
While a recent review (Shalabi et al., 2006) reported "a positive
effect on the bone response...from Ra/Sa of ~ 0.5 ␮m up to ~
8.5 ␮m", our narrow Sa range was at the lower end of that
range. Moreover, our ESCA data showed comparable values of
carbon on surfaces of all implants. (Commercial Ti implants
can also have high carbon levels on their surfaces; Wieland et
al., 2000; Massaro et al., 2002.) Ultimately, quantitative
analyses would complement our qualitative spatial and
temporal gene expressions. For example, our in situ data from
mice are consistent with Ogawa and Nishimura's (2006) RT-
PCR data from tissues near Ti implants and at osteotomy sites
Figure 3. Healing at the bone-implant interface. Schematic representations
in rat tibiae, which showed a 1.5- to two-fold up-regulation of of Ti-6Al-4V alloy and BioPin implants, and photomicrographs of these
collagen I, osteopontin, and osteocalcin expression (but not implants after placement (below). At post-surgical d5, longitudinal sections
Runx2 and Bmp2) at 3 and 7 days after surgery. through (A) the Ti-6Al-4V implant and the (B,C) BioPin implant show
evidence (by Trichrome staining) of new bone formation (arrowheads). (D-
Is Osseointegration Equivalent to Fracture Healing? F) Bone formation around the implants was increased at d7. (G-I) Bone
Concerning the oft-claimed analogy between fracture healing encasing the implants underwent remodeling until only a thin shell
remained in the bone marrow cavity. A schematic drawing representing a
and interfacial healing (Brånemark et al., 1977; Brånemark, direct interface and a minimal gap interface around an implant. Areas in
1985; Pilliar and Simmons, 2002; Schatzker, 2002), both the black box and the red box are illustrated on the tissue sections stained
begin with a breach in an intact skeletal element, an immune with Safranin-O Fast green (SO/FG) (J-M) and Trichrome (TC) (N-O). (J)
response, neo-vascularization, and recruitment of skeletal On d3, the direct bone-implant interface was not populated by cells,
whereas in (K), the gap interface was filled with a fibrous hematoma
progenitor cells. However, in a typical fracture, some skeletal
(bracket). (L) By d28, new bone was juxtaposed to the implant
progenitor cells differentiate into chondrocytes, while others (arrowheads indicate stained nuclei of new osteoblasts), while in the gap
differentiate into osteoblasts, followed by endochondral (M), a new bone interface has been created by the mineralization of the
ossification. Around an implant, all skeletal progenitor cells fibrous matrix (bracket), and stained nuclei distinguish new osteoblasts
differentiate directly into osteoblasts, followed by from old osteocytes. Scale bar in A and corresponding magnifications =
0.5 mm; C and other high-magnification images = 50 microns. (See color
intramembranous ossification. Perhaps the key difference version of this Fig. in the online APPENDIX.)
between fracture healing and interfacial healing is that the
latter involves cellular and molecular responses that may be
influenced by biomaterial surface texture, chemical
composition, and implant biomechanics. In that context, for detailed molecular analyses, and the use of mouse
advantages of the mouse model include its ability to allow mutants for the study of bone healing.
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Figure 4. The presence of an implant accelerated osteoblast differentiation. Cellular and molecular retention and bone-to-implant contact
analyses at the Ti-6Al-4V (A-C) and 303 SS (D-F) implant surfaces. (A) At post-surgical d5, Trichrome with fluoride-modified titanium
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(R01AG23218-01, Zena Werb); by an NIH/NIDCR grant (R03 bone-implant interfaces affects bone remodeling activity. In:
DE16701), and by the Musculoskeletal Transplant Foundation Transactions of the 41st Orthopaedic Research Society. Orlando, FL:
(C.C.) and FA9550-04-1-0075 for J.H. We thank S. Hadwin Orthopaedic Research Society, p. 188.
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implants, L. Gamble (NESAC/BIO, University of Washington, subjected to occlusal overload or plaque accumulation. Clin Oral
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