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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.
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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
... We have demonstrated that our novel autologous bone graft substitute (ABGS) containing rhBMP6 within autologous blood coagulum (ABC) with or without compression resistant matrix (CRM) restores large bone defects and achieves successful lumbar fusion in rabbits and sheep [2,[12][13][14][15][16][17][18][19]. Importantly, newly induced bone successfully fused with native bone, achieving complete osseointegration, which can be defined as functional fusion between new and old bone [20]. ...
... In addition, ABC serves as a physiological biocompatible carrier [12], promoting the tight binding of rhBMP6 to plasma proteins within the fibrin meshwork. ABC further allows a sustained release of rhBMP6 and suppresses foreign body responses elicited by the mineral-rich ceramics used as CRM [2,13,14]. Furthermore, synthetic ceramics are more preferred CRM than allografts [16,17,23] due to the reduced risk of viral transmission and immunogenicity [24]. Furthermore, biphasic calcium phosphate contains beta-tricalcium phosphate (TCP) as a more resorbable component and more stable hydroxyapatite (HA) at various ratios, thus allowing the proper tuning of CRM resorbability [25]. ...
... Furthermore, biphasic calcium phosphate contains beta-tricalcium phosphate (TCP) as a more resorbable component and more stable hydroxyapatite (HA) at various ratios, thus allowing the proper tuning of CRM resorbability [25]. The biology of BMP-induced bone formation has been studied at various time points in a rat subcutaneous bone formation assay [12][13][14]17], rabbit segmental bone defects, and in a rabbit and sheep posterolateral and anterior lumbar spinal fusion models [13][14][15][16]. In addition, ABGS has been proven to be safe and efficacious in patients with distal radial fractures (DRF; EudraCT 2014-005101-21) [26] and high tibial osteotomy (HTO; EudraCT 2015-001691-21) [27]. ...
Article
Full-text available
Autologous bone graft substitute (ABGS) containing rhBMP6 in autologous blood coagulum (ABC) with synthetic ceramics is a novel therapeutic solution for bone repair. The aim of this study was to investigate whether the application of Zoledronate (ZOL) with ABGS might enhance the properties of newly formed bone. The effect of ZOL on bone induction was tested in a rat subcutaneous implant model. ZOL bound to synthetic ceramics was added into ABGS implants, and the quantity, quality, and longevity of the induced bone were assessed by micro-CT, histomorphometry, and histology over a period of 365 days. Local use of ZOL in the ABGS implants with ceramics had no influence on the bone volume (BV) on day 14 but subsequently significantly increased BV on days 35, 50, 105, 140, and 365 compared to the control implants. Locally applied ZOL had a similar effect in all of the applied doses (2–20 µg), while its systemic use on stimulating the BV of newly induced bone by ABGS depended on the time of application. BV was increased when ZOL was applied systemically on day 14 but had no effect when applied on day 35. The administration of ZOL bound to ceramics in ABGS increased and maintained the BV over a period of one year, offering a novel bone tissue engineering strategy for treating bone defects and spinal fusions.
... We have recently developed an autologous bone graft substitute (ABGS) comprised of BMP6 delivered within an autologous blood coagulum to which a compression-resistant matrix, such as allograft or synthetic ceramics, can be added [22,73,74,76,[86][87][88][89][90][91][92]. Moreover, the volume of newly induced bone increased with the elevation of the CRM amount, which might be attributed to the enlargement in an overall surface area [73]. ...
... Rodent ectopic models have been extensively used for the initial evaluation of novel osteoinductive therapies. They might be also used for investigating the biology of ectopic bone induction and the formation of a bone organ or ossicle, including bone and bone marrow [31,32,39,[48][49][50][51][52][53][54][55][56][57]71,73,76,86,87,[93][94][95][96][97][98][99][100][101][102][103][104][105]. Rodent ectopic models (Tables 1 and 2) are further subdivided according to the species (mouse, rat) and the implantation site (subcutaneous or intramuscular). ...
... Rodent ectopic models (Tables 1 and 2) are further subdivided according to the species (mouse, rat) and the implantation site (subcutaneous or intramuscular). Implantation under the skin (Figure 2A-D) or into the muscle does not affect the bone formation outcome, and the bone formation occurs in the first two weeks following implantation of an osteoinductive device [76,86,87]. The later time points are needed for the evaluation of the bone longevity and maintenance of the ectopic bone structure. ...
Article
Full-text available
Bone morphogenetic proteins (BMPs) possess a unique ability to induce new bone formation. Numerous preclinical studies have been conducted to develop novel, BMP-based osteoinductive devices for the management of segmental bone defects and posterolateral spinal fusion (PLF). In these studies, BMPs were combined with a broad range of carriers (natural and synthetic polymers, inorganic materials, and their combinations) and tested in various models in mice, rats, rabbits, dogs, sheep, and non-human primates. In this review, we summarized bone regeneration strategies and animal models used for the initial, intermediate, and advanced evaluation of promising therapeutical solutions for new bone formation and repair. Moreover, in this review, we discuss basic aspects to be considered when planning animal experiments, including anatomical characteristics of the species used, appropriate BMP dosing, duration of the observation period, and sample size.
... Bone morphogenetic proteins (BMPs) are potent osteoinductive agents that recruit mesenchymal stem cells and promote their proliferation and differentiation into bone and cartilage during embryonic development Vukicevic and Grgurevic, 2009). Autologous blood coagulum (ABC) is a novel physiological BMP carrier that supports tight BMP binding to plasma proteins within the fibrin meshwork, suppresses foreign body response, and allows a sustained in vitro release of rhBMP6 (Vukicevic et al., 2014;Grgurevic et al., 2019;Vukicevic et al., 2020a;Vukicevic et al., 2020b). To improve biomechanical properties of implants, compression resistant matrix (CRM) could be added to implants in selected indications . ...
... We have recently demonstrated that an autologous bone graft substitute (ABGS) containing recombinant human bone morphogenetic protein 6 (rhBMP6) within ABC with and without allograft as a CRM was safe and efficacious in the rabbit ulnar segmental defect model (Grgurevic et al., 2019), rabbit and ovine posterolateral spinal fusion (PLF) and sheep anterior lumbar interbody fusion (ALIF) models Grgurevic et al., 2020). In continuation, the ABGS was tested in Phase I clinical trials in patients with distal radial fracture (Durdevic et al., 2020), and Phase I/II trial in patients undergoing high tibial osteotomy (Chiari et al., 2020) and was proven safe and efficacious as compared to placebo and standard of care treated patients. ...
... A complete lack of the new bone formation following primary surgery prompted us to use the novel ABGS with synthetic ceramics as an experimental therapy in an attempt to prevent the front leg amputation. The efficacy and safety of this ABGS device for the treatment of critical size bone defects has been previously demonstrated in rabbits (Grgurevic et al., 2019). To improve the implant biomechanical properties, we supplemented ABGS with allograft as a compression resistant matrix ). ...
Article
Full-text available
Background Management of large segmental defects is one of the most challenging issues in bone repair biology. Autologous bone graft substitute (ABGS) containing rhBMP6 within autologous blood coagulum (ABC) with synthetic ceramics is a novel biocompatible therapeutic solution for bone regeneration. Case presentation. A 2-year old dog was brought to the veterinary clinics due to pain and bleeding from the right front leg after being unintendedly hit by a gunshot. Radiological examination revealed a large, 3 cm long multisegmental defect of the humerus on the right front leg with a loss of anatomical structure in the distal portion of the bone. The defect was treated surgically and an external fixator was inserted to ensure immobilization. Complete lack of bone formation 3 months following surgery required a full reconstruction of the defect site with a novel ABGS (rhBMP6 in ABC with ceramic particles) to avoid front leg amputation. The healing was then followed for the next 16 months. The callus formation was observed on x-ray images 2 months following ABGS implantation. The bone segments progressively fused together leading to the defect rebridgment allowing removal of the external fixator by 4 months after the reconstruction surgery. At the end of the observation period, the function of the leg was almost fully restored while analyses of the humeral CT sections revealed restoration and cortices rebridgment with a renewal of uniform medullary canal including structural reconstruction of the distal humerus. Conclusion This large humeral gunshot segmental defect of the front leg in a dog was saved from amputation via inducing bone regeneration using a novel ABGS osteoinductive device containing BMP6 in ABC.
... Pre-clinical studies showed that BMP6 can be used in a much lower quantity than BMP2 and BMP7. We demonstrated that 50 µg BMP6 in the ABGS implant is more effective than 3.5 mg of BMP7 in the Osigraft in studies on rabbit ulna segmental bone defect 131 . When BMP2, BMP6 and BMP7 were used in similar amounts in a biocompatible ABGS in rabbits with a segmental bone defect, BMP6 showed the best healing properties without evident osteolysis. ...
... When BMP2, BMP6 and BMP7 were used in similar amounts in a biocompatible ABGS in rabbits with a segmental bone defect, BMP6 showed the best healing properties without evident osteolysis. In rat, fibrosis and an inflammatory response were not present at a subcutaneous site 131,132 (Fig. 1) due to the higher BMP6 potency associated to a lesser sensitivity to Noggin 43 . BMP6 dissociates from Noggin, following binding of the BMP-Noggin complex to the cell surface receptors type I 43 . ...
... ABGS is non-immunogenic, non-inflammatory and without bovine collagen, opposite to commercially available devices. It is a flexible, compact, malleable, cohesive, and easy to inject material, not disassembling into parts for at least 7 days after implantation 115,131 . In preclinical animal models of bone regeneration ABGS successfully bridged the segmental bone defects 12,115,131 . ...
Article
Full-text available
Bone Morphogenetic Proteins (BMPs) are growth and differentiation factors within the TGFβ superfam- ily of proteins. They induce ectopic and orthotopic endochondral bone formation and are involved in the regulation of cell proliferation, differentiation, apoptosis and mesenchymal-epithelial interactions in critical morphogenetic processes of tissues beyond bone. BMP2 and BMP7 osteogenic devices have been approved for enhancing healing in patients with long bone defects and anterior spinal fusion proce- dures. However, due to a high price and various serious adverse events including heterotopic ossifica- tion, retrograde ejaculation and pain their clinical use have been limited. In this review we discuss the BMP discovery, biology and their use in clinical studies with particular reference to the newly developed BMP6 based autologous bone graft substitute (ABGS). A novel ABGS consisting of an autologous bone coagulum (ABC) carrier with dispersed BMP6 to initiate the differentiation of mesenchymal cells into endochondral bone. The ABC met the conditions for an optimal delivery system for BMP6 due to han- dling simplicity, without an immunogenic and inflammatory response at the implantation site. Addition of allograft or synthetic ceramics to ABGS demonstrated in animal models significantly increased volume and better microarchitecture of the newly formed bone. The first clinical study was conducted in patients with distal radial fractures (Phase I study) and the second in patients undergoing high tibial osteotomy (Phase I/II study) and no serious adverse events have been observed. Finally, in the ongoing OSTEO- proSPINE study ABGS enforced with allograft bone is evaluated in patients with chronic back pain due to degenerative disc diseases. The novel ABGS bone mimetic is a major breakthrough and contribution to bone biology and regenerative medicine of skeletal repair.
... Autologous blood coagulum (ABC) is a preferred physiological native carrier for BMPs [7]. ABC presents several advantages that include eliciting little or no foreign body response, a tight BMP binding to plasma proteins within the fibrin meshwork and sustained release of BMP over the time [7][8][9]. Autologous bone graft substitute (ABGS) containing rhBMP6 in ABC has been recently tested for safety and efficacy in patients undergoing high tibial osteotomy (HTO) [10] and distal radial fracture (DRF) therapy [11]. ...
... RhBMP6 was mixed with the blood immediately at withdrawal and added to a sterile syringe (5 mL, BD) containing cylinder segment and left five to 15 minutes to coagulate. All implants were implanted within one hour following coagulation as described [8,9,24]. The dose of rhBMP6 was 20 μg per implant. ...
... Biomechanical properties of the implants (stiffness, elasticity, and work-to-break) were evaluated performing cut test (TA.HDPlus, Stable Micro Systems, UK) in which implants were cut through the middle of the implant as described [8,9]. Stiffness and elasticity of the implant as well as the work needed to cut the implant were calculated. ...
Article
Full-text available
Purpose: We have recently developed an autologous bone graft substitute (ABGS) containing recombinant human bone morphogenetic protein 6 (rhBMP6) in autologous blood coagulum (ABC) that induces new bone formation in vivo. In order to improve biomechanical properties of the implant, compression resistant matrix (CRM) consisting of synthetic ceramics in the form of macroporous cylinders was added to the ABGS and we evaluated the biomechanical properties and the quantity and quality of bone formation following subcutaneous implantation in rats. Methods: ABGS implants containing rhBMP6 in ABC with cylindrical ceramic blocks were implanted subcutaneously (n = 6 per time point) in the axillary region of Sprague-Dawley rats and removed at specified time points (7, 14, 21, 35, and 50 days). The quantity and quality of newly formed bone were analyzed by microCT, histology, and histomorphometric analyses. Biomechanical properties of ABGS formulations were determined by employing the cut test. Results: MicroCT analyses revealed that ABGS implants induced formation of new bone within ceramic blocks. Histological analysis revealed that on day seven following implantation, the endochondral ossification occupied the peripheral part of implants. On days 14 and 21, newly formed bone was present both around the ceramic block and through the pores inside the block. On both days 35 and 50, cortical bone encircled the ceramic block while inside the block, bone covered the ceramic surface surrounding the pores. Within the osseous circles, there were few trabeculae and bone marrow containing adipocytes. ABGS containing cylindrical ceramic blocks were more rigid and had significantly increased stiffness compared with implants containing ceramic particles as CRM. Conclusion: We demonstrated that macroporous ceramic blocks in a form of cylinders are promising CRMs with good handling and enhanced biomechanical properties, supporting bone formation with ABGS containing rhBMP6 within autologous blood coagulum. Hence, ABGS containing ceramic blocks should be tested in preclinical models including diaphyseal segmental defects and non-unions in larger animals.
... Osteogrow is a novel osteoinductive device comprised of recombinant human bone morphogenetic protein 6 (rhBMP6) delivered within autologous blood coagulum (ABC) as a BMP carrier [1]. Osteogrow has been extensively evaluated in preclinical studies including animal models such as rat subcutaneous assay and spinal fusions in rabbit and sheep models [2][3][4]. Moreover, Osteogrow has been proven safe and effective in phase I/II clinical trials in patients with distal radial fracture (DRF) and patients undergoing high tibial osteotomy (HTO) [5,6]. ...
... The skin over the thoracic region was sterilized and a vertical skin incision was made in the median line. Subcutaneous pockets were created bilaterally in the axillary region, and prepared Osteogrow-C implants were placed in the pockets as described [2,3,7,31]. Animals were euthanized 21 days after surgery according to the experimental design. ...
Article
Full-text available
PurposeOsteogrow, an osteoinductive device containing recombinant human Bone Morphogenetic Protein 6 (rhBMP6) in autologous blood coagulum, is a novel therapeutic solution for bone regeneration. This study aimed to evaluate different commercially available calcium phosphate synthetic ceramic particles as a compression-resistant matrix (CRM) added to Osteogrow implants to enhance their biomechanical properties.Methods Osteogrow implants with the addition of Vitoss, ChronOs, BAM, and Dongbo ceramics (Osteogrow-C, where C stands for ceramics) were evaluated in the rodent subcutaneous ectopic bone formation assay. Osteogrow-C device was prepared as follows: rhBMP6 was added to blood, and blood was mixed with ceramics and left to coagulate. Osteogrow-C was implanted subcutaneously in the axillary region of Sprague–Dawley rats and the outcome was analyzed 21 days following implantation using microCT, histology, morphometric analyses, and immunohistochemistry.ResultsOsteogrow-C implants with all tested ceramic particles induced the formation of the bone-ceramic structure containing cortical bone, the bone between the particles, and bone at the ceramic surfaces. The amount of newly formed bone was significant in all experimental groups; however, the highest bone volume was measured in Osteogrow-C implants with highly porous Vitoss ceramics. The trabecular number was highest in Osteogrow-C implants with Vitoss and ChronOs ceramics while trabeculae were thicker in implants containing BAM and Dongbo ceramics. The immunological response and inflammation were comparable among ceramic particles evaluated in this study.Conclusion Osteogrow-C bone regenerative device was effective with a broad range of commercially available synthetic ceramics providing a promising therapeutic solution for the regeneration of long bone fracture nonunion, large segmental defects, and spinal fusion surgeries.
... Despite that, large dose rhBMP2 based bovine collagen device has been commonly used off-label in PLF indication 29 resulting in immunogenicity of the collagen carrier, postoperative inflammation, radiculopathy, heterotopic ossification, vertebral bone resorption and retrograde ejaculation 30-32 . ABGS containing rhBMP6 within autologous blood coagulum (ABC) named Osteogrow is a novel therapeutic solution for various clinical indications, including spinal fusions and segmental bone defect management evaluated both in preclinical [33][34][35][36][37][38][39][40][41] and clinical studies 42-44 . BMP6 is more potent than BMP2 and BMP7 in promoting osteoblast differentiation in vitro and promoting bone regeneration in vivo due to its resistance to Noggin 45,46 . ...
... BMP6 is more potent than BMP2 and BMP7 in promoting osteoblast differentiation in vitro and promoting bone regeneration in vivo due to its resistance to Noggin 45,46 . ABC is a physiological native BMP carrier which suppresses foreign body response, promotes BMP binding to plasma proteins within the fibrin meshwork to allow a sustained in vitro BMP release 33,34,36 . ...
Article
Full-text available
Autologous bone graft substitute (ABGS) containing rhBMP6 in autologous blood coagulum (Osteogrow) is a novel therapeutic solution for bone regeneration. This study is aimed to investigate the long-term outcome of ABGS with synthetic ceramics (Osteogrow-C) in rabbit posterolateral spinal fusion (PLF) model. Osteogrow-C implants were implanted bilaterally between rabbit lumbar transverse processes. We compared the outcome following implantation of ABGS with ceramic particles of different chemical composition (TCP and biphasic ceramics containing both TCP and HA) and size (500–1700 µm and 74–420 µm). Outcome was analyzed after 14 and 27 weeks by microCT, histology, and biomechanical analyses. Successful bilateral spinal fusion was observed in all animals at the end of observation period. Chemical composition of ceramic particles has impact on the PLF outcome via resorption of TCP ceramics, while ceramics containing HA were only partially resorbed. Moreover, persistence of ceramic particles subsequently resulted with an increased bone volume in implants with small particles containing high proportion of HA. ABGS (rhBMP6/ABC) with various synthetic ceramic particles promoted spinal fusion in rabbits. This is the first presentation of BMP-mediated ectopic bone formation in rabbit PLF model with radiological, histological, and biomechanical features over a time course of up to 27 weeks.
... Recently, two studies have demonstrated this presumption in treating critical segmental defects and posterolateral lumbar spine fusion in rabbits [29,30]. Furthermore, in combination with synthetic ceramics, rhBMP-6 has shown new bone formation and ceramic particles integrated with the transverse processes resulting in superior biomechanical properties [31]. ...
Article
Full-text available
Objective With the advances in biological technologies over the past 20 years, a number of new therapies to promote bone healing have been introduced. Particularly in the spinal surgery field, more unprecedented biological therapeutics become available to enhance spinal fusion success rate along with advanced instrumentation approaches. Yet surgeons may not have been well informed about their safety and efficacy profiles in order to improve clinical practices. Therefore there is a need to summarize the evidence and bring the latest progress to surgeons for better clinical services for patients. Methods We comprehensively reviewed the literatures in regard to the biological therapeutics for enhancement of spinal fusion published in the last two decades. Results Autograft bone is still the gold standard for bone grafting in spinal fusion surgery due to its good osteoconductive, osteoinductive, and osteogenic abilities. Accumulating evidence suggests that adding rhBMPs in combination with autograft effectively promotes the fusion rate and improves surgical outcomes. However, the stimulating effect on spinal fusion of other growth factors, including PDGF, VEGF, TGF-beta, and FGF, is not convincing, while Nell-1 and activin A exhibited preliminary efficacy. In terms of systemic therapeutic approaches, the osteoporosis drug Teriparatide has played a positive role in promoting bone healing after spinal surgery, while new medications such as denosumab and sclerostin antibodies still need further validation. Currently, other treatment, such as controlled-release formulations and carriers, are being studied for better releasing profile and the administration convenience of the active ingredients. Conclusion As the world's population continues to grow older, the number of spinal fusion cases grows substantially due to increased surgical needs for spinal degenerative disease (SDD). Critical advancements in biological therapeutics that promote spinal fusion have brought better clinical outcomes to patients lately. With the accumulation of higher-level evidence, the safety and efficacy of present and emerging products are becoming more evident. These emerging therapeutics will shift the landscape of perioperative therapy for the enhancement of spinal fusion.
Article
Full-text available
At present, bone nonunion and delayed union are still difficult problems in orthopaedics. Since the discovery of bone morphogenetic protein (BMP), it has been widely used in various studies due to its powerful role in promoting osteogenesis and chondrogenesis. Current results show that BMPs can promote healing of bone defects and reduce the occurrence of complications. However, the mechanism of BMP in vivo still needs to be explored, and application of BMP alone to a bone defect site cannot achieve good therapeutic effects. It is particularly important to modify implants to carry BMP to achieve slow and sustained release effects by taking advantage of the nature of the implant. This review aims to explain the mechanism of BMP action in vivo, its biological function, and how BMP can be applied to orthopaedic implants to effectively stimulate bone healing in the long term. Notably, implantation of a system that allows sustained release of BMP can provide an effective method to treat bone nonunion and delayed bone healing in the clinic.
Article
Study design: A case-control genome-wide association study (GWAS) on spondylosis. Objective: Leveraging Geisinger's MyCode initiative's multimodal dataset, we aimed to identify genetic associations with degenerative spine disease. Summary of background data: Degenerative spine conditions are a leading cause of global disability; however, the genetic underpinnings of these conditions remain under-investigated. Previous studies using candidate-gene approach suggest a genetic risk for degenerative spine conditions, but large-scale GWASs are lacking. Methods: We identified 4434 patients with a diagnosis of spondylosis using ICD diagnosis codes with genotype data available. We identified a population-based control of 12,522 patients who did not have any diagnosis for osteoarthritis. A linear-mix, additive genetic model was employed to perform the genetic association tests adjusting for age, sex, and genetic principal components to account for the population structure and relatedness. Gene-based association tests were performed and heritability and genetic correlations with other traits were investigated. Results: We identified a genome-wide significant locus at rs12190551 (odds ratio = 1.034, 95% confidence interval 1.022-1.046, P = 8.5 × 10-9, minor allele frequency = 36.9%) located in the intron of BMP6. Additionally, NIPAL1 and CNGA1 achieved Bonferroni significance in the gene-based association tests. The estimated heritability was 7.19%. Furthermore, significant genetic correlations with pain, depression, lumbar spine bone mineral density, and osteoarthritis were identified. Conclusion: We demonstrated the use of a massive database of genotypes combined with electronic health record data to identify a novel and significant association spondylosis. We also identified significant genetic correlations with pain, depression, bone mineral density, and osteoarthritis, suggesting shared genetic etiology and molecular pathways with these phenotypes.Level of Evidence: N/A.
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.
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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.
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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.