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Synthetic ceramic macroporous blocks as a scaffold in ectopic bone formation induced by recombinant human bone morphogenetic protein 6 within autologous blood coagulum in rats

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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.
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ORIGINAL PAPER
Synthetic ceramic macroporous blocks as a scaffold in ectopic bone
formation induced by recombinant human bone morphogenetic
protein 6 within autologous blood coagulum in rats
Nikola Stokovic
1,2
&Natalia Ivanjko
1,2
&Marina Milesevic
1,2
&Ivona Matic Jelic
1,2
&Kristian Bakic
1,2
&
Viktorija Rumenovic
1,2
&Hermann Oppermann
3
&Larry Shimp
4
&T. Kuber Sampath
5
&Marko Pecina
6
&
Slobodan Vukicevic
1,2
Received: 1 October 2020 /Accepted: 2 October 2020
#SICOT aisbl 2020
Abstract
Purpose We have recently developed an autologous bone graft substitute (ABGS) containing recombinant human bone mor-
phogenetic 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 bothdays 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 contain-
ing 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.
Keywords Bonemorphogeneticproteins (BMPs) .BMP6 .Autologousblood coagulum .Autologousbone graft substitute .BMP
carrier .Macroporous blocks .Synthetic ceramics
*Slobodan Vukicevic
slobodan.vukicevic@mef.hr
1
Laboratory for Mineralized Tissues, Center for Translational and
Clinical Research, University of Zagreb School of Medicine,
Zagreb, Croatia
2
Scientific Center of Excellence for Reproductive and Regenerative
Medicine, Zagreb, Croatia
3
Genera Research, Kalinovica, Rakov Potok, Croatia
4
CaP Biomaterials, East Troy, WI, USA
5
perForm Biologics Inc., Holliston, MA, USA
6
Department of Orthopaedic Surgery, University of Zagreb School of
Medicine, Zagreb, Croatia
https://doi.org/10.1007/s00264-020-04847-9
/ Published online: 14 October 2020
International Orthopaedics (2021) 45:1097–1107
Introduction
Bone morphogenetic proteins (BMPs) were originally isolated
from demineralized bone matrix [1,2] and represent a large
subgroup of the TGFβsuperfamily members [3]. BMP2
(Infuse®) and BMP7 (OP-1®) with bovine collagen as a car-
rier have been shown to promote new bone formation in clin-
ical trials of several orthopaedic indications [4,5]. We have
recently shown that BMP6 is more potent in promoting oste-
oblast differentiation due to its resistance to noggin, a BMP
antagonist abundant in bone, and interacts with the BMP type
I and type II receptors [6]. BMP6 in autologous blood coagu-
lum as a carrier induced new bone regeneration in vivo at
lower doses than BMP2 and BMP7 used on bovine collagen.
Autologous blood coagulum (ABC) is a preferred physio-
logical native carrier for BMPs [7]. ABC presents several
advantages that include eliciting little or no foreign body re-
sponse, a tight BMP binding to plasma proteins within the
fibrin meshwork and sustained release of BMP over the time
[79]. Autologous bone graft substitute (ABGS) containing
rhBMP6 in ABC has been recently tested for safety and effi-
cacy in patients undergoing high tibial osteotomy (HTO) [10]
and distal radial fracture (DRF) therapy [11].
In indications requiring larger implants (e.g., posterolateral
spinal fusion (PLF) and segmental bone defects), the biome-
chanical resistance of implants was addressed with the addi-
tion of compression resistant matrix (CRM) to ABGS [9]. We
first reported usage of devitalized allograft as CRM and dem-
onstrated the efficacy and safety of ABGS containing
rhBMP6 within ABC with allograft in preclinical PLF rabbit
and sheep models [9,12]. Following successful outcome, the
same formulation is currently being evaluated in the human
PLIF clinical trial (EudraCT number 2017-000860-14).
However, allograft has several disadvantages including the
risk of viral transmission, immunogenicity, and regulatory
issues through different states which urged us to find a safe
and effective alternative [13]. Calcium phosphate ceramics,
most notably tricalcium phosphate (TCP) and hydroxyapatite
(HA), are well known osteoconductive materials [1420]. The
main difference between TCP and HA is their reactivity and
resorbability as TCP is more resorbable than HA. Therefore,
TCP and HA are combined to form biphasic calcium phos-
phate (BCP) ceramics in various TCP/HA ratios in order to
obtain the desired CRM resorbability [14].
Synthetic ceramics are formulated into various particle
sizes as granulates or preformulated blocks of different geo-
metrical shapes (e.g., cylinders, slabs) [21,22]. Due to the
aforementioned disadvantages of allograft, we reported the
use of ceramic particles as CRMs with ABGS containing
rhBMP6 and ABC in the rat subcutaneous assay and PLF
rabbit model [23,24]. Advantages of particle use include a
broad range of available sizes and geometrical shapes as well
as adaptability of implants to desired shapes in a specific
clinical condition. However, particles are by definition unor-
ganized and would assume their position and distribution
within ABGS during implant preparation. On the other hand,
preformulated blocks already have a desired shape and might
be an alternative to particles to design uniform osteogenic
ABGS devices for selected indications.
In the present study, we evaluated the biology of bone
induction by ABGS containing rhBMP6 within ABC and ce-
ramic macroporous cylinders as a CRM. In addition, we ex-
amined the biomechanical properties of ABGS containing ce-
ramic blocks and compared them with biomechanical proper-
ties of ABGS with and without ceramic particles.
Material and methods
Test items
The test items were rhBMP6 produced by Genera Research
(Zagreb, Croatia) and various synthetic ceramic macroporous
blocks in the form of porous ceramic cylinders produced by
CaP Biomaterials (East Troy, WI, USA) (Fig. 1a). The main
determinants of the porous ceramic cylinders were the foam
template structure, chemical composition, sintering tempera-
ture, and slurry weight/foam weight (S/F) ratio. The chemical
composition of the ceramic cylinders used in this study was a
biphasic calcium phosphate ceramic containing beta-
tricalcium phosphate and hydroxyapatite in an 80/20 ratio.
The sintering temperature was 1175 °C. Polyurethane foam
templates had a pore structure averaging 60 pores/in. (2.4
pores/mm) and with the S/F ratio either 30:1 or 35:1.
Properties of the cylindrical ceramic blocks are summarized
in Table 1.
Experimental design
The study consisted of testing in which we compared biome-
chanical properties of ABGS containing ceramic blocks and
ceramic particles as CRM and rat subcutaneous implantation
assays in which we evaluated the time course of bone forma-
tion as well as bone microarchitecture of newly formed bone
by ABGS containing rhBMP6 in ABC with ceramic blocks
(Fig. 1b).
We tested biomechanical properties of five different ABGS
formulations: ABC, ABC with small and medium particles
(74420 μm and 5001700 μm) [24] and ABC with two types
of cylindrical ceramic blocks (30:1 and 35:1) (Table 2). The
rat subcutaneous implant assay was used in order to define the
cascades of biological events following subcutaneous implan-
tation of ABGS containing rhBMP6 in ABC with two similar
cylindrical ceramic blocks (Table 2). The bone formation out-
come was evaluated 7, 14, 21, 35, and 50 days following
implantation using histological and microCT analyses.
1098 International Orthopaedics (SICOT) (2021) 45:1097–1107
Implant preparation
Preparation of implants for biomechanical testing
Implants for biomechanical testing consisted of blood coagu-
lum with or without CRM in the form of cylindrical ceramic
blocks and ceramic particles. Blood (2.5 mL) was withdrawn
into tubes without an anticoagulant substance. Cylindrical ce-
ramic blocks and ceramic particles were placed in appropriate
syringes (BD 5 mL). Blood was mixed with ceramic blocks/
particles and left to coagulate at room temperature as previ-
ously described [9].
Preparation of implants for rat subcutaneous assay
To prepare implants suitable for implantation into the axillary
region of rats, cylindrical ceramic blocks were cut into smaller
segments and each implant contained one uniform segment.
Blood was collected into tubes without an anticoagulant sub-
stance in a volume of 500 μL. RhBMP6 was mixed with the
blood immediately at withdrawal and added to a sterile sy-
ringe (5 mL, BD) containing cylinder segment and left five
to 15 minutes to coagulate. All implants were implanted with-
in one hour following coagulation as described [8,9,24]. The
dose of rhBMP6 was 20 μg per implant.
Fig. 1 Microarchitecture and biomechanical properties of the cylindrical
ceramic blocks. aFrontal (left) and longitudinal (right) microCT section
of cylindrical ceramic blocks used in in vivo experiments. b3D recon-
struction of ABGS implant containing rhBMP6 in autologous blood co-
agulum (ABC) with a cylindrical ceramic block. The height and diameter
of the ceramic block were around 5 mm. cSubcutaneous implantation of
ABGS in axillary region of Sprague-Dawley rats. dBiomechanical
properties (stiffness, elasticity, and work) of autologous bone graft sub-
stitute (ABGS) with cylindrical ceramic blocks (cylinders with 30:1 and
35:1 S/F ratio (Cyl 30:1, Cyl 35:1, respectively) compared with ABGS
with ceramic particles (small, 74420 μm (S) and medium, 5001700 μm
(M)) or without ceramic particles (autologous blood coagulum - ABC).
All values were determined using the cut test and expressed as mean ± SD
as indicated. One-way analysis of variance with Tukeys multiple com-
parisons test was performed. Arrows at lines above graph bars indicate the
experimental group statistically compared with other experimental groups
marked by a vertical line. Pvalues are marked with asterisk indicating *
P0.05, ** P0.01, *** P0.001
Table 1 Properties of cylindrical
ceramic blocks used in the study Group Chemical
composition
S/F
ratio
Sintering
temperature
Average pore
size
Average density Average
porosity
A TCP/HA 80/20 30:1 1175° 374 ± 14 μm 0.71 ± 0.04 g/cm
3
77 ± 1%
B TCP/HA 80/20 35:1 1175° 376 ± 24 μm 0.71 ± 0.03 g/cm
3
77 ± 1%
1099International Orthopaedics (SICOT) (2021) 45:1097–1107
Biomechanical testing
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.
Experimental animals
In in vivo experiments, male and female Sprague-Dawley labo-
ratory rats (8 weeks old, body weight between 200 and 300 g, in
house bred - animal facility at the Laboratory for Mineralized
Tissues, University of Zagreb School of Medicine, Zagreb,
Croatia) were used. Rats were housed in poly sulfonic (PS)
cages with environmental enrichment in conventional laboratory
conditions at the temperature of 2024 °C, relative humidity of
4070%, noise level 60 dB, and illumination 12 hours per day
provided by fluorescent lighting. Standard GLP diet and fresh
water were provided ad libitum.
Ethical principles of the study ensured compliance with
European Directive 010/63/E, the Law on Amendments to
Animal Protection Act (Official Gazette 37/13), the Animal
Protection Act (Official Gazette 102/17), ordinance on the
protection of animals used for scientific purposes (Official
Gazette 55/13), FELASA recommendations, and recommen-
dations of the Ethics Committee at School of Medicine,
University of Zagreb.
Surgical procedures
Rats were anaesthetized before the surgical procedure by a
combination of xylazine 5 mg/kg (i.m.) and ketamine
100 mg/kg (s.c.). ABGS containing rhBMP6 within ABC
with cylindrical ceramic blocks was implanted into the axil-
lary region as previously described [8,9,24](Fig.1c).
Laboratory animals were euthanized at days seven, 14, 21,
35, and 50. The axillary regions were dissected and implants
were extracted.
MicroCT analyses
Implants were scanned by 1076 SkyScan μCT machine
(Bruker, Belgium) to visualize and quantify the bone volume
through different stages of bone formation. Scanning param-
eters were 18 μm resolution, 0.5 mm aluminum filter, frame
averaging set to a value of 2 while the scanning width was set
to 34 mm as described [25].
Reconstruction of acquired images was done using the
NRecon software (Bruker, Belgium) while the CTAn software
(Bruker, Belgium) was used for further analyses. On recon-
structed images, newly formed bone tissue was separated from
ceramics and the amount of bone (bone volume) was calcu-
lated [23,24].
Histology
Samples were fixed in 4% formalin for ten days. All samples
were then decalcified using 14% EDTA in 4% formalin solution
for 20 days with a solution change every two days. All samples
were embedded in paraffin and cut at 5-μm slice thickness and
stained by Goldner as described [9]. In addition, several samples
were processed undecalcified as described [24] for histological
examination of the extent and microarchitecture of the newly
formed bone.
Immunohistochemistry
Immunohistochemistry was performed after tissue
deparaffinization and rehydration. Heat-induced epitope re-
trieval (HIER) was performed in sodium citrate buffer
pH 6.0 in a microwave oven for 15 minutes, following cooling
for 30 min at room temperature. Mouse and rabbit specific
HRP/AEC IHC detection kit micro-polymer (Abcam, UK)
was used in all procedures. Slides were incubated with the
following primary antibodies: rabbit anti-SOX9 and rabbit
anti-OSX overnight at 4 °C in a moist chamber. All antibodies
were purchased from Abcam (UK). Goat anti-rabbit second-
ary antibody was used, coupled with horseradish peroxidase
(HRP), and detected using diaminobenzidine (DAB)
Table 2 The experimental design
of the study 1. Biomechanical testing description of groups
a) Blood coagulum (control group)
b) Ceramic particles 74420 μm + ABC
c) Ceramic particles 5001700 μm + ABC
d) Cylindrical ceramic block (30:1, 1175°) + ABC
e) Cylindrical ceramic block (35:1, 1175°) + ABC
2. Rat subcutaneous implant assay
Experimental groups Time points (days)
a) Cylindrical ceramic block (30:1) + ABC + rhBMP6 7, 14, 21, 35, and 50
b) Cylindrical ceramic block (35:1) + ABC + rhBMP6
1100 International Orthopaedics (SICOT) (2021) 45:1097–1107
chromogen. Slides were counterstained in hematoxylin and
mounted using ImmunoHistoMount(Sigma-Aldrich, MO,
USA). Images were acquired using Olympus BX53
microscope.
Histomorphometry
Bone histomorphometry was conducted to quantify the
amount of newly formed tissues including bone, CRM, and
the bone marrow distribution among the ectopic explants on
days 14, 21, 35, and 50 as previously described [23,24].
Shortly, histology sections were stained by modified
Goldners stain, staining the bone green/turquoise, while
CRM remained white. Goldners stained sections were im-
aged in × 10 (1.83 pixel/μm) magnification using an
Olympus BX53 upright microscope equipped with a DP27
camera (5 megapixels, 15 fps) and operated by the cellSens
Dimension software (Olympus, Japan). The Photoshop soft-
ware (Adobe System, CA, USA) and Fiji ImageJ software
(version 1.51r; NIH, MD, USA) were used for image analy-
ses. Results were expressed as area percentages.
Data analysis
Data distribution of continuous variables had been checked
with the Kolmogorov-Smirnov test. Values are expressed as
mean ± SD as indicated. Depending on the experiment, one-
way analysis of variance or two-way analysis of variance with
Tukeys multiple comparisons test was performed. All P
values below 0.05 were considered significant and they are
marked with asterisks; *(P0.05), **(P0.01), ***(P
0.001). Statistical programs IBM SPSS Statistics (v.25) and
GraphPad Prism (v.8) were used in all statistical calculations.
Results
Biomechanical testing
We used the cut test to determine the stiffness, elasticity and
load, and work-to-break of various ABGS formulations con-
taining CRM as cylindrical ceramic blocks (Fig. 1ab)orpar-
ticles. Stiffness and work-to-break were significantly higher in
Fig. 2 MicroCT analysis and
microCT/histological sections
through the implants. aMicroCT
analyses and reconstructed im-
ages (RI) of autologous bone graft
substitute (ABGS) with ceramic
blocks showing ceramics (white)
on day 7 and then newly formed
bone (green) and ceramics (white)
on days 14, 21, 35, and 50. Newly
formed bone was uniformly pres-
ent inside the cylinder while the
cortical bone encircled the cylin-
der outside. bBone volume
(mm
3
) among experimental
groups throughout the follow-up
period. The amount of newly
formed bone reached its peak on
day 14 and then decreased to-
wards day 50. Two-way analysis
of variance with Tukeys multiple
comparisons test was performed.
Values are expressed as mean ±
SD. Pvalues are marked with as-
terisk; * P0.05, ** P0.01,
*** P0.001. cHistological
sections through bone-ceramic
structure (BCS) consisting of
newly formed bone and various
cylindrical ceramic blocks (yel-
low asterisk) containing pores
(red asterisk) on day 35.
Pronounced cortical bone (green
arrow) formed boundaries of
newly formed BCS
1101International Orthopaedics (SICOT) (2021) 45:1097–1107
Fig. 3 Sequence of histological
events following subcutaneous
implantation of autologous bone
graft substitute (ABGS).
Modified Goldnersstained
histology sections. Newly formed
bone was stained green, black
asterisks indicated ceramic
cylinders, yellow arrows marked
endochondral ossification, and
blue arrows the bone marrow.
Scale bars were shown in the
lower right corner. On day 7, the
endochondral ossification was
present outside the cylindrical
block and penetrated to few pores.
On days 14 and 21, the newly
formed bone was present
throughout the pores inside the
ceramic block. On days 35 and
50, the bone covered the surface
of the ceramics encircling pores.
Within the ossicle, there were few
trabeculae and bone marrow with
adipocytes
1102 International Orthopaedics (SICOT) (2021) 45:1097–1107
ABGS formulations containing ceramic blocks as compared
with ABGS with ceramic particles or ABC alone (Fig. 1d).
There was no significant difference in neither stiffness nor
work-to-break between tested ceramic blocks. Elasticity was
significantly lower in groups containing ceramic blocks than
in groups containing particles or those without CRM (Fig. 1d).
The elasticity between the tested ceramic blocks which dif-
fered in S/F ratio was similar.
Rat subcutaneous assay
All tested ABGS formulations induced formation of bone ad-
jacent or inside the cylindrical ceramic blocks and formed a
vascularized, bone tissue-engineered construct previously
named as a bone-ceramic structure (BCS) [24](Figs.2and
3). Newly formed BCS on day 14 and onwards typically
consisted of cortical bone forming BCS boundaries, bone on
the ceramic surface, and trabeculae in the pores. The cascade
of biological events and microarchitecture of the newly
formed BCS were analyzed by microCT and on histology
sections (see following sections).
MicroCT analyses
Bone induction and formation of BCS were analyzed and
quantified on microCT sections. Newly formed bone and
ceramics were separated on microCT sections (Fig. 2a)
and the amount of newly formed bone was calculated
for each time point (Fig. 2b).Ondayseven,onlysmall
areas of newly formed bone were visible on the peripheral
parts of implants and therefore newly formed bone was
almost undetectable by microCT analyses. However, on
day 14, there was an extensive amount of newly formed
bone in all implants which decreased in time. Importantly,
from day 21 onwards, the cortical bone at the boundaries
of BCS was well defined and distinguishable from the
trabecular bone (Fig. 2a and c).
Histology
On day seven after implantation, the endochondral bone
was present on the periphery and in a few pores of cylin-
drical ceramic blocks (Figs. 3and 4) while the central part
of the implant contained pores with remnants of autolo-
gous blood coagulum. On day 14, the new bone was pres-
ent in all histological sections (Fig. 3). The newly formed
bone was divided into the cortical bone, bone on the outer
and inner surfaces of ceramics, and trabecular bone inside
the pores (Fig. 4). Cortical bone was present outside the
cylindrical ceramic blocks forming boundaries of the new-
ly formed BCS. Inside the ceramic block, bone was uni-
formly present in the pores regardless of the pore position
Fig. 4 Immunolocalization of SOX-9 and osterix during ectopic bone
formation. Immunolocalization of SOX-9 on day 7 (upper row) and
osterix (OSX) on day 14 (lower row) following implantation. On day 7,
SOX-9 was localized in the areas with endochondral ossification outside
the ceramic blocks and in some pores. On day 14, OSX was localized on
surfaces of the newly formed bone in the ceramic block pores. Black
asterisk indicates the ceramic block
1103International Orthopaedics (SICOT) (2021) 45:1097–1107
inside the block. In the pores, bone covered the surface of
the ceramics encircling pores with bone and dense trabec-
ular network. Bone marrow was present between bone tra-
beculae. At days 14 and 21, there were only few adipocytes
in the bone marrow and the microarchitecture of BCS was
similar (Fig. 3). Inside the pores, the number and thickness
of the trabeculae decreased, while the adipocyte numbers
wereincreasedinthebonemarrowascomparedwithday
14. Histology on day 35 and day 50 was similar and it
seemed that the ectopic osteogenesis reached its final pat-
tern (Fig. 3). Outside the ceramic block, the well-formed
cortical bone defined the boundaries of BCS (Fig. 2c),
while inside the pores, bone completely covered the sur-
face of the ceramics surrounding the pores with bone (Fig.
3). Inside the ossicle, there were few trabeculae, while in
the bone marrow, adipocytes were dominant.
Histomorphometry
Histomorphometric analysis (Fig. 5a) was performed in order
to determine changes in the amount of bone and bone marrow
through the follow-up period. The amount of bone in the pores
was highest while the amount of bone marrow was lowest on
day 14 (Fig. 5b). Towards the end ofthe experiment, there was
a trend of a decreased bone volume and increased bone mar-
row area in the pores. Subsequently, the bone/bone marrow
ratio decreased through time (Fig. 5b).
Discussion
In the present study, we demonstrated that ABGS implants
containing synthetic ceramic macroporous blocks had
Fig. 5 Histomorphometric
analysis. aStatic
histomorphometric analysis of
histological sections stained by
Goldner. In order to determine the
amount of bone (yellow arrow),
bone marrow (blue arrow), and
compression resistant matrix
(CRM) (black asterisk), regions
of interest were masked with dis-
tinctive color (black) and further
thresholded for measurement. b
The amount of bone (%) and bone
marrow as well as bone/bone
marrow ratio determined by
histomorphometric analysis on
histological sections. The per-
centage of bone reached its peak
on day 14 and then decreased in
time, while the bone marrow in
parallel increased in time.
Subsequently, the bone/bone
marrow (b/bm) ratio decreased in
time from day 14 to day 50. Two-
way analysis of variance with
Tukeys multiple comparisons
test was performed. Values are
expressed as mean ± SD as indi-
cated. Pvalues are marked with
asterisk; * P0.05, ** P0.01,
*** P0.001
1104 International Orthopaedics (SICOT) (2021) 45:1097–1107
significantly increased stiffness and load (work-to-break) than
ABGS implants with or without ceramic particles. Moreover,
ABGS with ceramic blocks were more rigid and their elastic-
ity was significantly decreased. ABGS containing
macroporous blocks as CRM induced endochondral differen-
tiation on day seven at the periphery as shown by
chondrocytes marker, SOX-9. By day 14 onwards, new bone
formation was evident with bone volume reaching its peak
and slowly decreasing in time as examined by histology and
microCT analyses. The newly formed bone was uniform in
the duration of the study and present in all pores through the
macroporous block. We demonstrated for the first time a se-
quence of ectopic osteogenesis following subcutaneous im-
plantation of this novel ABGS containing cylindrical ceramic
blocks as CRM.
The osteoinductive and osteoconductive properties of
implants were typically tested in small animals (mice or
rats), both in orthotopic models (e.g., calvarial critical size
defect) [19,2630] and ectopic bone model (subcutane-
ous or intramuscular implantation) [18,3134]. Rat sub-
cutaneous bone induction model is routinely used for rap-
id evaluation of osteogenic properties of potential devices
for bone regeneration [35]. Here, we quantified and de-
scribed rhBMP6-induced bone formation in ABGS con-
taining macroporous blocks as CRM. MicroCT analyses
revealed that newly formed bone was uniform and as well
present in all pores in the macroporous block. However,
microarchitecture of the newly formed bone and the cel-
lular microenvironment significantly changed in time. The
number of bone trabeculae inside the pores decreased in
time and adipocytes became the predominant cell popula-
tion in the bone marrow. This phenomenon was ascribed
to biological activity of osteogenic BMPs in both osteo-
genic and adipogenic differentiation [28,3638]. It has
been demonstrated that osteogenic BMPs also possess
strong adipogenic activity, which is primarily regulated
through the activity of peroxisome proliferator-activated
receptor gamma 2 (PPARγ2) [37,38]. On days 35 and
50 following implantation, ectopic osteogenesis had un-
dergone extensive remodeling and exhibited a BCS
consisting of well-defined cortical bone on the bound-
aries, bone covering the ceramic surfaces, and few trabec-
ulae in the pores surrounded with abundant bone marrow
containing predominantly adipocytes. Pronounced cortical
bone surrounding the macroporous block is the most im-
portant feature of the newly formed BCS in ABGS con-
taining cylindrical macroporous blocks indicating that the
most appropriate indication for their further evaluation is
the treatment of diaphyseal segmental and non-union de-
fects in which the newly formed cortical bone should
establish a continuity along the cortices of long bones.
Implantation of ABGS containing rhBMP6, ABC, and ce-
ramic scaffold induced a formation of a defined, tissue-
engineered bone construct which we previously described as
BCS [24]. Unlike ABC which rapidly resorbs in time, the
ceramics provide a long-lasting scaffold whose resorbability
is determined by chemical composition of ceramics, specifi-
cally the tricalcium phosphate/hydroxyapatite ratio [14,16,
18]. The main advantage of ABGS containing organized,
one-part ceramic block is that they might be available in a
broad range of geometrical shapes adjusted for specific con-
ditions. Moreover, ceramic determinants such as overall po-
rosity, pore size, and wall thickness could be engineered dur-
ing manufacturing processes [14].
Conclusions
We demonstrated that macroporous ceramic blocks in a form
of cylinders are promising CRMs with enhanced biomechan-
ical properties and good handling, supporting bone formation
with ABGS containing rhBMP6 within autologous blood co-
agulum. Therefore, ABGS containing ceramic blocks should
be tested in preclinical models such as diaphyseal segmental
defects and non-unions in larger animals.
Acknowledgments For animal studies, we thank Mirjana Marija Renic
and Djurdjica Car for their excellent technical assistance. For biomechan-
ical testing, we thank Sven Karlovic and Goran Bosanac (Faculty of Food
Technology and Biotechnology, University of Zagreb, Zagreb, Croatia)
for the help in the conduction of experiment and data analysis. Special
thanks to Jack Ratliff (Ratliff Histology Consultants LLC) for the excel-
lent preparation of undecalcified histology sections.
Funding This program was funded by the FP7 Health Program (FP7/2007-
2013) under grant agreement HEALTH-F4-2011-279239 (Osteogrow),
H2020 Health GA 779340 (OSTEOproSPINE), and European Regional
Development Fund - Scientific Center of Excellence for Reproductive and
Regenerative Medicine (project Reproductive and regenerative medicine -
exploration of new platforms and potentials,GA KK.01.1.1.01.0008
funded by the EU through the ERDF).
Compliance with ethical standards
Conflict of interest SV, TKS, LG, CC, and HO have an issued patent
WO2019076484A1 licensed to perForm Biologics. HO received grants
and other from Genera Research during the study; LS is a part owner of
CaP Biomaterials. TKS received grants and other from perForm
Biologics during the study.
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1107International Orthopaedics (SICOT) (2021) 45:1097–1107
... Biomechanical properties of the Osteogrow device might be enhanced by the addition of compression-resistant matrix (CRM) to Osteogrow implants [2,[7][8][9]. During Osteogrow preclinical studies, allograft was the first tested CRM and Osteogrow implants with allograft (Osteogrow-A) were successfully tested in rabbit and sheep PLF studies [2,4]. ...
... Osteogrow-C implants were tested by conducting a wellestablished rat subcutaneous implant assay. The follow-up period was 21 days and it was chosen based on the findings from our previous studies which established bone formation parameters by undecalcified tissue histology, histomorphometry, microCT analyses, and immunolocalization Porosity Up to 90% 60% n/a n/a n/a of various molecules involved in osteogenesis [7,9]. The number of implants was 5 per experimental group, and it was determined based on the known variability of this assay and recommendations for conducting studies in relevant animal models [33,34]. ...
... To analyze newly formed bone-ceramic structures, extracted implants were scanned using a 1076 SkyScan µCT device (Bruker SkyScan, Billerica, Massachusetts, USA) using our standard protocol [7,9,31]. In brief, scanning parameters were set to a resolution of 18 µm, frame averaging of 2 with 0.5 mm aluminum filter, and rotation step of 0.5. ...
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... 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]. ...
... Histomorphometric analyses were conducted to evaluate the effect of ZOL on the number of osteoclasts, bone microarchitecture, and the amount of bone and bone/bone marrow ratio as previously described [16][17][18]. Briefly, osteoclasts were localized by histochemical acid phosphatase detection on HE-stained sections and were counted in five fields (one field = 0.4 mm 2 ) on each section. ...
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... The resorbability might be adjusted by varying HA/TCP ratios in BCP ceramics [75]. Moreover, CaP ceramics might be formulated into particles or blocks in a broad range of sizes and geometrical shapes while porosity, pore size, and interconnectivity are adjusted during the sintering process [73,75,76]. We demonstrated that particle size affects the volume of newly formed bone; smaller particles (74-420 µm) combined with rhBMP6 resulted in higher bone volume than larger particles (1000-4000 µm) [73]. ...
... 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). ...
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... 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 . ...
... ABGS comprised of rhBMP6 within autologous blood coagulum as a physiological BMP carrier and synthetic ceramics as a compression resistant matrix is a novel osteoinductive device for bone regeneration [33][34][35]37 . In our previous studies on rat subcutaneous model we addressed several unresolved issues regarding ABGS formulation, including the optimal dose and method of rhBMP6 application as well as the optimal combination of size and chemical composition of ceramic particles 38,41,56 . Moreover, in our first PLF study with ceramics as CRM we have demonstrated that ABGS containing synthetic ceramics named Osteogrow-C promotes spinal fusion and confirmed the finding from rat studies that there are two equally efficient methods of BMP6 application: rhBMP6 might be lyophilized on ceramic particles or directly added to the autologous blood 37,56 . ...
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... For signaling BMP6 uses ALK2, ALK3 and ALK6 BMP type I receptors and it increases the expression of bone differentiation markers in cell cultures 131,140 . ABC also decreases inflammation and foreign body cell accumulation when combined with allograft or calcium phosphate ceramics in PLF animal studies 132,135,136,153 . BMP6 in blood remains active for approximately 7 days and could not be detected in the remaining serum after the formation of the autologous blood coagulum 12,115,131 . ...
... Synthetic ceramics might be formulated into varioussize particles or blocks. We have demonstrated that addition of synthetic ceramics to ABGS significantly increased the amount of newly formed bone and decreased the outcome variability 137,153 . Moreover, we have demonstrated that size and shape of ceramic particles determines the quantity and microarchitecture of the newly formed bone 137 . ...
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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.
... It has been shown that autologous blood clots applied to a rabbit 2.7 mm non-critical-sized defect promotes bone formation when compared to hemostasis reagent microfibrillar collagen (8-fold increase), oxidized regenerated cellulose (1.5-fold increase), and equivalent to soluble alkylene oxide copolymer [27].The OSTEOGROW device, which utilizes a blood coagulum carrier to bind BMP6, has been found to stimulate mesenchymal stem cell differentiation and enhance healing of critical-sized ulnar bone defects without incurring bone resorption and inflammation. The device has demonstrated efficacy in regenerating bone in a canine bone defect healing model [24,28,29]. Furthermore, a recent study has shown that autologous blood clots can sustain-release BMP2 in vitro and promote osteogenic differentiation of stem cells. ...
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Background. Fibrin sealant has been used as a scaffold to deliver genetically modified human muscle-derived stem cells (hMDSCs) for bone regeneration. Alternatively, autologous blood clots are safe, economic scaffolds. This study compared autologous blood clot (BC) with fibrin sealant (FS) as a scaffold to deliver lenti-BMP2/GFP-transduced hMDSCs for bone regeneration. Methods. In vitro osteogenic differentiation was performed using 3D pellet culture and evaluated using microCT and Von Kossa staining. The lenti-GFP transduced cells were then mixed with human blood for evaluation of osteogenic differentiation. Furthermore, a murine critical- sized calvarial defect model was utilized to compare BC and FS scaffolds for lenti-BMP2/GFP-transduced hMDSCs mediated bone regeneration and evaluated with micro-CT and histology. Results. Lenti-BMP2/GFP transduced hMDSCs formed significantly larger mineralized pellets than non-transduced hMDSCs. hMDSCs within the human blood clot migrated out and differentiated into ALP+ osteoblasts. In vivo, BC resulted in significantly less new bone formation within a critical-sized calvarial bone defect than FS scaffold, despite no difference observed for GFP+ donor cells, osteoclasts, and osteoblasts in the newly formed bone. Conclusions. Human lenti-BMP2/GFP-transduced hMDSCs can efficiently undergo osteogenic differentiation in vitro. Unexpectedly, the newly regenerated bone in BC group was significantly less than the FS group. The autologous blood clot scaffold is less efficacious for delivering stem cells for bone regeneration than fibrin sealant.
... Particularly worth mentioning are two articles in our Basic Research section. In the first of them titled: "Synthetic ceramic macroporous blocks as a scaffold in ectopic bone formation induced by recombinant human bone morphogenetic protein 6 within autologous blood coagulum " [30] , provides the latest discoveries made by professor Vukičević's scientific teamthe so-called Z a g r e b B M P G r o u p , a n d t h e s e c o n d a r t i c l e " Macrophages' contribution to ectopic osteogenesis in combination with blood clot and bone substitute: possibility for application in bone regeneration strategies "is submitted by professor Živković's scientific team from Niš, Serbia [31]. ...
... Since the use of allograft has several disadvantages including regulatory issues, immunogenicity, and viral transmission risk (Mroz et al., 2009), we recently tested ABGS with synthetic calcium phosphate ceramics (tricalcium phosphate (TCP), hydroxyapatite (HA), and biphasic ceramics containing TCP and HA in various ratios) as a CRM. Superior osteoinductive and osteoconductive properties of ABGS with synthetic ceramics were confirmed in a rat subcutaneous assay Stokovic et al., 2020b), and in a rabbit spine PLF model due to successful new bone integration with native transverse processes of lumbar vertebrae (Stokovic et al., 2020c). ...
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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.
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Bone morphogenetic proteins (BMPs) are potent osteoinductive agents for bone tissue engineering. In order to define optimal properties of a novel autologous bone graft substitute (ABGS) containing rhBMP6 within the autologous blood coagulum (ABC) and ceramic particles as a compression resistant matrix (CRM), we explored the influence of their amount, chemical composition and particle size on the quantity and quality of bone formation in the rat subcutaneous assay. Tested ceramic particles included tricalcium phosphate (TCP), hydroxyapatite (HA) and biphasic calcium phosphate ceramic (BCP), containing TCP and HA in 80/20 ratio of different particle sizes (small 74-420 μm, medium 500-1700 μm and large 1000-4000 μm). RhBMP6 was either mixed with ABC or lyophilized on CRM prior to use with ABC. The experiments were terminated on day 21 and implants were analysed by microCT, histology and histomorphometry. Addition of CRM to ABGS containing rhBMP6 in ABC significantly increased the amount of newly formed bone and the optimal CRM/ABC ratio was found to be around 100mg/500 μL. MicroCT analyses revealed that all tested ABGS formulations induced an extensive new bone formation and there were no differences between the two methods of rhBMP6 application as determined by the bone volume. However, the particle size played a significant role in the quantity and quality of bone formed. ABGS containing small particles induced new bone forming a dense trabecular network, cortical bone at the rim, bone and bone marrow in apposition to and in between ceramic particles. ABGS containing medium and large particles also resulted in new bone on the surface of particles as well as inside the pores. Histomorphometrical analysis revealed that the ceramics particle size correlated with the quality of trabecular pattern of newly formed bone, bone/bone marrow ratio as observed in apposition and between particles, and the ratio between the cortical and trabecular bone. By employing rat subcutaneous implant assay, we showed for the first time that the size of synthetic ceramics particles affected the osteogenesis as defined by both the quantity and quality of ectopic bone.
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BMPs were purified from demineralized bone matrix based on their ability to induce new bone in vivo and they represent a large member of the TGF-β superfamily of proteins. BMPs serve as morphogenic signals for mesenchymal stem cell migration, proliferation and subsequently differentiation into cartilage and bone during embryonic development. A BMP when implanted with a collagenous carrier in a rat subcutaneous site is capable of inducing new bone by mimicking the cellular events of embryonic bone formation. Based on this biological principle, BMP2 and BMP7 containing collagenous matrix as carrier have been developed as bone graft substitutes for spine fusion and long bone fractures. Here, we describe a novel autologous bone graft substitute that contains BMP6 delivered within an autologous blood coagulum as carrier and summarize the biology of osteogenic BMPs in the context of bone repair and regeneration specifically the critical role that carrier plays to support osteogenesis.
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Posterolateral lumbar fusion (PLF) is a commonly performed surgical procedure for the treatment of pathological conditions of the lumbosacral spine. In the present study, we evaluated an autologous bone graft substitute (ABGS) containing rhBMP6 in autologous blood coagulum (ABC) and synthetic ceramics used as compression resistant matrix (CRM) in rabbit PLF model. In the pilot PLF rabbit experiment, we tested four different CRMs (BCP 500-1700 μm, BCP 1700-2500 μm and two different TCP in the form of slabs) which were selected based on achieving uniform ABC distribution. Next, ABGS implants composed of 2.5 mL ABC with 0.5 g ceramic particles (TCP or BCP (TCP/HA 80/20) of particle size 500-1700 μm) and 125 μg rhBMP6 (added to blood or lyophilized on bioceramics) were placed bilaterally between transverse processes of the lumbar vertebrae (L5-L6) following exposition and decortication in 12 New Zealand White Rabbits observed for 7 weeks following surgery. Spinal fusion outcome was analysed by μCT, palpatory segmental mobility testing and selected specimens were either tested biomechanically (three-point bending test) and/or processed histologically. The total fusion success rate was 90.9% by both μCT analyses and by palpatory segmental mobility testing. The volume of newly formed bone between experimental groups in of TCP or BCP ceramics and the method of rhBMP6 application was comparable. The newly formed bone and ceramic particles integrated with the transverse processes on histological sections resulting in superior biomechanical properties. The results were retrospectively found superior to allograft devitalized mineralized bone as a CRM as reported previously in rabbit PLF. Overall, this novel ABGS containing rhBMP6, ABC and the specific 500-1700 μm synthetic ceramic particles supported new bone formation for the first time and successfully promoted posterolateral lumbar fusion in rabbits.
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Bone morphogenetic proteins (BMPs) are known to induce new bone formation in vivo but treating trabecular bone defects with a BMP based therapeutic remains controversial. Here, we evaluated the safety and efficacy of a novel Autologous Bone Graft Substitute (ABGS) comprised of recombinant human BMP6 (rhBMP6) dispersed within an autologous blood coagulum (ABC) as a physiological natural carrier in patients with a closed distal radial fracture (DRF). We enrolled 32 patients in a randomized, standard of care (SoC) and placebo (PBO) controlled, double-blinded Phase I First in Human (FiH) clinical trial. ABGS was prepared from peripheral blood as 250 μg rhBMP6/mL ABC or PBO (1 mL ABC containing excipients only) and was administered dorsally via a syringe injection into the fracture site following closed fracture fixation with 3 Kirschner wires. Patients carried an immobilization for 5 weeks and were followed-up for 0 to 26 weeks by clinical examination, safety, serial radiographic analyses and CT. During the 13 weeks follow-up and at 26 weeks post study there were no serious adverse reactions recorded. The results showed that there were no detectable anti-rhBMP6 antibodies in the blood of any of the 32 patients at 13- and 26-weeks following treatment. Pharmacokinetic analyses of plasma from patients treated with ABGS showed no detectable rhBMP6 at any time point within the first 24 hours following administration. The CT image and radiographic analyses score from patients treated with AGBS showed significantly accelerated bone healing as compared to PBO and SoC at 5 and 9 weeks (with high effect sizes and P=0.027), while at week 13 all patients had similar healing outcomes. In conclusion, we show that intraosseous administration of ABGS (250 μg rhBMP6/mL ABC) into the distal radial fracture site demonstrated a good tolerability with no serious adverse reactions as well as early accelerated trabecular bone healing as compared to control PBO and SoC patients.
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BMPs are potent osteogenic proteins that induce new bone formation in vivo. However, their effect on bone healing in the trabecular bone surfaces remains challenging. We evaluated the safety and efficacy of recombinant human BMP6 (rhBMP6) applied within an autologous blood coagulum (ABC) in a surgically created wedge‐defect of the proximal tibia in patients undergoing high tibial osteotomy (HTO) for Varus deformity and medial osteoarthritis of the knee. We enrolled 20 HTO patients in a randomized, placebo controlled, double‐blinded Phase I/II clinical trial. RhBMP6/ABC (1.0 mg/10 mL ABC prepared from peripheral blood) or placebo (10 mL ABC containing excipients) were administered into the tibial wedge defects. Patients were followed for 0 to 24 months by clinical examination (safety) and CT and serial radiographic analyses (efficacy). The results show that there were no detectable anti‐rhBMP6 antibodies in the blood of any of the 20 patients at 14 weeks following implantation. During the 24 months follow‐up there were no serious adverse reactions recorded. The CT scans from defects of patients treated with rhBMP6/ABC showed an accelerated bone healing as compared to placebo at 9‐weeks (47.8 ± 24.1 vs. 22.2 ± 12.3 mg/cm2; P=0.008) and at 14‐weeks (89.7 ± 29.1 vs. 53.6 ± 21.9 mg/cm2; P=0.006) follow‐up. Radiographic analyses at weeks 6 and 24, and months 12 and 24, suggested the advanced bone formation and remodelling in rhBMP6/ABC treated patients. In conclusion, we show that rhBMP6/ABC at a dose of 100 μg/mL accelerated bone healing in patients undergoing HTO without serious adverse events and with a good tolerability, as compared to placebo alone. Overall, for the first time, a BMP‐based osteogenic implant was examined against a placebo for bone healing efficacy in the trabecular bone surface, using an objective BMD measurement system. This article is protected by copyright. All rights reserved.
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In the present study, we evaluated an autologous bone graft substitute (ABGS) composed of recombinant human BMP6 (rhBMP6) dispersed within autologous blood coagulum (ABC) used as a physiological carrier for new bone formation in spine fusion sheep models. The application of ABGS included cervical cage for use in the anterior lumbar interbody fusion (ALIF), while for the posterolateral lumbar fusion (PLF) sheep model allograft devitalized bone particles (ALLO) were applied with and without use of instrumentation. In the ALIF model, ABGS (rhBMP6/ABC/cage) implants fused significantly when placed in between the denuded L4-L5 vertebrae as compared to control (ABC/cage) which appears to have a fibrocartilaginous gap, as examined by histology and micro CT analysis at 16 weeks following surgery. In the PLF model, ABGS implants with or without ALLO showed a complete fusion when placed ectopically in the gutter bilaterally between two decorticated L4-L5 transverse processes at a success rate of 88% without instrumentation and at 80% with instrumentation; however the bone volume was 50% lower in the instrumentation group than without, as examined by histology, radiographs, micro CT analyses and biomechanical testing at 27 weeks following surgery. The newly formed bone was uniform within ABGS implants resulting in a biomechanically competent and histologically qualified fusion with an optimum dose in the range of 100 μg rhBMP6 per mL ABC, while in the implants that contained ALLO, the mineralized bone particles were substituted by the newly formed remodeling bone via creeping substitution. These findings demonstrate for the first time that ABGS (rhBMP6/ABC) without and with ALLO particles induced a robust bone formation with a successful fusion in sheep models of ALIF and PLF, and that autologous blood coagulum (ABC) serves as a preferred physiological native carrier to induce new bone at low doses of rhBMP6 and to achieve a successful spinal fusion.
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In the present study, we describe autologous blood coagulum (ABC) as a physiological carrier for BMP6 to induce new bone formation. Recombinant human BMP6 (rhBMP6), dispersed within ABC and formed as an autologous bone graft substitute (ABGS), was evaluated either with or without allograft bone particles (ALLO) in rat subcutaneous implants, and in a posterolateral lumbar fusion (PLF) model in rabbits. ABGS induced endochondral bone differentiation in rat subcutaneous implants. Coating ALLO by ABC significantly decreased the formation of multinucleated foreign body giant cells (FBGCs) in implants, as compared to ALLO alone. However, addition of rhBMP6 to ABC/ALLO induced a robust endochondral bone formation with little or no FBGCs in the implant. In rabbit PLF model, ABGS induced new bone formation uniformly within the implant resulting in a complete fusion when placed between two lumbar transverse processes in the posterolateral gutter with an optimum dose of 100 μg rhBMP6 per mL ABC. ABGS containing ALLO also resulted in a fusion where the ALLO was replaced by the newly formed bone via creeping substitution. Our findings demonstrate for the first time that rhBMP6, with autologous blood coagulum (ABC) as a carrier, induced a robust bone formation with a complete spinal fusion in a rabbit PLF model. RhBMP6 was effective at low doses with ABC serving as a physiological substratum providing a permissive environment by protecting against foreign body reaction elicited by ALLO.
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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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Bone fractures represent a significant medical morbidity among aged population with osteoporosis. Bone morphogenetic proteins (BMPs) are suggested to have therapeutic potential to enhance fracture healing in such patients. Though BMP-mediated fracture healing has been well-documented in preclinical models, there has been no clinical study that demonstrated unequivocally that indeed a BMP when presented with an appropriate scaffold could provide basis for robust outcome for delayed or non-union diaphyseal bone fractures. This review presents a comprehensive insight towards the existing knowledge on the role of BMP signaling in bone formation and maintenance. Also therapeutic options based on BMP biology are discussed.
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Bone morphogenetic proteins (BMPs) were purified from demineralized bone matrix by their ability to induce new bone formation in vivo. BMPs represent a large sub-family of proteins structurally related to TGF-beta and activins. Two BMP bone graft substitutes, BMP2 (InFuse®) and BMP7 (OP1®) have been developed as products for the repair of long bone non-union fractures and lumbar spinal fusion in humans. The approval of BMP2 and BMP7 based products for use in the clinic supports that the signals responsible for bone formation at ectopic sites can form a basis as therapeutics for bone repair and regeneration. This article describes a historical perspective of the discovery BMPs.