Anabolic agents and gene expression around the bone implant interface.
- [Show abstract] [Hide abstract]
ABSTRACT: Controversy exists over the potency of bone healing in the aged skeleton, and there is concern that enhancement of bone regeneration after use of bone-stimulating growth factors may not be effective in the aged. In this study, 30 skeletally mature beagles (1-2 or 10-12 years old) had titanium implants placed bilaterally in the proximal humerus for a period of 4 weeks in a model of intramembranous bone regeneration. A bony defect made at the time of surgery created a 3-mm gap between the implant surface and the host bone. Some of the implants were treated with recombinant human TGFbeta2 (rhTGFbeta2) at various does (0.32-35 microg per implant), and some served as paired controls. The dose response was similar in young and old animals. The most effective dose, 35 microg, led to a 3-fold increase in the volume fraction of new bone within the gap in both the young (p = 0.001) and old (p = 0.002) animals. At this dose, there was a 5-fold increase in osteoblast surface. While age did not significantly affect the quantity of new bone formed as assessed by backscatter scanning electron microscopy, the older animals had thinner regenerated trabeculae that tended to be spaced more closely than the younger animals. Coupled with the finding that the increase in osteoid was greater in the old animals compared with the young animals, these qualitative differences suggest that there may have been a slight delay in the rate or a defect of mineralization in the old animals.Journal of Bone and Mineral Research 05/2003; 18(4):730-6. · 6.13 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The purpose of the present study was to determine if recombinant human bone morphogenetic protein-2 (rhBMP-2) enhances bone ingrowth into porous-coated implants and gap healing around the implants. In the presence of a 3-mm gap between the implant and host bone, porous-coated implants were placed bilaterally for four weeks in the proximal humeri of skeletally mature, adult male dogs. In three treatment groups, the test implant was treated with HA/TCP and rhBMP-2 in buffer at a dose of 100 microg/implant (n=5), 400 microg/implant (n=6), or 800 microg/implant (n=5) and placed in the left humerus. In these same animals, an internal control implant was treated only with HA/TCP and buffer and placed in the right humerus. These groups were compared with a previously reported external control group of seven animals in which no growth factor was delivered [J. Orthop. Res. 19 (2001) 85]. The BMP treated implants in the two lower dose groups had significantly more bone ingrowth than the external controls with the greatest effect in the 100 g/implant group (a 3.5-fold increase over the external control, p=0.008). All three dose groups had significantly more bone formation in the 3-mm gap surrounding the BMP treated implants than the external controls with the greatest effect in the 800 microg group (2.9-fold increase, p<0.001). Thus, application of rhBMP-2 to a porous-coated implant stimulated local bone ingrowth and gap healing. The enhancement of bone formation within the implant (bone ingrowth) was inversely related to dose.Journal of Orthopaedic Research 01/2004; 22(1):58-65. · 2.88 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The repair of hemopoietic bone marrow following evacuation of the tibial or femoral cavity of the rat was sequentially studied with the light microscope. A stereotyped train of histologic events occurred. These were capillary invasion of the cavity, appearance of primitive mesenchymal cells, osteoblastic proliferation, cancellous bone formation, development of sinusoids, reappearance of hemopoietic tissue and resorption of cancellous bone. The studies suggest that restoration of marrow sinusoids takes place only in the interstices of cancellous bone. Mechanical disruption of the sinusoidal system is one method of triggering cancellous bone formation. The cancellous bone which appeared after injury was thought to be produced by endosteal osteoblasts and osteoblasts derived from cells residing in normal hemopoietic tissue. Localized radiation of the tibia followed by mechanical disruption of hemopoietic tissue demonstrated that cancellous bone production and the repair process were unimpaired by 1,000 r but were completely blocked by 4,000 r. This would imply that the cell which can differentiate into an osteoblast is resistant to 1,000 r.The Anatomical Record 06/1969; 164(1):101-11.
Joint replacement using implants is a common surgical pro-
cedure in orthopaedics. Approximately 200,000 total hip
replacement procedures alone were performed in 2001 in the
OAHip_replace.pdf). Cementless implant fixation is cur-
rently the preferred option for the acetabular component
and is commonly used on the femoral side as well. While the
vast majority of orthopaedic implants succeed in restoring
function, a significant proportion fail due primarily to asep-
tic loosening brought about by wear particle-induced osteol-
ysis – this topic will be discussed in detail elsewhere in the
Workshop. One of the main reasons for late stage loosening
is thought to be the consequence of early loosening resulting
from failure to achieve biological fixation in the initial phas-
es of repair. It is now well known that implant fixation can be
enhanced by various means which includes the use of exoge-
nous applied anabolic agents such as growth factors1.
Over the past number of years our laboratory has shown
the positive effects of TGF-‚ and BMP-2 on bone ingrowth
and ongrowth around an implant in a canine gap model2-5.
While these studies have demonstrated the action of these
growth factors in terms of bone volume over tissue volume
and the strength of fixation, the findings are limited to a phe-
nomenon rather than mechanistic interpretation. In addition
to the fact that canine models are inherently costly, they are
also less amenable to molecular analysis due mainly to the
lack of genetic information available for this species. In
order to circumvent these limitations and better understand
the underlying molecular mechanisms in growth factor
enhanced bone tissue regeneration around an implant we
have sought to utilize a rat bone marrow ablation model.
This model was originally described for studying hematopoe-
sis6,7and was later characterized for osteogenesis and bone
remodeling8-10. We have modified the model by placing an
implant in the ablated space in the medullary cavity to emu-
late the biological environment surrounding the implant in
our canine model. Due to the significant reduction in cost
for the rat model, it provides greater flexibility in experiment
design to accommodate time course studies. We have used
the rat bone marrow ablation model to characterize the
molecular events occurring during the reparative process in
the absence of an implant (ablation alone), in the presence
of an implant and with implants carrying TGF-‚2.
We examined the time-dependent gene profiles in the rat
model at 1, 3, 5, 7, 10 and 14 days post-ablation. Expression
levels of a total of 39 genes related to osteogenesis and
GAPDH were measured by real-time PCR11. One group was
used as an intact control (time point 0 day). During the
inflammatory phase between days 1 and 5 there was down-
regulation of several cytokine genes, including COX-1 and -2,
although TNF-· and IL-1 showed upregulation. The repair
phase of days 3 to10 was characterized by upregulation of a
number of growth factor, receptor and inhibitor genes relat-
ed to all three TGF‚1,2and 3, bFGF, BMP-2, -4 and -7,
VEGF and IGF-1. During this time, Cbfa-1, alkaline phos-
phatase, collagen type I, osteonectin, osteopontin and osteo-
calcin exhibited peak expression at days 5 or 7. At later time
points of days 10 to 14 when remodeling begins, there were
peak levels of RANK, RANKL, cathepsin K, IL-6 and COX-2.
Principal component analysis used to identity 8 underlying
components that together explained over 80% of the vari-
ance in the data. When components are arranged chrono-
logically, there is a clear picture of the molecular cascade of
sets of genes which may be co-regulated in vivo during bone
In another study using the rat model, we examined the
ability of TGF-‚2to enhance implant fixation by comparing
J Musculoskel Neuron Interact 2004; 4(4):388-389
Anabolic agents and gene expression around
the bone implant interface
A.S. Virdi1, A. De Ranieri1, S. Kuroda1,2, Y. Dai3, D.R. Sumner1
1Department of Anatomy and Cell Biology, Rush Medical College, Rush University Medical Center,Chicago, IL, USA,
2Oral Implantology and Regenerative Dental Medicine, Department of Masticatory Function Rehabilitation, Tokyo Medical and Dental
University, Tokyo, Japan, 3Department of Bioengineering, University of Illinois at Chicago, Chicago, IL, USA
Keywords: Implant Interface, Bone Regeneration, Marrow Ablation, Gene Expression
The authors have no conflict of interest.
Corresponding ·uthor: Amarjit S. Virdi, Department of Anatomy and Cell Biol-
ogy, 600 S. Paulina Street AcFac 507, Chicago, Illinois 60126, USA
Accepted 4 August 2004
A.S. Virdi et al.: Gene modulation around implant
three doses (0.1Ìg, 1Ìg and 10Ìg) to a control group (0Ìg) by
placing HA/TCP treated rod coated with TGF-‚2in the
ablated femoral marrow space12. Bone volume per tissue vol-
ume (BV/TV) for the region between the endocortical enve-
lope and surface of the implant was calculated ÌCT scans
which showed that the 10Ìg group had noticeably more bone
formed in the vicinity of the implant. The mechanical pull-
out testing performed to measure implant strength of fixa-
tion also showed the same picture. In order to determine the
molecular events responsible for the TGF-‚2mediated
enhancement of bone formation and implant fixation, we
examined the temporal gene expression profiles of 21 genes
at 1, 3, 5, 7, 10, 14 and 28 days post-operatively using real-
time PCR13. When compared to time point matched controls
(no TGF-‚2) the locally applied TGF-‚2accelerated, delayed
and in some cases augmented gene expression. For example,
the expression of IGF-1, VEGF, BMP-4, IGF-1R, T‚RI,
T‚RII, osteocalcin and osteonectin occurred at earlier times
in the TGF-‚2treated animals than in controls. TGF-‚1,
TGF-‚3, alkaline phosphatase and osteopontin expression
was delayed while TGF-‚2, BMP-2, BMP-7 and Flt-1 had up-
regulation without a change in timing of peak expression.
Principal components analysed identified four factors which
explained 93% of the variance in the data in the control
group and four factors explaining 94% of the variance in the
TGF-‚2treatment group. Closer examination of the genes
grouped in each component revealed that sets of genes that
were co-expressed in the control group were generally also
co-expressed in the treatment group. In all, 14 of the 21
genes examined exhibited this pattern. This observation
strongly suggests that the molecular events during bone
regeneration are governed by strict expression of certain sets
of genes whether or not an anabolic agent is used. However,
the presence of an anabolic agent may enhance the regener-
ative process by altering their expression profile.
1. Sumner DR. Fixation of implants. In: Callaghan JJ,
Rosenberg AG, Rubash HE, Simonian PT, Wickiewicz
TA (eds) The Adult Knee. Lippincott Williams &
Wilkins, Philadelphia; 2003:289-296.
Sumner DR, Turner TM, Purchio AF, Gombotz WR,
Urban RM, Galante JO. Enhancement of bone
ingrowth by transforming growth factor beta. J Bone
Joint Surg Am 1995; 77-A:1135-1147.
Sumner DR, Turner TM, Urban RM, Leven RM,
Hawkins M, Nichols EH, McPherson JM, Galante JO.
Locally delivered rhTGF-‚2enhances bone ingrowth
and bone regeneration at local and remote sites of
skeletal injury. J Orthop Res 2001; 19:85-94.
Sumner DR, Turner TM, Cohen M, Losavio P, Urban RM,
Nichols EH, McPherson JM. Aging does not lessen the
effectiveness of TGF-‚2enhanced bone regeneration. J
Bone Miner Res 2003; 18:730-736.
Sumner DR, Turner TM, Urban RM, Turek T,
Seeherman H, Wozney JM. Locally delivered rhBMP-2
enhances bone ingrowth and gap healing in a canine
model. J Orthop Res 2004; 22:58-65.
Amsel S, Maniatis A, Tavassoli M, Crosby WH. The sig-
nificance of intramedullary cancellous bone formation
in the repair of bone marrow tissue. Anat Rec 1969;
Patt HM, Maloney MA. Bone marrow regeneration
after local injury: a review. Exp Hemat 1975; 3:135-148.
Suva LJ, Seedor JG, Endo N, Quartuccio HA,
Thompson DD, Bab I, Rodan GA. Pattern of gene
expression following rat tibial marrow ablation. J Bone
Miner Res 1993; 8:379-388.
Liang CT, Barnes J, Seedor JG, Quartuccio HA,
Bolander M, Jeffrey JJ, Rodan GA. Impaired bone
activity in aged rats: alterations at the cellular and
molecular levels. Bone 1992; 13:435-441.
10. Tanaka H, Wakisaka A, Ogasa H, Kawai S, Liang CT.
Local and systemic expression of insulin-like growth fac-
tor-I (IGF-I) mRNAs in rat after bone marrow ablation.
Biochem Biophys Res Commun 2001; 287:1157-1162.
11. Kuroda S, Virdi AS, Sumner DR. Expression profiling
of regenerate after bone marrow ablation. J Bone
Miner Res 2003; 18:S305.
12. De Ranieri A, Kuroda S, Virdi AS, Sumner DR. Locally
delivered TGF-‚ enhances implant fixation and bone
regeneration in rat model. J Bone Miner Res 2003;
13. De Ranieri A, Virdi AS, Kuroda S, Sumner DR.
Exogenously applied rhTGF-‚2enhances bone regener-
ation and implant fixation by accelerating and up-regu-
lating the innate gene expression profile in a rat model.
Trans ORS 2004; 29:1418.