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Recombinant Human Bone Morphogenetic Protein 6 Delivered Within Autologous Blood Coagulum Restores Critical Size Segmental Defects of Ulna in Rabbits: BMP6 DELIVERED WITHIN ABC AS A BONE GRAFT SUBSTITUTE

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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.
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 μm (magnification 4×); middle column 200 μm (magnification 10×); right column upper (top) image 20 μm (magnification 60×) and middle and lower (bottom) image 50 μm (magnification (40×). (B) Photomicrographs of histology of ABGS (25 μg rhBMP6 per implant) at days 1, 3, 7, and 35. Size marker: left column 500 μm (magnification 4×); middle column upper (top) and lower (bottom) image 200 μm (magnification 10×), middle images 500 μm and 200 μm (magnification 4× and 10×, respectively); right column upper (top) image 20 μm (magnification 60×) and middle and lower (bottom) images 50 μm (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 μg rhBMP2/300 mg bovine Achilles tendon, 20 μg rhBMP7/300 mg bovine bone collagen carrier, or 20 μg rhBMP6/300 μL ABC, respectively. Mean ± SEM (n = 10), *p < 0.01 versus rhBMP6, **p < 0.05 versus rhBMP6. (D) Myeloperoxidase (MPO) activity. Mean ± SEM (n = 8), *p < 0.01 versus rhBMP6, **p < 0.05 versus rhBMP6.
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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 tendonderived 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 brin 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 neurobromatosis
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,
(13)
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.
(1012)
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 113
DOI: 10.1002/jbm4.10085
© 2018 American Society for Bone and Mineral Research
1
osteoconductive substratum.
(1315)
Advances made in the
isolation of bone morphogenetic proteins (BMP) from DBM
(16)
and subsequently the identication of the corresponding
genes
(17,18)
allowed the production of recombinant human
BMPs in promoting fracture healing at compromised set-
tings.
(1921)
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.
(2224)
BMP alone cannot form bone unless it is delivered with an
appropriate scaffold and responding cells are available in a
permissive environment.
(2530)
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 precalcied cellulose matrix as scaffolds in
preclinical studies.
(3133)
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.
(6,3638)
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
inammatory processes due to its autologous nature, provides
functional and physiological carrier for rhBMP6, and offers a
exibility 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
afnity 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 quantied 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 identication and characterization, lyophilized
protein samples were subjected to SDS-PAGE electrophoresis,
transferred to the nitrocellulose membrane, and analyzed by
Western blot using the rhBMP6-specic 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-specic antibody.
For determination of the release prole 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 prole 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-specic ELISA
(R&D Systems, DY 507).
Binding affinity of rhBMP6 for plasma proteins
Autologous brinogen 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 afnity of
99m
Tc-labeled
rhBMP6 to FC and other extracellular matrix (ECM) molecules,
including brinogen, 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 veried.
(41)
Briey, 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
tted 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 scientic 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)
Briey, 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
quantied by micro-CT analysis.
The neutrophil inltration 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 nally 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 certicate, 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%, uorescent
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
scientic purposes (ETS 123).
In the critical size ulna defect experiments, rabbits were
prepared as described
(14,39,45)
and treated with rhBMP6 amounts
to conrm previous rhBMP6 efcacy results. Briey, after
acclimatization period, enrooxacin (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 lled with ABC
only; B) defect lled with ABC þrhBMP6 (25 mg/mL); C) defect
lled with ABC þrhBMP6 (50 mg/mL); and D) defect lled 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%
isourane 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 xation 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,
occulent radio density with spots of calcication 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
lter, 0.8° rotational shift throughout the 198° and frame
averaging value set at 2. Obtained images were reconstructed
using NRecon (Bruker) and the quantication was performed
using CTAn (Bruker) software. For quantication 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 xed 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 Studentst
test was used and p<0.05 was considered signicant. Two-way
analysis of variance with Duncans multiple range test was
performed to determine the effect of treatment and time on
biochemical and bone repair parameters. Additional specic
data analyses are presented in gure 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 puried to near homogeneity (>99%) from the
medium by subjecting to conventional ionic, hydrophobic, and
metal-chelated chromatography with nal 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-specic antibody (Fig. 1B). BMP6-
specic 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
puried 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 specic 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. Purication 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) Specic 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% condence intervals (left-
sided 97.5% at 60 minutes). (B) Release prole 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
brinogen, 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 rst 24 hours was lower than 0.2%.
Examination of the time to achieve the formation of ABC
consistently with dened 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 ndings 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 inammation, brosis, or
edema, and by day 35, it completely disappeared with no sign of
the implant visible. The absence of inammatory 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 zoneslowly
penetrated by cells from outside the ABC. This interconnected
area of ABC composed of MSCs under the rhBMP6 inuence
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 signicant (Q1, Q3quartiles). (C) The effect of CaCl
2
.
6GRGUREVIC ET AL. JBMR Plus (WOA)
degree of inammation, 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 rats axilla is shown in Fig. 5Aand indicates absence of a
brous capsule. Quantication 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. 7AC). 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 conrmed 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 ll 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 (magnication 4); middle column 200 mm (magnication 10); right column upper (top) image 20mm
(magnication 60) and middle and lower (bottom) image 50 mm (magnication (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 (magnication 4); middle column upper (top) and lower (bottom) image 200 mm
(magnication 10), middle images 500 mm and 200 mm (magnication 4and 10, respectively); right column upper (top) image 20 mm
(magnication 60) and middle and lower (bottom) images 50 mm (magnication 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 inltration 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 conrmed 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
(5456)
applications; however, safety issues and limitation in their use
were reported.
(5759)
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
brotic 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 (06) 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
quantication, 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 specic 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) brin 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 inammation 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
dened 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 inammation 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 reects 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) Magnied 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 efcacy
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 Pzer, 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 conict 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 Scientic Center of Excellence for
Reproductive and Regenerative Medicine (project Reproduc-
tive and regenerative medicineexploration of new platforms
and potentials,GA KK01.1.1.01.0008 funded by the EU through
the ERDF).
Authorsroles: 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 nal
version of the manuscript: TKS and SV. SV takes responsibility for
the integrity of the data analysis.
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JBMR® Plus (WOA) BMP6 DELIVERED WITHIN ABC AS A BONE GRAFT SUBSTITUTE 13
... These constructs have demonstrated the ability to induce endogenous endochondral ossification healing processes and promote the formation of cartilage resembling hyaline cartilage in caprine models. Moreover, the use of recombinant human bone morphogenetic protein 6 (rhBMP6) and autologous blood coagulum (ABC) as carriers for autologous bone graft substitutes has shown potential in enhancing the repair of critical bone defects and promoting bone formation in animal models (Fig. 5e) [91]. ...
... [88]); c. mix between 3D printed RGD-Alginate hydrogel functionalized with FPSC coculture and chondrocytes with BMSCs tested in osteochondral defects of nude mice (Adapted from Ref. [89]); d. Acceleration through whole-body vibration was tested in the number 8 ribs of Wistar Hannover rats (Adapted from Ref. [94]) e. Osteoinductive autologous bone graft substitute with recombinant human BMP6 was tested subcutaneously in Rattus norvegicus and Oryctolagus cuniculus ulna segmental defect (Adapted from Ref. [91]). ...
... In vitro studies suggested that rhBMP6 is superior to rhBMP2 and rhBMP7 due to its reversible binding to noggin [21,22]. OSTEOGROW and its formulation with allograft (OSTEOGROW-A) and synthetic biocompatible ceramics based on calcium phosphate (OSTEOGROW-C) as a compression-resistant matrix (CRM) have been tested in various preclinical studies [18,[23][24][25][26][27][28][29][30]. The efficacy and safety of OSTEOGROW have been evaluated in clinical trials in patients undergoing high tibial osteotomy (HTO) and distal radius fracture (DRF) procedures [31,32]. ...
... The activity of rhBMP2 and rhBMP6 was determined by C2C12-BRE-Luc cell assay [24,34]. Mouse C2C12-BRE-Luc cells were cultured in complete medium -DMEM with high glucose (Merck, Germany) with 10% fetal bovine serum (FBS) (Merck, Germany) at 37 • C in 5% CO 2 with humidification. ...
... This finding is especially important since reported side effects in previous use of BMP2-containing device including ectopic bone formation, osteolysis, swelling of adjacent tissue and radiculitis resulted from the use of large and supraphysiological doses of BMP2 in these devices 1 . Moreover, it has been previously shown that ABC promotes tight rhBMP6 binding with plasma proteins in the fibrin meshwork and allows a sustained rhBMP6 release 51 . Therefore, the aforementioned side effects might be avoided with the use of this novel osteoinductive device efficacious with a relatively small rhBMP6 dose (500 µg) as demonstrated in this study. ...
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Posterolateral spinal fusion (PLF) is a procedure used for the treatment of degenerative spine disease. In this study we evaluated Osteogrow-C, a novel osteoinductive device comprised of recombinant human Bone morphogenetic protein 6 (rhBMP6) dispersed in autologous blood coagulum with synthetic ceramic particles, in the sheep PLF model. Osteogrow-C implants containing 74–420 or 1000–1700 µm ceramic particles (TCP/HA 80/20) were implanted between L4–L5 transverse processes in sheep (Ovis Aries, Merinolaandschaf breed). In the first experiment (n = 9 sheep; rhBMP6 dose 800 µg) the follow-up period was 27 weeks while in the second experiment (n = 12 sheep; rhBMP6 dose 500 µg) spinal fusion was assessed by in vivo CT after 9 weeks and at the end of the experiment after 14 (n = 6 sheep) and 40 (n = 6 sheep) weeks. Methods of evaluation included microCT, histological analyses and biomechanical testing. Osteogrow-C implants containing both 74–420 and 1000–1700 µm ceramic particles induced radiographic solid fusion 9 weeks following implantation. Ex-vivo microCT and histological analyses revealed complete osseointegration of newly formed bone with adjacent transverse processes. Biomechanical testing confirmed that fusion between transverse processes was complete and successful. Osteogrow-C implants induced spinal fusion in sheep PLF model and therefore represent a novel therapeutic solution for patients with degenerative disc disease.
... Six weeks post-treatment, BMP-2-loaded autologous BCs resulted in >95% closure of skull defects in mouse models (Fan et al., 2021). Similarly, Grgurevic et al. found autologous BCs to be an effective delivery vehicle for BMP-6, resulting in improved healing of critical size segmental defects of the ulna in rabbit models (Grgurevic et al., 2019). Furthermore, a case report by Girad et al. showed autologous blood mixed with biphasic calcium phosphate microparticles to be an effective therapeutic for sizable mandibular bone defects in dog models (Girard et al., 2020). ...
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Repair after injury in mammalian tissue involves a complex cascade of events, with the formation of local blood clots being essential for the initial phases of wound healing. As a result, emerging research has sought to harness this biological activity to generate a pro-regenerative biomaterial to speed up wound healing. According to recent studies, “blood clots” created in vitro can be employed as an orthobiologic-based biomaterial for promoting tissue regeneration. Even though such research is still in its early phases, numerous studies show encouraging results that suggest autologous blood clots created in vitro might be a valuable treatment for soft tissue and orthopedic injuries. In this article, we discuss the function of blood clots in physiologic healing, how exogenous material can affect this process, and the most recent clinical research that proposes the use of autologous blood clots as a therapeutically beneficial biomaterial.
... Recombinant human bone morphogenetic protein-7 (BMP-7), also called osteogenic protein-1 (OP-1), was a FDA-approved biologic for the treatment of bone nonunions and spine fusion. Interleukin-1β and β-Nerve growth factor as monoclonal antibody are also used to alleviate pain and inflammation of OA (Grgurevic et al., 2018). Fibroblast growth factor is considered ideal biologic agents for OA treatment should alleviate pain, relieve symptoms and restore the normal structure of the joint. ...
Research Proposal
Full-text available
Osteoarthritis (OA) is a chronic degenerative disorder of the joint. OA is currently the fourth leading cause of disability worldwide. The prevalence of OA in Malaysia was 10-20% among the elderly population, aged 55 years or above. At present, there is no prescribed medicine or treatment strategies to cure this disease. Conventional medicine are used to manage pain and inflammation, but joint degeneration process couldn't be halted using these medicines. Mesenchymal stem cells (MSCs) have self-renewal capacity and can give rise to osteocytes, chondrocytes and tendocytes cells which could restore degenerative bone joint. Thus, this article will highlight Mesenchymal stem cell therapy that could be a promising treatment method to restore joint homeostasis which is lost due to OA. Consequently, patients will get relief from inflammation and pain and go back to their normal life.
... The coagulation time for rabbit blood is around 5 to 10 min, which is similar to the coagulation time in humans. In addition, they are often considered the best animal model to study human platelets due to their similarities in platelet function and regulation [88][89][90][91][92][93][94][95]. Regarding platelet count, rabbits have a similar range of platelet numbers compared to humans, with an average platelet count ranging from 150,000 to 450,000 platelets per microliter of blood. ...
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Full-text available
Experimental research is critical for advancing medical knowledge and enhancing patient outcomes, including in vitro and in vivo preclinical assessments. Platelet-rich fibrin (PRF) is a blood by-product that has garnered attention in the medical and dental fields due to its potential for tissue regeneration and wound healing. Animal models, such as rabbits and rats, have been used to produce PRF and examine its properties and applications. PRF has demonstrated potential in the dental and medical fields for reducing inflammation, promoting tissue repair, and accelerating wound healing. This narrative review aims to compare existing evidence and provide guidelines for PRF animal research, emphasizing the importance of standardizing animal models, following ethical considerations, and maintaining transparency and accountability. The authors highlight the necessity to use the correct relative centrifugal force (RCF), standardize centrifugal calibration, and report detailed information about blood collection and centrifuge parameters for reproducible results. Standardizing animal models and techniques is crucial for narrowing the gap between laboratory research and clinical applications, ultimately enhancing the translation of findings from bench to bedside.
... To improve the biomechanical properties of Osteogrow implants might be supplemented with allograft bone (Osteogrow A), host bone (Osteogrow B) and synthetic ceramics (Osteogrow C) (144)(145)(146). Osteogrow family of products have been proven safe and efficacious in rat subcutaneous assay, rabbit segmental defect model as well as rabbit and sheep posterolateral spinal fusion (PLF) model (144,145,(147)(148)(149)(150)(151)(152)(153)(154)(155). Moreover, Osteogrow has been evaluated in the phase 1 study in patients with distal radius fractures (156), in phase 2 in patients with high tibial osteotomy (157) and in phase 2 in patients treated by posterolateral lumbar interbody fusion for degenerative disc disease. ...
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Full-text available
The 13th International BMP Conference was held in October 2022 in Dubrovnik. The conference was attended by more than 240 participants from North America, Europe, Asia, and Australia who got an insight into the latest achievements in basic, translational, and clinical research of BMP molecules through 75 lectures categorized into several scientific sections. This review paper provides the most important novel findings on the structure, function, and signaling of BMPs, the role of BMPs in patterning and organoids as well as the role of BMP in metabolism. Moreover, we discussed the role of BMPs in various diseases including cancer pathogenesis, pulmonary arterial hyperten-sion, and fibrodysplasia ossificans progressiva (FOP). Finally, we provided an overview of the new BMP-based therapies in regenerative medicine that are currently in different stages of preclinical and clinical trials. Sažetak: Nova dostignuća u području koštanih morfogenetskih proteina: 13. međunarodna BMP konferencija, Dubrovnik 2022 13. međunarodna BMP konferencija održana je u listopadu 2022. godine u Dubrovniku. Na kon-ferenciji je sudjelovalo više od 240 sudionika iz Sjeverne Amerike, Europe, Azije i Australije koji su kroz 75 predavanja u različitim znanstvenim sekcijama dobili uvid u najnovija dostignuća u bazič-nim, translacijskim i kliničkim istraživanjima BMP molekula. U ovom radu smo prikazali najvažnije nove spoznaje o strukturi, funkciji i signalizaciji BMP molekula, njihovoj ulozi u razvoju te meta-bolizmu. Također, raspravili smo ulogu BMP molekula u patogenezi različitih bolesti uključujući karcinome, plućnu hipertenziju i progresivnu osificirajuću fibrodisplaziju. Konačno, u radu smo dali
Article
The management of bone defects and nonunions creates unique clinical challenges. Current treatment alternatives are often insufficient and frequently require multiple surgeries. One promising option is bone morphogenetic protein-2 (BMP-2), which is the most potent inducer of osteogenesis. However, its use is associated with many side effects, related to the delivery and high doses necessary. To address this need, we developed an ex vivo biomimetic hematoma (BH), replicating naturally healing fracture hematoma, using whole blood and the natural coagulants calcium and thrombin. It is an autologous carrier able to deliver reduced doses of rhBMP-2 to enhance bone healing for complex fractures. More than 50 challenging cases involving recalcitrant nonunions and bone defects have already been treated using the BH delivering reduced doses of rhBMP-2, to evaluate both the safety and efficacy. Preliminary data suggest the BH is currently the only clinically used carrier able to effectively deliver reduced doses (∼70% less) of rhBMP-2 with high efficiency, rapidly and robustly initiating the bone repair cascade to successfully reconstruct complex bone injuries without side effects. The presented case provides a clear demonstration of this technology's ability to significantly alter the clinical outcome in extremely challenging scenarios where other treatment options have failed or are considered unsuitable. A favorable safety profile would portend considerable promise for BH as an alternative to bone grafts and substitutes. Although further studies regarding its clinical efficacy are still warranted, this novel approach nevertheless has tremendous potential as a favorable treatment option for bone defects, open fractures, and recalcitrant nonunions.
Article
Full-text available
Bone morphogenetic proteins (BMPs) have been used for orthopedic and dental application due to their osteoinductive properties; however, substantial numbers of adverse reactions such as heterotopic bone formation, increased bone resorption and greater cancer risk have been reported. Since bone morphogenetic proteins signaling exerts pleiotropic effects on various tissues, it is crucial to understand tissue-specific and context-dependent functions of bone morphogenetic proteins. We previously reported that loss-of-function of bone morphogenetic proteins receptor type IA (BMPR1A) in osteoblasts leads to more bone mass in mice partly due to inhibition of bone resorption, indicating that bone morphogenetic protein signaling in osteoblasts promotes osteoclast function. On the other hand, hemizygous constitutively active (ca) mutations for BMPR1A (caBmpr1a wt/+ ) in osteoblasts result in higher bone morphogenetic protein signaling activity and no overt skeletal changes in adult mice. Here, we further bred mice for heterozygous null for Bmpr1a (Bmpr1a +/− ) and homozygous mutations of caBmpr1a (caBmpr1a +/+ ) crossed with Osterix-Cre transgenic mice to understand how differences in the levels of bone morphogenetic protein signaling activity specifically in osteoblasts contribute to bone phenotype. We found that Bmpr1a +/− , caBmpr1a wt/+ and caBmpr1a +/+ mice at 3 months of age showed no overt bone phenotypes in tibiae compared to controls by micro-CT and histological analysis although BMP-Smad signaling is increased in both caBmpr1a wt/+ and caBmpr1a +/+ tibiae and decreased in the Bmpr1a +/− mice compared to controls. Gene expression analysis demonstrated that slightly higher levels of bone formation markers and resorption markers along with levels of bone morphogenetic protein-Smad signaling, however, there was no significant changes in TRAP positive cells in tibiae. These findings suggest that changes in bone morphogenetic protein signaling activity within differentiating osteoblasts does not affect net bone mass in the adult stage, providing insights into the concerns in the clinical setting such as high-dose and unexpected side effects of bone morphogenetic protein application.
Article
Successful repair of large bone defects remains a clinical challenge. Following fractures, a bridging hematoma immediately forms as a crucial step that initiates bone healing. In larger bone defects the micro-architecture and biological properties of this hematoma are compromised, and spontaneous union cannot occur. To address this need, we developed an ex vivo Biomimetic Hematoma that resembles naturally healing fracture hematoma, using whole blood and the natural coagulants calcium and thrombin, as an autologous delivery vehicle for a very reduced dose of rhBMP-2. When implanted into a rat femoral large defect model, complete and consistent bone regeneration with superior bone quality was achieved with 10-20× less rhBMP-2 compared to that required with the collagen sponges currently used. Moreover, calcium and rhBMP-2 demonstrated a synergistic effect enhancing osteogenic differentiation, and fully restored mechanical strength 8 weeks after surgery. Collectively, these findings suggest the Biomimetic Hematoma provides a natural reservoir for rhBMP-2, and that retention of the protein within the scaffold rather than its sustained release might be responsible for more robust and rapid bone healing. Clinically, this new implant, using FDA-approved components, would not only reduce the risk of adverse events associated with BMPs, but also decrease treatment costs and nonunion rates.
Chapter
Full-text available
Cell-based implants with or without osteoinductive biomolecules on optimal carrier materials as an advanced therapeutic medicinal product (ATMP) are a promising strategy for poorly healing long-bone defects. This chapter will focus on ATMPs combining bone morphogenetic proteins (BMPs) and progenitor cells for the clinical treatment of large bone defects in compromised environments. We describe BMP signaling involved in the process of bone fracture healing with specific emphasis on clinically relevant BMP ligands, followed by characterization and BMP responsiveness of progenitor cells obtained from different sources. Then we explore different biomaterials and their contribution to achieve optimal BMP release and osteoinduction. Finally, we provide a perspective on the applicability of ATMPs in bone repair by reviewing the preclinical studies carried out so far in various animal models. We believe the era of regenerative medicine has just started. First-generation BMP and stem cell technologies have demonstrated that in the postnatal environment, one can successfully enhance the healing of damaged tissues by recapitulating the principles of developmental tissue formation. A second generation of products is needed that leads to successful bone healing in compromised environments.
Book
From the basic science to potential and approved clinical applications the most recent data in the rapidly growing field of bone morphogenetic proteins (BMPs) are summarized in this topical volume. Distinguished scientists present reviews on a range of scientific topics, including biochemistry, biology, molecular biology and preclinical animal studies on spinal fusion, cartilage repair, craniofacial and dental reconstruction using BMPs, as well as approved clinical applications in human bone non-unions. This book provides a resource not only for experts in the field, but also for undergraduate students, newcomers and clinicians worldwide, given that the use of BMPs in orthopedic reconstruction has been already approved in Europe, Australia, Canada and the USA.
Article
Objective: This study was to compare the effectiveness of Masquelet technique versus Ilizarov bone transport in the treatment of lower extremity bone defects following posttraumatic osteomyelitis. Patients and methods: We retrospectively reviewed 39 patients who had been treated at our department for lower extremity bone defects following posttraumatic osteomyelitis. They were 30 males and 9 females with a mean age of 39.18 (range, 12-63 years). The infected bone defects involved 26 tibias and 13 femurs. The mean length of the bone defects after radical debridement was 6.76cm (range, 2.7-15.7cm). Masquelet technique (MT, group A) was used in 20 patients and Ilizarov bone transport (IBT, group B) in 19 ones. The measurements were bone outcomes (union, deformity, infection and leg-length discrepancy) and functional outcomes (significant limping, joint contracture, soft tissue dystrophy, pain and inactivity). Results: The mean follow-up after removal of the apparatus was 25.26 months (range, 14-51 months). The mean finite fixator time was 10.15 months (range, 8-14 months) in group A versus 17.21 months (range, 11-24 months) in group B. The bone outcomes were similar between groups A and B [excellent (5 vs. 7), good (10 vs.9), fair (4 vs. 2) and poor (1 vs. 1)]; group A showed better functional outcomes than group B [excellent (8 vs. 3), good (9 vs. 6), fair (3 vs. 8) and poor (0 vs. 2)]. Conclusions: In the treatment of segmental lower extremity bone defects following posttraumatic osteomyelitis, both IBT and MT can lead to satisfactory bone results while MT had better functional results, especially in femoral cases. IBT should be preferred in cases of limb deformity and MT may be a better choice in cases of periarticular bone defects.
Chapter
BMPs are originally identified based on their ability to induce new bone in vivo and represent large members of the TGF-β superfamily of proteins. BMPs serve as inductive signals for cell migration, growth, and subsequently differentiation in many organ developments during embryogenesis and are shown to modulate inflammation, angiogenesis, and immune responses and thus provide biological cues for adult tissue repair, protection, and regeneration. BMP-2- and BMP-7-containing osteogenic devices have been approved for use as bone graft substitutes for spine fusion and long bone fractures. BMP-7 biology has been considered positively against parenchymal tissue fibrosis to improve function. In this chapter, I summarize the biology of BMPs to emphasize its (1) morphogenic role in skeletal tissue repair and regeneration; (2) modulatory role in curtailing inflammation, governing angiogenesis, suppressing apoptosis, and reducing fibrosis following immunological and mechanical insults; (3) metabolic role in glucose, calcium, and phosphate and iron homeostasis; and (4) cytoprotective role to maintain skeletal and vascular integrity. The importance of BMP biology is further corroborated in rare genetic disorders (e.g., pulmonary arterial hypertension, hemochromatosis, fibrodysplasia ossificans progressiva, and osteogenesis imperfecta) and in cancer.
Chapter
Over the last 50 years the concept of inducing bone formation, using biologically active signalling molecules, has evolved significantly. The most potent of these osteoinductive molecules remain the Bone Morphogenetic Proteins, with established role on the chemotaxis, proliferation and differentiation of mesenchymal progenitor cells to form cartilage and bone. The initial enthusiasm following the synthetic production of BMP2 and BMP7 using recombinant gene technology, was followed by an expansion of their use “in-” and “off-label” in clinical practice, on parallel to a large number of basic science and translational medicine studies attempting to define further their effect. The key role of BMPs in bone repair stimulated their widespread use in the orthopaedic discipline including the management of delayed union and non-union of fractures, bone defects, open fractures, fusion of joints, spinal fusions, as well as treatment of osteoarthritis and intervertebral disc cartilage degeneration. It is quite evident that rhBMPs in humans have a different dose–response relationship in comparison to animal species, as well as that the final outcome of their use is also relevant to the specifics of their carrier and delivery system, their containment, the timing of their application, as well as the state of the recipient host local environment. The different effect of different BMPs, and their variable interaction with inhibiting molecules and negative feedback mechanisms, are nowadays better understood, widening further the horizon of contemporary research of bone, as well as of cartilage regeneration. The reputation of BMPs has been negatively affected lately due to the recent redraw from the market of their recombinant synthetic forms, which is however attributed mostly to strategic commercial planning rather than their performance. New osteoinductive molecules emerge attempting to fill in the gap, whilst the challenge of evidence based clinical practice remains. This article presents the contemporary understanding, as well as a summary of selected published evidence on the roles of the BMPs in bone and cartilage regeneration.
Chapter
Complications associated with the clinical use of BMP2 and BMP7 result from the limited understanding of their molecular mechanisms in bone remodeling. Recently, a novel BMP6-based approach has been developed with superior healing results and reduced side effects in preclinical studies. BMP6-containing osteogenic medicinal product called Osteogrow, which is aimed to induce and accelerate bone formation, is currently being tested in clinical studies. It comprises of a biologically compatible autologous carrier made from the patient’s peripheral blood and of rhBMP6 as an active ingredient. Such formulation circumvents the use of animal-derived materials, significantly limits inflammatory processes common in commercial bone devices, and renders the carrier flexible and injectable ensuring the ease of use. The ongoing clinical trial results will provide a more detailed insight into the safety, tolerability, pharmacokinetics, and bone healing effects in humans and hopefully provide novel and valuable therapeutic options in the field of bone regeneration.