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Recombinant Human Bone Morphogenetic Protein 6
Delivered Within Autologous Blood Coagulum Restores
Critical Size Segmental Defects of Ulna in Rabbits
Lovorka Grgurevic,
1
Hermann Oppermann,
2
Marko Pecin,
3
Igor Erjavec,
1
Hrvoje Capak,
4
Martina Pauk,
1
Sven Karlovic,
5
Vera Kufner,
1
Marija Lipar,
3
Jadranka Bubic Spoljar,
1
Tatjana Bordukalo-Niksic,
1
Drazen Maticic,
3
Mihaela Peric,
1
Reinhard Windhager,
6
T Kuber Sampath,
7
and Slobodan Vukicevic
1
1
Laboratory for Mineralized Tissues, School of Medicine, University of Zagreb, Zagreb, Croatia
2
Genera Research, Kalinovica, Sveta Nedelja, Croatia
3
Clinics for Surgery, Orthopedics, and Ophthalmology, School of Veterinary Medicine, University of Zagreb, Zagreb, Croatia
4
Department of Radiology, School of Veterinary Medicine, University of Zagreb, Zagreb, Croatia
5
Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia
6
Department of Orthopedics and Trauma Surgery, Medical University of Vienna, Vienna, Austria
7
perForm Biologics Inc., Holliston, MA, USA
ABSTRACT
BMP2 and BMP7, which use bovine Achilles tendon–derived absorbable collagen sponge and bovine bone collagen as scaffold,
respectively, have been approved as bone graft substitutes for orthopedic and dental indications. Here, we describe an
osteoinductive autologous bone graft substitute (ABGS) that contains recombinant human BMP6 (rhBMP6) dispersed within
autologous blood coagulum (ABC) scaffold. The ABGS is created as an injectable or implantable coagulum gel with rhBMP6 binding
tightly to plasma proteins within fibrin meshwork, as examined by dot-blot assays, and is released slowly as an intact protein over 6
to 8 days, as assessed by ELISA. The biological activity of ABGS was examined in vivo in rats (Rattus norvegicus) and rabbits
(Oryctolagus cuniculus). In a rat subcutaneous implant assay, ABGS induced endochondral bone formation, as observed by histology
and micro-CT analyses. In the rabbit ulna segmental defect model, a reproducible and robust bone formation with complete
bridging and restoration of the defect was observed, which is dose dependent, as determined by radiographs, micro-CT, and
histological analyses. In ABGS, ABC scaffold provides a permissive environment for bone induction and contributes to the use of
lower doses of rhBMP6 compared with BMP7 in bovine bone collagen as scaffold. The newly formed bone undergoes remodeling
and establishes cortices uniformly that is restricted to implant site by bridging with host bone. In summary, ABC carrier containing
rhBMP6 may serve as an osteoinductive autologous bone graft substitute for several orthopedic applications that include delayed
and nonunion fractures, anterior and posterior lumbar interbody fusion, trauma, and nonunions associated with neurofibromatosis
type I. © 2018 American Society for Bone and Mineral Research.
KEY WORDS: BONE MORPHOGENETIC PROTEIN 6; AUTOLOGOUS BLOOD COAGULUM CARRIER; CRITICAL SIZE DEFECT; AUTOLOGOUS BONE
GRAFT SUBSTITUTE; FRACTURE HEALING
Introduction
Bone heals spontaneously upon fracture by recapitulating the
cellular events associated with embryonic bone develop-
ment,
(1–3)
except when compromised by smoking, use of steroids,
pseudoarthrosis in NF-1 patients, and osteoporosis.
(1,4,5)
The large
bone defects causedby tumors, trauma, congenital disorders,and
infection also fail to heal and over time become a nonunion
representing challenging issues in orthopedic medicine and a
burden for the health care system.
(1,6)
In approximately 10% of
cases, bone fractures heal slowly or fail to heal and require
additional medical interventions.
(7,8)
For example, tibial nonunion
can potentially lead to the loss of the function or even loss of
limb.
(9)
The medical need in this clinical area warrants new and
effective therapies to be developed and introduced in clinical
practice.
Autograft is a gold standard to promote bone healing and
restore function for segmental long bone defects, delayed and
nonunion fractures, and spinal fusion procedures. The availabil-
ity of autologous bone tissue, limited in quantity and morbidity,
is associated with harvested site, which demands an alternative
to autograft bone.
(10–12)
Allogenic demineralized bone matrix
(DBM) has been used widely as an option to induce new bone
formation because it provides osteoinductive signals and
Received in original form June 28, 2018; revised form August 29, 2018; accepted September 16, 2018. Accepted manuscript online September 24, 2018.
Address correspondence to: Slobodan Vukicevic, MD, PhD, Laboratory for Mineralized Tissues, School of Medicine, University of Zagreb, Salata 11, HR-10000
Zagreb, Croatia. E-mail: slobodan.vukicevic@mef.hr
ORIGINAL ARTICLE
JBMR® Plus (WOA), Vol. xx, No. xx, Month 2018, pp 1–13
DOI: 10.1002/jbm4.10085
© 2018 American Society for Bone and Mineral Research
1
osteoconductive substratum.
(13–15)
Advances made in the
isolation of bone morphogenetic proteins (BMP) from DBM
(16)
and subsequently the identification of the corresponding
genes
(17,18)
allowed the production of recombinant human
BMPs in promoting fracture healing at compromised set-
tings.
(19–21)
Recombinant BMP2 soaked in absorbable collagen
sponge (InFuse) and recombinant BMP7 combined with bovine
bone collagen (OP1-Putty) have been approved as biologic bone
graft substitutes to bridge the gap and restore delayed and
nonunion fractures.
(22–24)
BMP alone cannot form bone unless it is delivered with an
appropriate scaffold and responding cells are available in a
permissive environment.
(25–30)
Studies thus far have used
animal-derived collagens as scaffold for BMP2 and BMP7 in
approved products and collagens, synthetic calcium phosphate-
based ceramics, and precalcified cellulose matrix as scaffolds in
preclinical studies.
(31–33)
In clinical studies, rhBMP2 containing
collagen has been used in reconstruction of mandibular bone
defects
(34,35)
and delayed diaphysis fractures. Evaluation of
rhBMP2 containing ceramic-collagen composite device in
posterolateral lumbar fusion (PLF), however, has not met
primary endpoint and failed to get an FDA approval. This
device was also reported to have unwanted side effects likely
attributed to the use of a high dose of rhBMP2 and its weak
binding to collagen-ceramics.
(6,36–38)
Here we describe an autologous bone graft substitute (ABGS)
that is composed of recombinant human BMP6 (rhBMP6)
combined with autologous blood coagulum (ABC) to guide the
formation of new bone to promote bone healing in a large
diaphysis segmental defect and bridge the gap. ABC circum-
vents the use of animal-derived collagen, limiting possible
inflammatory processes due to its autologous nature, provides
functional and physiological carrier for rhBMP6, and offers a
flexibility to mold to the desired shape, thus facilitating its use
within different anatomical structures.
(9)
We also describe the
method of formulation using ABC and rhBMP6, binding and
release characteristics of rhBMP6 from plasma proteins, and
rheological properties for in vivo bone-inducing activity in rat
subcutaneous implants and rabbit critical size ulna defect.
(39)
The preclinical data generated represent a solid foundation to
progress the ABGS toward further stages of drug development
and its use in human orthopedic indications.
Materials and Methods
Recombinant human BMPs
The manufacturing process for rhBMP6 was developed and is
conducted by Genera Research (Kalinovica, Croatia). Engi-
neered Chinese Hamster Ovary (CHO) cell line was used to
produce and purify rhBMP6 from the media using heparin
affinity and hydrophobic interaction chromatography,
followed by the reverse phase HPLC. It was then lyophilized
and stored at 20°C in vials containing 0. 5 mg >99% pure
rhBMP6. rhBMP2 and rhBMP7 used in in vitro experiments
were from R&D Systems (Abingdon, UK). rhBMP7 for in vivo
experiments was used as OP-1 Putty commercial device
(Ossigraft) for human use.
Mouse C2C12-BRE-Luc reporter gene assay
The activity of rhBMP6 was determined in mouse C2C12-BRE-Luc
BMP reporter cell assay, stably transfected with a reporter
plasmid consisting of a BMP response element (BRE) from the
Id-1 promoter fused to a luciferase reporter gene (kindly
provided by Dr Gareth Inman).
(40)
To measure the luciferase
activity, 20 mL of the cell lysate was added to 100 mL luciferase
assay reagent (Promega, Madison, WI, USA) and luminescence
was then quantified by Wallac Victor luminometer (PerkinElmer,
Waltham, MA, USA).
Formulation of rhBMP6 within ABC
Blood samples were collected from rabbit marginal ear veins
into tubes without anticoagulant substance in a volume of
1.5 mL. rhBMP6 was added into the blood in the amounts of
25 mg, 50 mg, and 100 mg with 50 mM concentration of calcium
chloride and mixed by rotating the tubes. ABC þrhBMP6 were
prepared in a syringe and left at room temperature to coagulate
for 60 to 90 minutes. The liquid portion (serum) was removed
and the homogeneous, cohesive, injectable and malleable ABGS
gel was ready for use.
rhBMP6 characterization and release studies
For rhBMP6 identification and characterization, lyophilized
protein samples were subjected to SDS-PAGE electrophoresis,
transferred to the nitrocellulose membrane, and analyzed by
Western blot using the rhBMP6-specific monoclonal antibody
(available in the rhBMP6 DuoSet ELISA kit, R&D Systems, DY507).
To demonstrate rhBMP6 stability in the coagulum after ABGS
formation, ABGS was homogenized 60 and 90 minutes after
preparation in 2% SDS, insoluble particles were removed by
centrifugation, and supernatants were analyzed by Western blot
using the same rhBMP6-specific antibody.
For determination of the release profile of rhBMP6 from the
ABGS, rhBMP6 was added to the blood in two concentrations, 2
and 5 mg/mL. After the coagulum was formed, it was placed in
the basal medium and the release profile of the rhBMP6 was
determined during a period of 14 days. The medium was
changed every 2 days, and samples of medium for rhBMP6
measurement were collected on days 1, 3, 5, 8, 10, and 12 after
ABGS formation. rhBMP6 released into the medium was
measured by the commercially available rhBMP6-specific ELISA
(R&D Systems, DY 507).
Binding affinity of rhBMP6 for plasma proteins
Autologous fibrinogen concentrate (FC) was prepared from
human plasma, obtained from healthy volunteers. rhBMP6 was
radiolabeled with radioactive technetium (
99m
Tc) using the
IsoLink Kit Mallinckrodt (Covidien Pharmaceuticals; gift from
Dr Hector H Knight). The binding affinity of
99m
Tc-labeled
rhBMP6 to FC and other extracellular matrix (ECM) molecules,
including fibrinogen, albumin, thrombin, heparan sulfate
proteoglycan, collagen I and IV, as well as the retention of
BMP6 in the plasma sample added into the blood before
precipitation, was semiquantitatively verified.
(41)
Briefly, 1 mL
of human blood (healthy volunteers) was drawn into a 2 mL
syringe and, using a syringe connector, blood, CaCl
2
,and
200 mLof
99m
Tc-rhBMP6 were mixed and left at room
temperature over 90 minutes in the 2 mL syringe. After 90
minutes, each ABC was expelled from the syringe and the
syringe was washed with 2 mL of 2% SDS and the wash was
pooled together. Radioactivity of the samples was measured
after a 2-hour incubation period using a gamma counter and
was expressed as counts per minute (cpm). All values were
corrected for the half-life factor of
99m
Tc.
2GRGUREVIC ET AL. JBMR Plus (WOA)
Testing of ABC biomechanical parameters
For evaluation of ABC biomechanical properties, forward
extrusion test (FET) and cut test were developed and validated
using Texture Analyzer TA.HD Plus (Texture Technologies,
Hamilton, MA, USA).
(9)
Sixteen samples were initiated from
each individual blood sample; 8 for each of the tests. Of these, 5
were used to investigate the effect of coagulation time on ABC
biomechanical properties (ie, standing at room temperature for
30, 45, 60, 75, or 90 minutes); 1 to investigate the effect of
hemolysis, which was induced by vigorous shaking (tested after
60 minutes); and 2 to investigate the effect of different CaCl
2
concentrations (high 15 mM and low 1 mM) after 30 and 60
minutes. Overall, 9 different healthy donors (including both men
[n¼7] and women [n¼2]) contributed with 15 blood samples in
total. In both tests, stiffness, elasticity, and work (required for
forward extrusion or cutting, respectively) were determined. The
effect of time was estimated by comparing values at 30 minutes,
45 minutes, and 60 minutes to each other (30 to 60 minutes is
the anticipated coagulum formation time) and by comparing
values at 75 and 90 minutes to the average for 30 to 60 minutes
to evaluate changes in ABGS properties after 60 minutes. The
effect of blood shaking (hemolysis) was estimated by comparing
the properties with and without vigorous shaking at 60 minutes.
Evaluation of time effect and the CaCl
2
effect included multiple
comparisons. The effect of CaCl
2
was estimated by comparing
time-averaged values between coagula formed with 0 (no), 1
(low), or 15 (high) mM CaCl
2
. General linear mixed models were
fitted using SAS 9.3 (SAS Institute, Cary, NC, USA).
Animal models
The number of rats in s.c. bone formation assays was determined
based on a well-known ectopic bone formation cascade in time,
as previously described.
(16,42)
The number of rabbits per
experimental group was determined by well-characterized
percent of CSD bone repair in time, as previously described.
(39)
In brief, using general linear mixed models (GLMMs) with
restricted maximum likelihood (REML) estimation to produce
time-averaged difference between groups and with using four
repeated assessments and assuming variability around 20% at
each time point, autocorrelation 0.6, and autoregressive
covariance structure, 7 animals per group per time point
provide 80% power to detect such a difference at a two-sided
a¼0.05. Because there was no healing of the defect in the
control animals (variability ¼0%), we applied the 3R principle
and decreased the number of animals to 5 per each dose group
with a possibility of gradually increasing the number of rabbits
up to 7 per group, if applicable.
Rat subcutaneous implant assay
Assay was performed on 12-week-old male Sprague Dawley
laboratory rats (body weight 320 to 350 g). Laboratory animals
were housed in polysulfonic cages in conventional laboratory
conditions at 20°C to 24°C, relative humidity of 40% to 70%,
and noise level 60 dB. Standard GLP diet and fresh water was
provided ad libitum, without environmental enrichment.
Animal care was in compliance with SOPs of animal facility;
the European convention for the protection of vertebrate
animals used for experimental and other scientific purposes
(ETS 123).
rhBMP6 osteogenic activity was tested at different doses in
the rat subcutaneous assay, as previously described.
(16,42)
ABC
was prepared from 0.5 mL of rat full blood, which was mixed
with an appropriate amount of rhBMP6 and left for 60 minutes to
coagulate in a 1 mL syringe. After removing the serum, 370 mLof
the ABGS was implanted. The osteogenic response of rhBMP6
doses (2.5 mg, 5 mg, 10 mg, and 20 mg per mL blood) was tested
in two rats for each dose, as previously described.
(14,43)
Briefly, a
small pocket was created under the skin in the axial regions to
implant ABC prepared with rhBMP6. The ABC was implanted and
sealed with a single suture to the fascia and three sutures for the
skin. To analyze ectopic bone formation, animals were scanned
using the 1076 micro-CT device (SkyScan, Bruker microCT,
Kontich, Belgium) 14 days after implantation. Ectopic bone
formation was observed in all groups of animals and was
quantified by micro-CT analysis.
The neutrophil infiltration was determined using naphtol AS-
D chloroacetate esterase staining (Sigma, St. Louis, MO, USA) on
histological sections. Neutrophils were counted in the vicinity
and inside ABGS using an ocular grid. Neutrophil accumulation
was expressed as number of neutrophils per mm
2
in two
implants from 4 rats at day 3 after implantation. Implants
contained 20 mg rhBMP2/300 mg bovine Achilles tendon–
derived absorbable collagen sponge; 20 mg rhBMP7/300 mg
bovine bone collagen carrier; and 20 mg rhBMP6/300 mL ABC. In
addition, neutrophil activity was determined at day 3 after
implantation by myeloperoxidase activity (MPO), as previously
described.
(44)
Implants (two from 4 rats) were extracted in 50 mM
KPO
4
buffer, pH 6.0, homogenized for 10 minutes, sonicated for
5 minutes, and finally the lysate was centrifuged for 60 minutes
at 20,000g.
The differential sensitivity to increasing concentration of
Noggin was tested in C2C12 cells transfected with BRE-Luc
reporter, in which rhBMP6 (5 mg) was more resistant to noggin-
mediated inhibition compared with rhBMP7 (5 mg). Recombi-
nant human Noggin was obtained courtesy of Dr Aris
Economides (Regeneron Corp., Tarrytown, NY, USA).
Rabbit ulna segmental defect model
Study protocols were conducted in male laboratory rabbits
(Oryctolagus cuniculus), New Zealand strain, 10 weeks old, with
health certificate, body weight from 2.3 to 2.5 kg. Animal facility
is registered by Directorate for Veterinary; reg. no. HR-POK-001.
An approval for the animal studies was given by the Directorate
for Veterinary and Food Safety, Ministry of Agriculture, Republic
of Croatia (approval no. 525-10/0255-14-3). Rabbits were
acclimated for 5 days and randomly assigned to their respective
treatment group. Animals were housed by standard rabbit cages
in conventional laboratory conditions at the temperature of
18°C to 22°C, relative humidity of 50% to 70%, fluorescent
lighting provided illumination 12 hours per day, and noise level
was 60 dB. Standard GLP diet (Mucedola srl, Milan, Italy),
bedding, and environmental enrichment were available and
fresh water was provided ad libitum. Animal care was in
compliance with SOPs of registries Croatian Animal facility HR-
POK-001; using 3R principle, pain and suffering were minimized
during the experiment. European convention for the protection
of vertebrate animals was used for experimental and other
scientific purposes (ETS 123).
In the critical size ulna defect experiments, rabbits were
prepared as described
(14,39,45)
and treated with rhBMP6 amounts
to confirm previous rhBMP6 efficacy results. Briefly, after
acclimatization period, enrofloxacin (10 mg/kg) was given to
the animals by intramuscular injection 1 day before operation
JBMR® Plus (WOA) BMP6 DELIVERED WITHIN ABC AS A BONE GRAFT SUBSTITUTE 3
and then 10 days after surgery. Before the surgery, blood was
collected from rabbit marginal ear veins into tubes without
anticoagulant substance. ABC was prepared 60 and 90 minutes
before implantation from 1.5 mL of blood mixed with an
appropriate volume of rhBMP6. Animals were randomly divided
into four groups (n¼5 each): A) control, defect filled with ABC
only; B) defect filled with ABC þrhBMP6 (25 mg/mL); C) defect
filled with ABC þrhBMP6 (50 mg/mL); and D) defect filled with
ABC þrhBMP6 (100 mg/mL). In another experiment (n¼5 per
group), ABGS (1.5 mL ABC þrhBMP6 (100 mg/mL) was compared
with collagen (150 mg) þrhBMP7 (100 mg/100 mg;) at weeks 2
and 8 after implantation.
Each animal was premedicated with a mixture of ketamine
50 mg/kg, acepramazine 1 mg/kg, and xylazine 5 mg/kg.
Anesthesia was maintained using a mixture of 1% to 1.5%
isoflurane and oxygen deliver by mask. A lateral incision,
approximately 2.5 cm in length, was made, and the tissues
overlying the ulna were dissected (skin and musculature). A
segment of the ulna measuring 17 mm (large defect) was
removed and the device was implanted into the defect site, with
the radius left intact for mechanical stability, without use of
internal or external fixation devices, as previously described.
(9)
Radiological images of the right forelimb were taken immedi-
ately after surgery and during 23-week bone healing period.
During the experiment, there were no adverse effects. Rabbits’
euthanasia was conducted after sedation, premedication of
3 mg/kg xylapane, and 20 mg/kg ketamine i.m. and administra-
tion of T61 (1 mL/kg) intrapulmonary.
Radiography
To monitor the critical size defect healing, X-rays were taken at
weeks 6, 9, 13, 16, 19, and 23 after surgery. All obtained
radiographs from rabbit bones were interpreted and scored
using a radiographic grading score system
(46,47)
by a surgeon
and a radiologist blinded to the treatment protocol and
postoperative interval. Radiographic grading scores (from 0 to
6) were as follows: 0, no change from immediate postoperative
appearance; 1, trace of radio-dense material in defects; 2,
flocculent radio density with spots of calcification and no defect
bridging; 3, defect bridged at least one point with material of
nonuniform radio density; 4, defect bridged in medial, and
lateral sides with material of uniform radio density (cut ends of
cortex remain visible); 5, same as grade 3, at least one of four
cortices obscured by new bone; 6, defect bridged by uniform
new bone, cut ends of cortex not found.
Micro-CT quantification
In subcutaneous rat assay, bone formation was scanned in vivo
after 14 days using SkyScan 1076 micro-CT device (SkyScan/
Bruker) at 18 mm resolution, as previously described.
(48,49)
Scanning parameters were 50 kV/200 mA, 0.5 mm aluminum
filter, 0.8° rotational shift throughout the 198° and frame
averaging value set at 2. Obtained images were reconstructed
using NRecon (Bruker) and the quantification was performed
using CTAn (Bruker) software. For quantification of the
medullary canal volume, the defect site was approximated
and delineated manually after which parameters for bone
volume (BV) and newly formed endosteal/medullary volume
(MV) were calculated. 3D models of the scanned bones were
obtained using CTVox (Bruker) software. Quantitative micro-CT
results were analyzed by one-way ANOVA with post hoc test for
linear trend.
Histology
Soft tissue free bones were fixed in 4% formalin for 2 weeks, and
entire bone was embedded in plastic resin (Technovit 7200).
Samples were cut at 5 mm slices with a diamond saw and
stained using Masson Goldner Trichrome dye, as previously
described.
(50,51)
Images were obtained using Olympus BX51 Epi-
Fluorescence microscope.
Data analysis
Values are expressed as mean SEM or SD as indicated. For
statistical comparison of two samples, a two-tailed Student’st
test was used and p<0.05 was considered significant. Two-way
analysis of variance with Duncan’s multiple range test was
performed to determine the effect of treatment and time on
biochemical and bone repair parameters. Additional specific
data analyses are presented in figure legends. Analyses were
performed by SAS for Windows 9.3 (SAS Institute).
Results
rhBMP6 production and biological activity
rhBMP6 was produced as a secretory dimeric protein from a
stable CHO cell line generated by recombinant technology.
(9,14)
rhBMP6 was purified to near homogeneity (>99%) from the
medium by subjecting to conventional ionic, hydrophobic, and
metal-chelated chromatography with final reverse-phase C18-
HPLC columns. rhBMP6 behaves as a diffused 37 kDa in
nonreduced condition and 17 kDa under reduced condition, as
stained by Coomassie blue (Fig. 1A) and as cross-reacted species
by Western blot using BMP6-specific antibody (Fig. 1B). BMP6-
specific monoclonal antibody, which has been used for Western
blot, recognizes all forms of nonreduced protein and mostly the
larger form of reduced species. The biological activity of the
purified rhBMP6 was assessed by using mouse C2C12-estab-
lished myoblast cell line transfected with BMP Response
Elements (BRE)–Luciferase (Leu) construct at varying doses
(Fig. 1C).
rhBMP6 binding and release studies using blood
coagulum
Blood samples taken randomly from 60 human subjects
(referred to Department of Laboratory Diagnostics of University
Hospital Center Zagreb) demonstrated that 75% of samples
coagulated in 30 minutes, and 100% of samples achieved
coagulation in 60 minutes from blood sampling (Fig. 2A). Studies
with radioactive technetium (
99m
Tc)-labeled rhBMP6 performed
to evaluate the extent of rhBMP6 binding to coagulum show
that more than 99% of
99m
Tc-rhBMP6 was retained in the
coagulum at 30, 45, and 60 minutes after mixing with blood.
Examination of the ratio of released/retained rhBMP6 from the
blood coagulum over a 14-day period shows that it is released
slowly from the coagulum with a mean residence time of
approximately 5 to 7 days as determined by a specific ELISA
(Fig. 2B). The analysis of the stability of rhBMP6 during the
preparation of ABGS demonstrated no signs of degradation after
60 to 90 minutes and overall loss in syringes was around 5% as
examined by Western blot (Fig. 2C). Studies on binding and bio-
distribution of rhBMP6 to blood proteins using radio-labeled
rhBMP6, dot-blot, and immunoblot analysis demonstrated
that more than 95% of the rhBMP6 was captured within
the coagulum and sequestered to blood components like
4GRGUREVIC ET AL. JBMR Plus (WOA)
Fig. 1. Purification and biological activity of rhBMP6. SDS-PAGE analysis of rhBMP6: Coomassie-stained (A) and Western blot (B). Nonreduced (37 kDa)
forms are presented in lanes 1 and 6 and reduced (17 kDa) forms are in lanes 2 and 5, respectively. Molecular weight marker (ColorBurst, Sigma-Aldrich) is
indicated in lanes 3 and 4, respectively. (C) Specific rhBMP6 activity in C2C12-BRE-Luc reporter gene assay: comparison between two different clinical
batches. The activity was tested in a range of concentrations as in xaxis and expressed in relative light units (RLU) as in yaxis. Data represent mean SEM
of 3 independent measurements.
Fig. 2. Characterization of coagulation and binding, release, and stability of rhBMP6 in autologous bone graft substitute (ABGS). (A) Cumulative
frequency of coagulation formation over time. Raw data (n/N) and percentages are shown. Bracketed values are exact 95% confidence intervals (left-
sided 97.5% at 60 minutes). (B) Release profile of rhBMP6 from ABGS using two different concentrations of rhBMP6 over a 14-day period, as determined
by ELISA. Data represent mean SEM of 3 independent measurements. (C) The stability of rhBMP6 during preparation of the ABGS implant was
maintained over 60 minutes (lane 4) and 90 minutes (lane 5) with a loss of around 5% during preparation (lane 3). Arrow indicates 35 kDa band, which
corresponds to the mature rhBMP6 under nonreduced condition. (D) Semiquantitative analysis of
99m
Tc-labeled rhBMP6 binding to blood components
in a dot blot assay. Mean þSEM (n¼3) are shown.
JBMR® Plus (WOA) BMP6 DELIVERED WITHIN ABC AS A BONE GRAFT SUBSTITUTE 5
fibrinogen, alpha 2-macroglobulin, beta-2-microglobulin, and
thrombin (Fig. 2D). A high level of rhBMP6 saturation (99.9%)
was achieved within coagulum and the release of rhBMP6 from
the coagulum within the first 24 hours was lower than 0.2%.
Examination of the time to achieve the formation of ABC
consistently with defined rheological properties (stiffness,
elasticity, and work load) suggests a requirement of 45 to 60
minutes (Fig. 3) to achieve a uniform coagulation, and it
maintained its shape for 5 days, then reduced its size, dimension,
and consistency at day 8 and dissolved by day 14. Although all
the results described here are obtained using human blood
samples, comparable findings were observed for rat and rabbit
blood coagulum (data not shown).
Biological activity of ABGS at rat subcutaneous sites
The biological activity of ABGS was assessed in the rat
subcutaneous implantation assay. The formation of endochon-
dral bone was examined at days 1, 2, 3, 7, and 35 post-
implantation by histology and micro-CT analyses. Fig. 4Ashows
the photomicrographs of ABC alone implants harvested at days
1, 3, and 7. ABC alone formed a solid pluglike implant on day 1
surrounded by a thin membrane of extracellular matrix and
external mononuclear cells. Inside ABC, a layer of mesenchymal
osteoprogenitor-like cells formed a zone that toward day 3
merged with external cell condensations. By day 7, the ABC
showed the sign of dissolution and was replaced by normal
connective tissue without evident inflammation, fibrosis, or
edema, and by day 35, it completely disappeared with no sign of
the implant visible. The absence of inflammatory cells and no
granuloma tissue was noted. Fig. 4Bshows the photomicro-
graphs of ABC with rhBMP6 (25 mg/implant) implants harvested
at days 1, 3, 7, and 35. Day 1 implant composed of
osteoprogenitor cells (mesenchymal stem cells [MSCs]) stained
positive for alkaline phosphatase. By day 3, MSCs underwent
condensation with extracellular matrix expansion and sign of
early chondrocytes within the “osteoprogenitor zone”slowly
penetrated by cells from outside the ABC. This interconnected
area of ABC composed of MSCs under the rhBMP6 influence
rapidly differentiated into chondrocytes. By day 7, differentiated
chondrocytes underwent hypertrophy, resulting in endochon-
dral bone formation. By day 35, a dense trabecular bone was
evident with a broad outside cortexlike structure. The cellular
response elicited by ABGS (rhBMP6/ABC) was compared with
rhBMP2/Bovine Absorbable Collagen Sponge and rhBMP7/
Bovine Bone Collagen implants at an early time point. The
Fig. 3. Mechanical properties of the coagulum stiffness (N), elasticity (mm), and work load. (A) The effect of time. LS ¼least squares (mean). (B) The effect
of hemolysis. p<0.025 considered statistically significant (Q1, Q3–quartiles). (C) The effect of CaCl
2
.
6GRGUREVIC ET AL. JBMR Plus (WOA)
degree of inflammation, as determined by neutrophil accumu-
lation and myeloperoxidase activity (MPO), on day 3 implants
suggests that ABGS had a reduced neutrophil accumulation
(Fig. 4C) and a lower total MPO activity (Fig. 4D) compared with
BMP2- or BMP7-containing xenogeneic collagen implants.
Macroscopically, visualization after removal of implants from
the rat’s axilla is shown in Fig. 5Aand indicates absence of a
fibrous capsule. Quantification of the ectopic bone formation as
represented as bone volume (BV) showed a dose response,
assessed by micro-CT analysis (Fig. 5B).
Evaluation of ABGS in rabbit ulna defect models
The ABC implanted alone did not result in the formation of new
bone and failed to achieve rebridgement of the defect (Fig. 6).
ABC containing rhBMP6 (ABGS), however, reproducibly induced
new bone formation and restored the defect as assessed by
radiography. The new bone formation was induced in a dose-
dependent manner as represented at weeks 6, 9, 13, 16, 19, and
23 (Fig. 6), and all rabbit ulna are shown at the week 23
(Fig. 7A–C). Micro-CT analyses showed a dose-dependent
increase in bone quantity as examined by bone volume (BV)
and medullary volume (MV), which are comparable to the intact
bone of the contralateral side (Fig. 7D). The bone quality was
further confirmed by histology, as shown in a representative
sample from each group (Fig. 8). The dose of 100 mg rhBMP6/mL
ABC resulted in the complete restoration with fully established
cortices and remodeled medullar canal. In three rabbits, a partial
synostosis between healed ulna and radius appeared due to the
lack of space separating the two bones (Fig. 7B). The limitation of
the current study is a lack of biomechanical analysis as we have
dedicated most of the animals for the radiographic, micro-CT,
and histologic analyses.
ABGS versus rhBMP7/bovine collagen
We compared side-by-side rhBMP7/bovine bone collagen
device with the ABGS (rhBMP6/ABC) device in the rabbit ulna
defect repair model. Collagen alone did not induce bone
formation (data not shown), but rhBMP7 containing collagen
induced new bone formation (Fig. 9A). The rhBMP7/bovine bone
collagen commercial device contains 3.5 mg rhBMP7/g of
collagen, and to fill the rabbit ulna defect, we used 300 mg
that accounts for the total amount of 1.06 mg rhBMP7 in a
Fig. 4. Evaluation of ABGS in rat subcutaneous implants harvested at days 1, 3, 7, and 35. (A) Photomicrographs of histology of ABC alone at days 1, 3, and
7. Size marker: left column 500 mm (magnification 4); middle column 200 mm (magnification 10); right column upper (top) image 20mm
(magnification 60) and middle and lower (bottom) image 50 mm (magnification (40). (B) Photomicrographs of histology of ABGS (25 mg rhBMP6 per
implant) at days 1, 3, 7, and 35. Size marker: left column 500 mm (magnification 4); middle column upper (top) and lower (bottom) image 200 mm
(magnification 10), middle images 500 mm and 200 mm (magnification 4and 10, respectively); right column upper (top) image 20 mm
(magnification 60) and middle and lower (bottom) images 50 mm (magnification 40). Asterisks denote clearly demarcated zone composed of
osteoprogenitor cells stained positive for alkaline phosphatase on day 1; with condensations of extracellular matrix (black arrows) and formation of early
chondrocytes within the osteoprogenitor zone (black arrowheads). On day 3, cells from outside the ABC slowly penetrated and hypertrophic
chondrocytes in endochondral bone formation appeared on day 7 (yellow arrowheads). On day 35, dense trabecular bone (yellow arrows) with a broad
cortexlike structure from outside demonstrated a solid persistent bone ossicle (green arrowhead). (C) The neutrophil infiltration on histological sections.
The implants were examined on day 3 after implantation of 20 mg rhBMP2/300 mg bovine Achilles tendon, 20 mg rhBMP7/300 mg bovine bone collagen
carrier, or 20 mg rhBMP6/300 mL ABC, respectively. Mean SEM (n¼10), p<0.01 versus rhBMP6, p<0.05 versus rhBMP6. (D) Myeloperoxidase (MPO)
activity. Mean SEM (n¼8), p<0.01 versus rhBMP6, p<0.05 versus rhBMP6.
JBMR® Plus (WOA) BMP6 DELIVERED WITHIN ABC AS A BONE GRAFT SUBSTITUTE 7
collagen carrier. This rhBMP7/collagen-induced rebridgement of
the ulna defect was compared with ABGS (100 mg rhBMP6 in
1.5 mL blood) as shown in Fig. 9A. ABGS induced, at weeks 2 and
6, a formation of a new uniform bone and underwent a
remodeling to rebridge new cortices with adjacent host bone,
whereas the bone formation with rhBMP7/collagen was
delayed. Micro-CT analysis confirmed that the ABGS containing
rhBMP6 induced on week 8 after surgery around 2more bone
volume (Fig. 9B,C). The differential sensitivity to increasing
concentration of Noggin was tested in the C2C12 cell assay, in
which rhBMP6 (5 mg) was resistant to Noggin-mediated
inhibition compared with rhBMP7 (5 mg) (Fig. 9D).
Discussion
Bone morphogenetic proteins have been extensively explored
for their remarkable potential to regenerate new bone at
ectopic
(13)
and orthotopic sites.
(52)
Among BMPs, BMP2 and
BMP7 have been used in various clinical studies to promote
bone formation both in orthopedic
(21,53)
and dental
(54–56)
applications; however, safety issues and limitation in their use
were reported.
(57–59)
The bone devices consisting of a bovine
collagen matrix soaked with rhBMP2 or rhBMP7
(60,61)
were
approved by regulatory agencies for the treatment of tibial
fractures and nonunions but have also been used off-label for
different bone repair indications with an aim to overcome the
impaired healing.
(61,62)
In the present study, we demonstrate that an autologous
bone graft substitute containing rhBMP6 delivered within
autologous blood coagulum is capable of inducing new bone
formation in rat subcutaneous implants and can rebridge a large
segmental defect in ulna of mature rabbits. BMP6 was chosen
because it does not bind avidly to Noggin,
(63)
a natural BMP
antagonist abundant in bone and induces downstream
Fig. 5. Rat subcutaneous implants. (A) Rat subcutaneous implants in
vivo. The white arrow indicates the implant; white circle shows lack of
fibrotic tissue accumulation. (B) Bone volume (BV) calculated after micro-
CT scan in rat subcutaneous implants at day 35 with various rhBMP6
doses and accompanying 3D models of the newly formed bone. The
rhBMP6 dose used is represented as mg/implant. Mean SEM (n¼4 per
dose), p<0.01 versus 2.5 mg, 5 mg, and 10 mg; p<0.05 versus 2.5 mg.
Fig. 6. Radiographs of bone healing through the course of 0 to 23 weeks
for different doses of rhBMP6 (0, 25, 50 and 100 mg). Representative
rabbit from each treatment group is presented.
8GRGUREVIC ET AL. JBMR Plus (WOA)
Fig. 7. The effect of rhBMP6 dose on bone healing in rabbit ulna segmental defect. (A) Radiographs of all bone samples (n¼5 per group) at week 23. (B)
Radiographic grading scores (0–6) of all bone samples using an established scoring system.
(38,39)
(C) 3D models (longitudinal and cross-sectional) of bone
healing in rabbits after ex vivo micro-CT scan at week 23 after study termination. Three bone samples per dose group are shown, with corresponding X-
ray image on the left. Yellow arrows indicate initial defect size and introduced bone osteotomy sites. White asterisk indicates a partial synostosis between
ulna and radius. (D) Quantitative analysis of newly formed bone revealed a dose-dependent manner of rhBMP6 stimulation of bone defect healing. For
quantification, the defect site was approximated and delineated manually after which parameters for bone volume (BV) and newly formed endosteal/
medullary volume (MV) were calculated. 3D models of the scanned bones were obtained using CTVox (Bruker) software. Quantitative micro-CT
parameters were analyzed by one-way ANOVA with post hoc test for linear trend. Each bar represents mean SD.
JBMR® Plus (WOA) BMP6 DELIVERED WITHIN ABC AS A BONE GRAFT SUBSTITUTE 9
signaling by spanning across most of the BMP type I and type II
receptors that are present on the cell surface of responding
osteoprogenitors.
(64)
These BMP6 biological characteristics
support a high specific alkaline phosphatase activity in cultures
of established C2C12 mouse cell line
(41)
and rat osteosarcoma
osteoblastic cell line, ROS 17/2.8, compared with other BMPs
examined.
(63)
A wide range of carriers for BMPs have been investigated for
orthopedic indications comprising polymers (synthetic and of
natural origin), inorganic materials, and composites ranging
from nanoparticles to complex three-dimensional scaffolds,
membranes for tissue-guided regeneration, biomimetic surfa-
ces, and smart thermosensitive hydrogels.
(65,66)
Carrier systems
sustain the concentration of the rhBMP at the treatment site,
provide temporary scaffolding for osteogenesis, prevent ectopic
bone formation, and are ultimately absorbed over time.
Autologous blood coagulum was choosen as a preferred
scaffold because it provides: 1) fibrin meshwork for rhBMP6 to
bind tightly and release it slowly as intact protein over 6 to
8 days; 2) circulating osteoprogenitors for rhBMP6 to respond
readily during the fabrication of coagulum; and 3) a permissive
environment provoking lesser inflammation and devoid of
immune response. In ABGS, most (>95%) of rhBMP6 added to
autologous blood bound tightly to plasma proteins in the
coagulum, including albumin, thrombin, heparan sulfate, and
others. The addition of lower amounts of CaCl
2
ensured that the
coagulum remains homogeneous, cohesive, syringeable, in-
jectable, and malleable. The time required to achieve the
defined physical characteristic of the coagulum appears to be in
the range of 45 to 60 minutes. The time to achieve a uniform
coagulation of ABC was determined based on the rheological
properties (elasticity, stiffness) preferred for injection or
implantation to assure a shape at the pocket of segmental
defect. This was supported by the coagulum formation over
time in human population where 60 minutes provided a safer
and more reliable time frame for obtaining implant of desired
and necessary quality characteristics (Fig. 2A).
In the rat subcutaneous assay, rhBMP6/ABC induces the
cascade of cellular events that result in endochondral new bone
formation histologically comparable to that of rhBMP2 or
rhBMP7 with rat allogenic bone collagen as carrier and/or
allogenic DBM
(13,67,68)
with an accelerated chondrogenesis and
osteogenesis as examined at various time intervals after the
implantation. The newly formed bone undergoes a typical
remodeling that results in ossicles containing functional bone
marrow elements with cortices surrounding the implant outer-
space and fully maintained the volume of the implant by day 35.
The use of ABGS in the subcutaneous assay resulted in absence
of inflammation and immune responses compared with animal-
derived collagen as shown by a lower neutrophil accumulation
and lower total MPO activity.
ABGS (rhBMP6/ABC) induced new bone formation in rabbits
and restored ulna critical size defects in a dose-dependant
manner, as examined by ex vivo radiographs. The dose of
100 mg/mL ABC induced an optimal bone formation, whereas
25 mg/mL ABC showed a lesser response and at 50 mg/mL of ABC
showed an intermediate response. The ABGS-induced bone
formation is directly proportional to the dose used and this
dose-dependent response is comparable both in rat subcutane-
ous implants and rabbit ulna segmental defect models, as has
previously been shown for rhBMP2 in a gap healing defect
canine model.
(69)
A comparative study with rhBMP7/collagen
sponge showed that ABGS induced a new bone that is restricted
to the defect area and undergoes remodeling to achieve a
complete union quality comparable with native bone. Although
preclinical studies have limitations as no given animal model
mimics human skeletal and biomechanical conditions in
entirety,
(39)
diaphyseal segmental defect in preclinical studies
served as measurable outcome for bone formation toward
clinical studies.
(70)
One of the drawbacks of studies in small
animal models (rodents and rabbits) is retaining of notochordal
cells in adult life, so the regenerative effect of BMPs may be
different from that observed in humans.
(21)
Another limitation is
that four-legged animal loading and precise musculoskeletal
structure is mechanically dispositioned compared with humans.
The appearance of synostosis between ulna and radius was
observed in three rabbits, which reflects the nature of
osteogenic BMPs to induce bone formation and fusion upon
contact with new and old bone as the space of separation
between the ulna and radius is smaller in rabbits than
humans.
(39)
In summary, we present an ABGS that contains a low dose of
rhBMP6 delivered within ABC, a biocompatible native scaffold,
and may serve as a safe and robust biological osteoinductive
device, which is in contrast to other BMP-based therapies
Fig. 8. Evaluation ABGS in ulna critical size defect in rabbits. (A)
Photomicrographs of histology of representative slides from animals
treated with rhBMP6. Rabbits treated with 100 mg rhBMP6 showed a
complete restoration with cortical and trabecular bone in the diaphysis
and rabbits treated with 50 mg rhBMP6 showed a complete healing with
delayed remodeling, while rabbits treated with 25 mg rhBMP6 showed
an incomplete rebridgement of bone defect showing a dose-dependent
effect at 23 weeks after surgery. Yellow arrowheads denote the size of
surgical defects. (B) Magnified section of newly formed bone from yellow
rectangles in A. Black arrowheads indicate blood vessels, and arrows
indicate enlarged osteocyte lacunae in the new woven bone under
remodeling. Size marker indicates 200 mm.
10 GRGUREVIC ET AL. JBMR Plus (WOA)
(rhBMP2 and rhBMP7), employing bovine Achilles tendon and
bone-derived collagen, respectively, and a high BMP dose. More
preclinical studies and subsequent evaluation is ongoing,
(71)
and
future clinical trials are needed to address the safety and efficacy
of ABGS (rhBMP6/ABC)-based bone graft implant for its potential
use in orthopedic patients.
Disclosures
LG, HO and SV have an issued patent US8197840 and licensed to
Genera Research (GR). HO received grants and other from GR
during the study, RW is a consultant for Pfizer, Stryker, Takeda,
Depuy Synthes and Zimmer Biomet, TKS received grants and
other from perForm Biologics during the study; SV received
grants and other from (GR) and perForm Biologics during the
study. Other authors declare no conflict of interest.
Acknowledgments
This program was funded in part by the FP7/2007-2013 program
under GA HEALTH-F4-2011-279239 (Osteogrow), by the Horizon
2020 research and innovation program under GA No. 779340
Fig. 9. (A) Comparison of rhBMP7/bovine bone collagen with ABGS implant (rhBMP6/ABC) (right) in rabbit ulna segmental defect models at 2 and
6 weeks after administration. (B) In vivo X-ray analysis of critical size defects of rabbit ulna 2 and 6 weeks after surgery, treated with 100 mg BMP7 þbovine
bone collagen, 100 mg of BMP6 þABC, and a bovine collagen alone (control) (n¼8 rabbits per group). p<0.05 versus BMP7; p<0.01 versus control
(ANOVA, Dunnett test). (C) Ex vivo micro-CT analysis of critical size defects of rabbit ulnas 8 weeks after treatment (the same as in B,n¼8 per group).
p<0.05 versus BMP7; p<0.01 versus control (ANOVA, Dunnett test). (D) Effects of the inhibitory action of Noggin as examined for BRE-driven
luciferase activity in C2C12 cells for BMP6 and BMP7. Each data point is mean SD of 3 measured values. p<0.01 versus BMP6.
JBMR® Plus (WOA) BMP6 DELIVERED WITHIN ABC AS A BONE GRAFT SUBSTITUTE 11
(OSTEOproSPINE), and the Scientific Center of Excellence for
Reproductive and Regenerative Medicine (project “Reproduc-
tive and regenerative medicine—exploration of new platforms
and potentials,”GA KK01.1.1.01.0008 funded by the EU through
the ERDF).
Authors’roles: Study design: LG, TKS, RW, and SV. Study
conduct: LG, MP, IE, MP, TBN, VK, ML, HC, JBS, DM, and HO. Data
collection: MP, IE, MP, VK, HC, and MP. Data analysis: LG, MP, IE,
TBN, MP, and SV. Data interpretation: IE, MP, HO, RW, and SV.
Drafting manuscript: LG, IE, MP, TBN, VK, JBS, RW, and SV.
Revising manuscript content: TKS and SV. Approving final
version of the manuscript: TKS and SV. SV takes responsibility for
the integrity of the data analysis.
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