Smith&Nephew, Inc., provided titanium implants for this study. There are no further patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
Conceived and designed the experiments: YL JSL BKG RI RV MDM WLM. Performed the experiments: YL JSL BN. Analyzed the data: YL JSL KR RV MDM WLM. Contributed reagents/materials/analysis tools: JSL KR MDM WLM. Wrote the paper: YL. Proofread the manuscript: MDM, WLM.
Despite the potential for growth factor delivery strategies to promote orthopedic implant healing, there is a need for growth factor delivery methods that are controllable and amenable to clinical translation. We have developed a modular bone growth factor, herein termed “modular bone morphogenetic peptide (mBMP)”, which was designed to efficiently bind to the surface of orthopedic implants and also stimulate new bone formation. The purpose of this study was to coat a hydroxyapatite-titanium implant with mBMP and evaluate bone healing across a bone-implant gap in the sheep femoral condyle. The mBMP molecules efficiently bound to a hydroxyapatite-titanium implant and 64% of the initially bound mBMP molecules were released in a sustained manner over 28 days. The results demonstrated that the mBMP-coated implant group had significantly more mineralized bone filling in the implant-bone gap than the control group in C-arm computed tomography (DynaCT) scanning (25% more), histological (35% more) and microradiographic images (50% more). Push-out stiffness of the mBMP group was nearly 40% greater than that of control group whereas peak force did not show a significant difference. The results of this study demonstrated that mBMP coated on a hydroxyapatite-titanium implant stimulates new bone formation and may be useful to improve implant fixation in total joint arthroplasty applications.
Total joint replacement surgeries have been performed popularly because these surgeries can successfully relieve pain and improve functional outcomes. However, the failure rate of revision joint replacements is also dramatically higher than primary replacements due primarily to a challenging environment for new bone formation
Collectively, previous studies demonstrate the potential for growth factor delivery strategies to promote orthopedic implant healing. However, translation of growth factor delivery to clinical applications is plagued by significant challenges
Bone morphogenetic proteins (BMPs) are the key cytokines in bone formation and repair. A possible strategy to utilize BMPs’ activity in clinical applications is to enhance the activity of autologous BMPs. BMP-2 incorporated into biomimetic calcium phosphate coatings has been demonstrated to be capable of inducing bone formation at an ectopic site and sustaining osteogenic activity for a considerable period of time
We hypothesized that a modular bone morphogenetic peptide (mBMP) could be used to “dip-coat” the surface of an orthopedic implant, which in turn would be capable of promoting implant-native bone healing. Specifically, we synthesized a peptide with two functional units: i) an osteocalcin-inspired hydroxyapatite (HAP)-binding sequence; and ii) a peptide sequence previously shown to mimic some of the pro-osteogenic properties of the protein BMP-2
Custom implants (diameter: 8 mm, HAP coated length: 14 mm. Smith and Nephew Corp. Andover, MA) were “porous coated”, meaning that they incorporate a porous titanium surface that is plasma spray bound with hydroxyapatite. In addition, the implants were designed to include a 1 mm gap all around the perimeter of the cylindrical implants, so that there was a well-defined gap between the native bone and the implant surface (
Modular bone morphogenetic peptide (Amino acid sequence of mBMP; KIPKASSVPTELSAISTLYLAAAAγEPRRγEVAγEL) was synthesized using standard Fmoc solid phase peptide synthesis, as reported previously
Each experimental HAP-titanium implant was incubated in 4 mL of mBMP solution (1.9 mM) 4 hours at 37°C. The mBMP solution was sterilized by passage through sterile 0.22 µm filters. All incubation procedures were performed in aseptic conditions.
Twelve mature female sheep, ranging in age from 3.5 to 5 years and weighing between 70 to 110 kg (82.4±5.6 kg: mean±SD) were utilized in the study. All experimental protocols were approved by the Institutional Animal Use and Care Committee of University of Wisconsin-Madison.
In each of the 12 sheep, one of the stifles was randomized (block design) to receive an mBMP-coated HAP-titanium implant and the other (contralateral) received an un-treated implant (n = 12/treatment). The sheep were euthanized at 4 weeks after surgery. 8 sheep (16 stifles, 8/group) were subjected to C-arm computed tomography (DynaCT) and biomechanical testing, 4 sheep (8 stifles, 4/group) were used for histologic analysis.
Surgery was carried out under general anesthesia with isoflurane and oxygen inhalation via endotracheal intubation. Procaine Penicillin G (5 mls) was administered via muscle pre-operatively and an additional dose (5 mls) post-operatively. An 8 cm medial incision was made along the anterior edge of medial collateral ligament in one randomly selected stifle. The medial portion of medial condyle was exposed. An 8 mm diameter hole (14 mm deep) was drilled from the medial surface of the condyle near to collateral ligament origin by an 8 mm cannulated drill bit 5 mm proximal to the joint line taking care to avoid the joint. An mBMP-coated HAP-titanium implant was inserted in the hole (
After 4 weeks, all sheep were euthanized for biomechanical, DynaCT and histological analyses.
At the same day of sacrifice, the medial femoral condyles were cut into uniform bone blocks (3.0×3.0×2.0 cm3) and imaged using a Siemens Artis Zeego (Siemens Healthcare, Erlangen, Germany) C-arm computed tomography (DynaCT), which has a wide angle cone-beam x-ray tube and 40 cm × 30 cm flat-panel detector integrated with a robotic C-arm gantry. The acquisition parameters for DynaCT were as follows: 86 kV tube voltage, automatic tube current ranging from 70 to 125 mA, a 20 s rotation time (total of 496 projections over 200 degree sweep angle). The projection images were acquired in a 2 × 2 binning mode, providing a 154 µm detector element resolution. Acquired images were then transferred to a dedicated post-processing workstation (
The Dyna CT image of each specimen was analyzed with Image J software (National Institutes of Health, Bethesda, MA). The area of bone ingrowth in the gap between the implant and hosting bone was calculated and compared between mBMP-coated and control groups.
After DynaCT scan, the femoral condylar bone blocks were used for push out mechanical testing. The specimen was placed in a MTS 858 BIONIX Test System (MTS Systems Corp., Eden Prairie, MN) with a 1500 lb load cell. The titanium implant in the specimen was precisely aligned with a pushing shaft. A linear extensometer was attached to measure the displacement of the implant. The displacement rate was 5.0 mm/min as previously described
The remaining 8 condyles in 4 sheep were used for histological analysis. The specimens were fixed in neutral buffered 10% formalin for 1 week. The medial condyle was sectioned sagitally into 3 equal sections crossing the implant axis. The undecalcified specimens were embedded in polymethylmethacrylate (PMMA), sectioned (100 µm) and stained with Goldner’s Trichrome for the evaluation of implant to bone healing. The sections from each specimen were examined by three senior researchers blinded to group assignments. Fine detail contact microradiography (Hewlett-Packard Faxitron, McMinnville, OR. USA) combined with vacuum technique was also performed on 3 sections of each specimen as previously described.
The Shapiro-Wilk Test was first used to determine the normal distribution (Gaussian distribution) of histological, microradiographic, DynaCT and mechanical testing data sets. If the data sets were normally distributed, the Student’s Paired t-test was used for statistical analysis for histology, microradiography, DynaCT and mechanical testing data between the two treatment groups. If the data sets were detected to be not normally distributed, the Mann-whitney U test was used to compare these differences. Differences were considered to be significant at a probability level of 95% (p<0.05). All statistical analyses were performed with a commercially available software program (SAS Version 8e, SAS Institute Inc., Cary, NC).
The amount of mBMP initially bound to a HAP-titanium implant was 414.4±22.8 µg. The release profile of mBMP in SBF showed large initial release 51.1±2.9% of initially bound mBMP during first two days (
(A) Cumulative release of mBMP from HAP-titanium implant in SBF is over 60% at 4 weeks. (B) Fluorescent images of an implant after incorporation with fluorescently labeled mBMP (top) and after 4-week incubation in simulated body fluid (SBF) (bottom).
No postoperative complications were seen and all sheep were fully weight-bearing at the same day after surgery. No signs of infection were observed at the time of euthanasia.
DynaCT analysis demonstrated that the mBMP-treated group (4.7±2.0 mm2) had a significantly higher density of mineralized bone tissue filling the 1 millimeter bone-implant gap than the control group (3.7±2.0 mm2) (p<0.05) (
A: Control, DynaCT demonstrating that gap between the implant and host bone was still visible (white arrows). B: The gap between mBMP coated HAP-implant and host bone was filled with high density mineralized tissue.
Microradiographical data analyses showed the mBMP-coated HAP-titanium implants had significantly greater area of higher bone density in the bone-implant gaps compared to the controls (p<0.05) (
“*” means significant difference between treatments (p<0.05).
In C and D, areas between white arrows highlighted original 1-mm gap between the implant and host bone.
“*” means significant difference between treatments (p<0.05).
The results of mechanical push-out testing demonstrated that the stiffness of the mBMP-treated group (2157±651.9 N/mm) was significantly greater than that of control group (1545±480.5 N/mm) (p<0.05), whereas there was no significant difference in peak force between the mBMP-treated group (1022±371.4 N) and the control group (682.2±269.3 N) groups (p>0.05).
The mBMP peptides were efficiently bound to a HAP-titanium implant and released over 28 days in simulated body fluid.
mBMP release kinetics showed a substantial initial release of over 50% during the initial 2 days, followed by minimal release kinetics over more than 28 days. These distinct regimes of mBMP release can be attributed to an initial release of poorly bound peptide due to surface saturation, followed by slower release of mBMP that was strongly bound to the hydroxyapatite surface. In particular, the solution concentration used for mBMP binding here (1.9 mM) was much higher than the concentration previously shown to saturate binding to a hydroxyapatite surface
Sheep were selected for this study because they provide similar bone density and weight to humans, and they have been widely used for evaluation implant-bone healing in previous studies
DynaCT, histological and microradiographical results of this study demonstrated that significantly higher density tissue and new bone formation was present in the gap between mBMP-coated HAP-titanium implant and condylar bone compared to control. An interesting observation here was that endochondral ossification with the formation of cartilaginous tissues was not observed at 4 weeks post-surgery. Instead, it appeared that direct ossification of mesenchymal tissue as intramembraneous ossification was taking place, as reported in a previous study
New bone formation in the gap between the implant and native host bone can also increase rigidity and fixation of an implant, which has been validated in the current study. The push-out stiffness of mBMP coated HAP-titanium implant group was significantly greater than that of control although the peak force did not reveal a statistically significant difference. Few studies to date have performed mechanical push-out testing on HAP-titanium implants treated with biologic molecules. Elmengaard et al evaluated the effects of an Arg-Gly-Asp (RGD) peptide coating on tissue integration and titanium implant fixation across a bone-implant gap in dogs
It is noteworthy that specific aspects of this study were designed for relevance to potential clinical applications of these biomaterials. First, the bone-implant gap size of implant selected for this study (1 mm) was a critical size, which may be larger than the gap in actual human clinical applications, but may represent a challenging scenario for bone-implant healing in revision joint arthroplasty. Second, the four week evaluation time is a relatively early time point to characterize new bone formation, and may provide particular insights into the degree of accelerated bone formation. A later time point could provide insight into the ability of mBMP to promote stable long-term implant fixation, but would not indicate an ability to promote the rapid fixation that is needed. The purpose of this study was to characterize a challenging bone-implant gap and an early time point, to more directly represent the challenging environment present during revision arthroplasty, and the need to accelerate fixation and avoid development of fibrous tissue that can lead to pain and implant instability.
The mBMP peptides were efficiently bound to a HAP-titanium implant and released over 28 days in a sustained manner
The authors thank Vicki Kalscheur and Kari A. Pulfer for their technical support and Smith&Nephew Corp. for its providing HAP-titanium implants.