Autologous blood coagulum containing rhBMP6 induces new
bone formation to promote anterior lumbar interbody fusion
(ALIF) and posterolateral lumbar fusion (PLF) of spine in sheep
Lovorka Grgurevic, Igor Erjavec, Munish Gupta, Marko Pecin,
Tatjana Bordukalo-Niksic, Nikola Stokovic, Drazen Vnuk,
Vladimir Farkas, Hrvoje Capak, Milan Milosevic, Jadranka Bubic
Spoljar, Mihaela Peric, Mirta Vuckovic, Drazen Maticic, Reinhard
Windhager, Hermann Oppermann, T. Kuber Sampath, Slobodan
Reference: BON 115448
To appear in: Bone
Received date: 29 April 2020
Revised date: 19 May 2020
Accepted date: 20 May 2020
Please cite this article as: L. Grgurevic, I. Erjavec, M. Gupta, et al., Autologous blood
coagulum containing rhBMP6 induces new bone formation to promote anterior lumbar
interbody fusion (ALIF) and posterolateral lumbar fusion (PLF) of spine in sheep, Bone
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© 2018 Published by Elsevier.
Autologous blood coagulum containing rhBMP6 induces new bone
formation to promote anterior lumbar interbody fusion (ALIF) and
posterolateral lumbar fusion (PLF) of spine in sheep
Lovorka Grgurevic, MD, PhD1*, Igor Erjavec, PhD1*, Munish Gupta, MD2, Marko
Pecin, DVM, PhD3, Tatjana Bordukalo-Niksic, PhD1, Nikola Stokovic, MD1, Drazen
Vnuk, DVM, PhD3, Vladimir Farkas, DVM4, Hrvoje Capak, DVM, PhD5, Milan
Milosevic, MD, PhD6, Jadranka Bubic Spoljar, DVM, PhD1, Mihaela Peric, PhD7,
Mirta Vuckovic, DVM3, Drazen Maticic, DVM, PhD3, Reinhard Windhager, MD, PhD8,
Hermann Oppermann, MD9, T. Kuber Sampath, PhD10, Slobodan Vukicevic, MD,
* equal contribution
1Laboratory for Mineralized Tissues, Center for Translational and Clinical Research,
University of Zagreb School of Medicine, 10000 Zagreb, Croatia
2Department of Orthopedic Surgery, Washington University, St. Louis MO 63110,
3Clinics for Surgery, Orthopedics and Ophthalmology, School of Veterinary Medicine,
University of Zagreb, 10000 Zagreb, Croatia
4Division of Experimental Physics, Rudjer Boskovic Institute, 10000 Zagreb, Croatia
5Department of Radiology, School of Veterinary Medicine, University of Zagreb,
10000 Zagreb, Croatia
6Department of Environmental and Occupational Health and Sports, School of Public
Health „Andrija Stampar“, University of Zagreb School of Medicine, Rockefellerova 4,
10000 Zagreb, Croatia
7Center for Translational and Clinical Research, School of Medicine, University of
Zagreb, 10000 Zagreb, Croatia
8Department of Orthopedics and Trauma Surgery, Medical University of Vienna, 1090
9Genera Research, Kalinovica, 10431 Sveta Nedelja, Croatia
10perForm Biologics Inc., Holliston MA 01746, U.S.A.
Correspondence: Slobodan Vukicevic, MD, PhD, Laboratory for Mineralized
Tissues, Center for Translational and Clinical Research, University of Zagreb School
of Medicine, Salata 11, 10000 Zagreb, Croatia; e-mail: email@example.com;
ORCID ID 0000-0003-4076-0285
Declaration of interest: LG, HO and SV have an issued patent US8197840 licensed
to Genera Research (GR). HO received grants and other from GR during the study,
RW is a consultant for Pfizer, Stryker, Takeda, Depuy Synthes and Zimmer Biomet,
TKS received grants and other from perForm Biologics during the study. MG is a
consultant for Depuy Synthes, Innomed and Medtronic and receive royalties from
Depuy Synthes and Innomed.
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 g 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
Sheep anterior lumbar interbody fusion (ALIF), sheep posterior lateral lumbar fusion
(PLF), rhBMP6, autologous blood coagulum (ABC) as natural BMP carrier, allograft
(ALLO), compression resistant matrix (CRM)
Autografts and allografts containing autologous bone marrow are routinely
employed with or without instrumentation to stimulate osteogenesis and promote
spine fusion. They are applied either in the intervertebral disc space, as in anterior
lumbar interbody fusion (ALIF)  or in an ectopic site between two lumbar
transverse processes bilaterally as in the posterolateral lumbar fusion (PLF) . A
variety of disorders are treated with a spinal fusion, including, but not limited to,
degenerative disc disease (DDD), spondylolisthesis, spinal stenosis, scoliosis,
infections, spinal fractures, and various tumors, primarily to treat deformity or relieve
the source of back and leg pain [3-7].
Autograft from the patient’s Iliac crest bone is a “Gold Standard” for spine
fusion surgery as the harvested bone chips have live bone marrow cells and an
immunologically compatible extracellular matrix [8-10]. However, the use of autograft
presents disadvantages as it requires another incision that may result in post-
operative pain, infection and the amount of bone that can be harvested is limited [11-
18]. As an alternative to autograft, allograft (cadaver bone from a bone bank),
demineralized bone matrix (DBM) [19-22], various calcium-based ceramics in
conjunction with patients bone marrow [23-27], and bone morphogenetic proteins
(BMPs) with animal derived collagen [28-31] and/or with ceramic composite as
scaffolds [31-34] have been developed for clinical use [35-37]. The treatment efficacy
of lumbar arthrodesis in DDD is a complex clinical and economic issue for patients
and health care providers. The rate of nonunion is around 25-36% for non-
instrumented PLF and 10 % for single-level ALIF . The addition of instrumentation
decreased the nonunion rate to 4-12% for PLF, while the use of cages for spinal
fusion contributed to higher fusion rates for ALIF and PLF at 2-year follow up .
The ability of BMP to induce new bone at ectopic sites upon reconstitution with
an appropriate collagenous scaffold serves as a prototype for tissue engineering
where BMP serves as a signal and local site provides responding cells to allow bone
differentiation under a permissive vascular environment [36,38,39]. In accord with the
principles of tissue engineering, rhBMP2 applied within an absorbable collagen
sponge, ACS (InFUSE) has been shown to induce new bone formation and promote
spine fusion to treat DDD in skeletally mature patients  at one level fusion from L2
to S1 using Titanium LT cages via an ALIF  approach. Depending on the size of
the LT-CAGE the FDA recommended between 4.2 mg and 12 mg BMP per level .
However, the off-label use of rhBMP2/ACS in related interbody fusion procedures
(e.g., cervical) has resulted in unwanted safety issues likely due to the high dose
employed and the use of bovine collagen as a carrier [19,31,42-44]. The clinical
evaluation of rhBMP2 soaked in bovine-sourced collagen and synthetic ceramics
(hydroxyapatite and tri-calcium phosphate) composite as a scaffold (AMPLIFY) for
the PLF procedure  was not approved for use as it resulted in unwanted local and
systemic safety issues [31,46-49]. Similarly, bovine bone collagen dispersed with
additive carboxyl-methyl cellulose that contained rhBMP7 (OP-1 Putty) [50,51] also
resulted in disapproval. Subsequently a rise in off-label BMP applications and the
lack of guidelines ensued in spinal fusion procedures with a wide range of BMP
doses used (2.5 – 40 mg BMP per level) . This suggested a need for a
physiological native carrier instead of animal derived collagen to avoid foreign-body
reactions to high-mineral containing Ca-P based ceramics.
We have recently described autologous blood coagulum (ABC) to serve as a
physiological native carrier for rhBMP6 as an autologous bone graft substitute
(ABGS), which also might contain compressive resistant matrix like allograft. ABGS
when implanted at ectopic sites significantly reduced the foreign body giant cells
response and induced spinal fusion in rabbits without instrumentation following
decortication of transverse processes [53,54]. In the present study, we demonstrate
that recombinant human BMP6 (rhBMP6), a BMP with high specific bone forming
activity due to a low affinity for Noggin, an abundant endogenous BMP antagonist ,
is preferred in spinal surgery. RhBMP6, when delivered in a low dose with ABC alone
or with allograft (ALLO), was capable of inducing new bone formation and achieving
spinal fusion in sheep models of ALIF and PLF.
2. Materials and Methods
Study protocols were conducted in Female sheep (Ovis aries), Merinolaandschaf
breed, aged 3 to 4 years, with health certificate and weighing 50-70 kg. The animal
facility was registered by Directorate of Veterinary, Reg. No: HR-POK-020. Sheep
were acclimated for 3 days after transport to the animal facility and randomly
assigned to their respective treatment group. They were housed by standard corrals
in conventional climate conditions at the temperature of 16-20°C, relative humidity of
50-70%, and noise level up to 80 dB. Each corral was identified following animal
identification, animal strain, study number, group, dose, number and sex of each
animal. A standard sheep diet of oats, processed hey, added salt and fresh water
was provided ad libitum. Animal care was in compliance with standard operating
procedures of the Croatian Animal facility HR-POK-020 using 3R principle and
minimization of the pain suffering during the experiment. The guidelines of the
European convention for the protection of vertebrate animals used for experimental
and other scientific purposes (ETS 123) have been followed.
2.2. ABGS Implant Preparation
RhBMP6 used in experiments was prepared as follows. Lyophilized rhBMP6
(Genera Research, Zagreb) was dissolved in sterile water to a final concentration of 1
For the ALIF operation, a blood sample for implant preparation was collected from
the jugular vein of the animal in tubes without anticoagulant substance. Full blood in
the volume of 1.5 mL was supplemented with 0.025 mL of 15 mM CaCl2 solution and
150 µL of rhBMP6 or sterile water. The solution was mixed and transferred to a
sterile 10 mL syringe containing CFRP I/F cervical cage (Bengal, DePuy, USA), and
allowed to coagulate at room temperature. A typicaly prepared ABGS implant is
depicted in Figure 1A.
Prior to PLF surgery, blood samples were collected from sheep jugular veins into
tubes without anticoagulant. Full blood in the volume of 8 mL was supplemented with
0.1 mL of 15 mM CaCl2 solution and mixed with rhBMP6, according to dose, and
then left at room temperature to coagulate. Allogenic devitalized bone particles
(ALLO) of 70-420 µm size were prepared as described  and were added at 0.3 g
per mL of blood/rhBMP6 mix. The mineralized bone particles are distributed evenly
as evidenced by X-ray and micro CT images (see Figure 4A in the Results).
2.3. Anterior lumbar interbody fusion (ALIF) surgical procedure
Female sheep, aged 3–4 years, weighing 50-60 kg were used to test the efficacy
of rhBMP6 in the ALIF procedure after implantation of the CFRP cervical cage filled
with placebo ABC or 150 µg rhBMP6 ABC. Sheep were randomly assigned into two
groups: ABC alone (n=5) and ABGS containing 100 µg/mL rhBMP6 (n=5). The
surgeries were carried out under general anesthesia and performed on all animals by
the same surgical team. Upon excision of the intervertebral disc (L4-L5) and rasping
the cartilage of the end plate, prepared ABGS containing cage was implanted. Fascia
and skin were sutured and disinfected.
Sheep were clinically and radiographically supervised by a veterinarian at four
different stages: immediately after surgery and at weeks 7 and 11, and 16. During the
experiment no adverse effects in any of the animals were observed. The experiment
was terminated 16 weeks post-surgery after sedation and premedication with 5
mg/kg xylapane and 20 mg/kg ketamine i.m. and administration of T61 (0.1 mL/kg)
i.v. The spine segments were excised and fixed in 10% formalin for additional
analysis by X-ray, micro CT and histology.
2.4. Posterolateral lumbar fusion (PLF) surgical procedure
Two separate PLF experiments were performed in sheep. In the first experiment,
5 female sheep were surgically treated with ABC alone (n=1) and with ABGS
containing 62.5 µg/mL (0.5 mg/implant) rhBMP6 (n=4). In the second experiment, 12
sheep were administered with ABGS containing 187.5 µg/mL (1.5 mg/implant)
rhBMP6 and randomly assigned to three groups: 1) ABGS (n=3), 2) ABGS plus
devitalized sheep ALLO (2 g/implant) (n=4) and 3) ABGS plus devitalized sheep
ALLO (2 g/implant) with instrumentation (n=5). Blood samples for implant preparation
were collected as described and two implants per animal were prepared. The
surgeries were carried out under general anesthesia. Spinal fusion was carried out
bilaterally in the lumbar region between L4 and L5 vertebrae. Lateral aspect of
transverse processes was decorticated until bleeding by a high speed burr (Nouvag
AG, High Surg 30, Switzerland) and ABGS implants were placed into the lateral
gutter (see Figure 4A in the Results). Fascia and skin were sutured and disinfected.
Clinical and radiographical supervision was conducted by a veterinarian immediately
after surgery, and at weeks 8 and 27. The experiment was terminated 27 weeks post-
surgery after sedation and premedication with 5 mg/kg xylapane and 20 mg/kg
ketamine i.m. and i.v. administration of T61 (0.1 mL/kg). Spine segments were
excised and fixed in 10% formalin for additional analyses by X-ray, micro CT and
2.5. Methods of Evaluation
Radiographical images were taken before the surgery and at noted time points
after surgery. X-ray imaging of lumbar spine segments was performed using two
standard orthogonal views (lateral and dorsoventral). Samples were scanned by a
Eichermeyer EDR HP (IMD Generators s. r. l., Italy) X-ray machine using the 40 kV
and 8 mA settings with all ionization protection protocols respected during the
imaging and images were processed using an Agfa CR 30-X (Agfa, Japan). All
obtained radiographs from sheep bones were interpreted and scored using the
Denver Sheep Fusion Scale for PLF radiographic grading score system  by a
surgeon and a radiologist blinded to the treatment protocol and postoperative
interval. Denver Sheep Fusion Scale for PLF is based on scoring of new fusion from
0 (no bony response) to 5 (bilateral fusion) .
Micro CT analysis of sheep lumbar spine spanning from L4 to L7 was done using
the SkyScan 1076 micro CT device . Spine samples were prepared for scanning
by trimming the transverse processes in the ALIF experiments and sawing the spines
in half in the PLF experiments. Ex vivo lumbar spine was scanned at the resolution of
18 µm with concurrent analyses of the site of implantation by CTAn (Bruker)
software. Morphometric data for bone quantification included bone volume (BV),
trabecular thickness (Tb.Th) and separation (Tb.Sp). For ALIF experiments new
bone inside the cage and heterotopic ossification outside the cage were analyzed.
For PLF experiment the new bone formation and trabecular bone parameters were
depicted throughout the whole area of newly formed bone, as previously described
[59,60]. Quality of the newly formed bone was compared to the bone in transverse
processes (TP) and vertebral body (VB) by delineating a volume of interest (VOI) with
8.5 mm of height and width, and 0.9 mm of depth in each structure with concurrent
bone parameters analyses as previously described [53,54,60].
Undecalcified histological processing was performed on selected samples
following micro CT. The vertebral body on ALIF specimens was cut by oscillating saw
in the transversal plane to expose the intervertebral disc space, which permitted
central region of the interbody cage/implant and cranial/caudal endplates to avoid
orientational metal beads in the cage. This exposed the region of the cage and
interior of the new bone for further analysis. PLF specimens were dissected in the
transversal plane to expose newly formed bone and adjacent transverse processes.
Each specimen was then dehydrated in graded solutions of ethyl alcohol using an
automated tissue processing system (ASP300S, Leica Biosystems, Buffalo Grove,
Illinois, USA), and cleared manually with methyl salicylate and xylenes before being
polymerized into hardened acrylic resin blocks (MMA). Semi-thin microtome sections
were collected in the transversal plane at a thickness of five microns using tungsten-
carbide knives (D-profile, Delaware Diamond Knives, USA) and an automated sledge
microtome (SM2500, Leica Biosystems, USA). All microtome sections were collected
and mounted on custom prepared gelatin coated (Haupt’s adhesive) glass
microscope slides. Semi-thin microtome sections were deplasticized, hydrated, and
stained with hematoxylin and eosin (H&E), modified Goldner’s trichrome or Von
Kossa / MacNeal’s tetrachrome.
2.7. Biomechanical testing
Specimens of newly formed ectopic bone were randomly selected and dissected
together with transverse processes from sheep that underwent PLF and
biomechanical testing was conducted to determine the maximum force, work to
fracture, and elasticity of newly formed bone. Specimens were grouped to those with
and without instrumentation and then divided in two subgroups (unilateral and
bilateral fusion). The three-point bending instrument (TA.HDPlus, Stable Micro
Systems, UK) used in this study was set with a 50 kg load cell. The bone was placed
on two supports and force was applied perpendicular to the midpoint. Speed was
adjusted at 0.5 mms-1, and force was applied using a single-pronged loading device
with flat-tipped wedge [61,62].
2.8. Data management
Values are expressed as mean ± SEM or SD as indicated. Data distribution was
checked with the Kolmogorov-Smirnov test and according to the results and the small
sample size appropriate non-parametric tests and data description have been used.
For statistical comparison of two groups, a two-tailed Student t-test was used, while
for comparison of more groups two-tailed ANOVA with post hoc Tukey test was used
and P<0.05 was considered significant where indicated. Differences between groups
regarding force, elasticity and work were analyzed with the Kruskall-Wallis test (all
groups together) followed by post-hoc Mann-Whitney U test (comparison between
each two groups). All data have been shown in Box and Whisker's plots. All P values
below 0.05 were considered significant. Statistical software IBM SPSS Statistics,
version 25.0, have been used in all statistical procedures.
3.1. Anterior Lumbar Interbody Fusion (ALIF) Study
Upon termination, the lumbar spine segments were excised, fixed, cleaned of
soft tissue and vertically cut for further analyses. The gross anatomical structure,
presented in Figure 1B, indicated that implant containing ABC alone did not ossify
and fuse fully within the cage (left panel), while the ABGS implant showed a
significant area of ossification and fusion of the L4-L5 vertebral bodies (right panel).
The bone structure in the cage was further confirmed by micro CT and histology at
higher magnification as stained by Von Kossa/MacNeal’s tetrachrome (Figures 1C
and 1D). It was evident that sheep treated with rhBMP6 fused significantly the
vertebral bodies as compared to control ABC group.
Figure 1. Preparation and placement of ABGS implants and analyses of harvested
implants in sheep ALIF model. A. 5 mL of blood was drawn from the jugular vein and
mixed with rhBMP6. The cage was immersed into the blood/rhBMP6 mixture and
allowed to coagulate for 60 min after which it was implanted in between L4-L5
vertebrae. After 16 weeks, the experiment was terminated, and the lumbar segment
of interest was excised and vertically cut through the cage. B. Upon gross
examination, the newly formed bone within the cage (black) did not fuse in ABC
treated samples (yellow bidirectional arrow), while in ABGS filled cages the fusion
was almost complete (unidirectional yellow arrow). C. 3D model of the same
specimens scanned by micro CT. D. Histological analysis of the same specimens
stained by Von Kossa/MacNeal tetrachrome. Blue area marked by bidirectional
yellow arrow indicates a broad unfused area filled with fibrotic tissue in sheep treated
with ABC and almost complete fusion in ABGS treated cage as marked by
unidirectional yellow arrow. The scale bar indicates 2 mm.
In the sheep that had received ABGS, newly formed bone was present in and
outside the cage, and the new bone fused with both vertebral bodies (Figure 2A).
Micro CT quantitative analyses indicated the fused bone volume was significantly
higher in the ABGS treated sheep than in sheep treated with ABC alone. On the
other hand, the heterotopic bone observed adjacent to treated vertebral bodies was
comparable both in ABC and ABGS (Figure 2B). Histology of the ALIF spine samples
is shown in Figure 3A. In the control ABC group, though bone was formed inside and
outside the cage, an area of fibrocartilaginous tissue was present between the cranial
and caudal area of newly formed bone and the fusion between vertebral bodies was
not achieved. In the ABGS group, areas of newly formed bone both inside and
outside the cage were significantly higher and a successful fusion was achieved,
except in a few discrete areas in which clusters of dividing chondrocytes and more
intensively stained cartilaginous tissue were present (Figure 3B)
Figure 2. Micro CT and histological analyses of ALIF sheep specimens. A. In ABC
treated sheep, newly formed bone (yellow arrow) within the cage (red arrows) was
not fused, while in the sheep treated with ABGS the bone within the cage (red
arrows) is almost completely fused (yellow arrow). In the ABGS treated sheep
(middle panel), additional bone ingrowth through the cage (red arrows) gaps (green
arrow) was observed. In the right panels, heterotopic ossification (white arrows) was
observed both in ABC and ABGS specimens. B. Micro CT morphometric analyses
show significant increase of bone volume within the cage of ABGS treated sheep,
while no difference in heterotopic ossification was observed between groups. Data
were presented as box-and-whisker plots with mean, median and all values (n=6)
and analyzed by the Mann-Whitney test.
Figure 3. A. Histology sections of ABC and ABGS implants stained by
Hematoxylin/Eosin. Unfused area, both inside (left panels) and outside (right panels)
of the cage as shown by bidirectional yellow arrows was present in ABC treated
sheep (upper panels). Magnification is indicated with a scale bar (2 mm). In ABGS
treated sheep (lower panels) the newly formed bone was almost completely fused
(unidirectional yellow arrows). VB stands for vertebral body, while CAGE stands for
perpendicularly cut cage through the spine segment. B. Magnification of histology
sections of ABC and ABGS implants from Figure 3A stained by Goldner’s trichrome.
An abundant fibrocartilaginous area was present in the ABC group, while clusters of
dividing chondrocytes with more intensively stained cartilaginous tissue were present
in the narrow areas of progressing ossification in ABGS treated specimens. Black
arrow indicates fibrous tissue; blue arrows indicate cartilage composed of
chondrocytes embedded in cartilaginous extracellular matrix. Magnification is
indicated with a scale bar of 50 µm.
There were no side effects regarding mobility, partial or total paralysis, nerve
irritation and/or pain, decreased food intake and weight loss recorded in animals
undergoing ALIF procedure. One sheep from the control group died due to
pneumonia. The autopsy, performed by a trained veterinary pathologist after 16
weeks, observed no ectopic ossifications, edema, swelling, tumors or any other
visible gross changes. During the entirety of the study, no incidence of infection was
noted. The use of ABGS resulted in significant fusion of two adjacent lumbar
vertebrae, as compared to incomplete fusion in control animals.
3.2. Posterolateral Lumbar Fusion (PLF) Study
ABGS without ALLO at a dose of rhBMP6 62.5 g/mL ABC (0.5 mg/implant) or
rhBMP6 187.5 g/mL ABC (1.5 mg/implant) induced new bone formation and
achieved a complete fusion when harvested at 27-weeks post implantation. ABC
alone implants have failed to induce bone and achieve spine fusion (data not shown).
ABGS with ALLO at 187.5 g/mL rhBMP6 (1.5 mg/implant) implanted with and
without instrumentation also induced new bone formation. Success rate was 88%
without instrumentation, and 80% with instrumentation, as determined using the
Denver Sheep Fusion Scale for PLF . Figure 4B shows the images of radiographs
(top panel) and micro-CT (bottom panel). Morphometric parameters, bone volume
(BV), trabecular number (Tb.N), and trabecular separation (Tb.Sp) of the fused bone
as determined from micro-CT analyses were comparable between the two dose
groups (Figure 4C) of ABGS without ALLO. ABGS/ALLO implants at 187.5 g/mL
showed significantly higher bone volume when implanted without instrumentation as
compared to ABGS without ALLO. However, in ABGS with ALLO implants with
instrumentation the bone volume was reduced significantly (50%) but was
comparable to ABGS without ALLO. Similar to ALIF animals, no side effects were
recorded in sheep undergoing PLF, including any visible morphological changes.
Figure 4. Preparation and placement of ABGS implants and analyses of harvested
implants at 27 weeks after surgery in sheep PLF model. A. ABGS implants with
ALLO particles were produced as described in Materials and Methods; a
representative implant shows that allograft bone particles are distributed uniformly
within ABGS implants as shown by gross, X-ray and micro CT images (left panel). A
photograph of ABGS/ALLO implant (white asterisk) placement in the gutter between
two transverse processes is shown without instrumentation (middle panel) and with
interpeduncular screws and rods (right panel, yellow arrowhead). B. Radiographs
(upper panels) and micro CT (lower panels) analyses from representative ABGS
implants are shown. Note that ABGS implants induced new bone (yellow asterisks)
which achieved a complete fusion between two transverse processes both at a dose
of 62.5 µg and 187.5 µg of rhBMP6/mL of ABC without ALLO, or with ALLO
respectively. ABGS implants also induced new bone formation and lumbar fusion at
a dose of 187.5 µg rhBMP6/mL of ABC with ALLO and using instrumentation (the
utmost right panels). C. Micro CT morphometric analysis of bone volume, trabecular
number and trabecular separation of the treatment regimens as indicated. Results
are shown as mean ± SD (n=6). * P < 0.05 vs. 187.5 µg/mL rhBMP6+ALLO+INS, **P
< 0.05 vs. 62.5 µg/mL (one-way ANOVA with Tukey post-hoc test).
Figure 5A left shows the gross examination of new bone fused between two
transverse processes bilaterally. Figure 5A middle and right panels show the fusion
sites at high magnification. The osseointegration is evident and indistinguishable
without any demarcation at the juncture of the new bone with native bone transverse
processes. Micro-CT analysis further confirmed the quality of osseointegration with
the transverse process as observed by gross anatomy examination (Figure 5B left).
At higher magnification (Figure 5B middle and right panels), it was evident that the
trabeculae of new bone were merging and connecting with the trabeculae of native
transverse bone processes. Bone volume and trabecular separation as assessed by
quantitative micro CT analysis showed the new bone was stronger than the native
bone of transverse process or the vertebral body (Figure 5C).
Successful osseointegration of the newly formed bone with the transverse
processes was further confirmed on histological sections (Figure 5D). Bone marrow
of new bone and bone in the transverse processes differed because the predominant
cells in the bone marrow of new bone were adipocytes, while hematopoietic cells
dominated the bone marrow of transverse processes (Figure 5D). The border
between them was sharp. Based on cell population present in the bone marrow, it
was possible to visualize the fusion line between the new bone and the bone of the
transverse processes. Implanted ALLO was completely resorbed in implants and
there was no pronounced histological difference among the experimental groups.
Figure 5. Photographs of fused bones between two transverse processes from a
representative sheep treated with ABGS/ALLO containing 187.5 µg/mL rhBMP6
bilaterally in PLF model. A specimen was macerated from soft tissues and subjected
to micro CT analysis. A. Gross-anatomical structure showing the complete fusion
between transverse processes and newly formed bone is shown bilaterally (Left
panel). Higher magnification shows the integration of fused bone (white arrows) at
the juncture of transverse processes (middle and right panels). B. Micro CT
confirmation of the fully fused newly formed bone between the transverse processes
at low magnification (left panel). Representative sites for morphometric analysis by
micro CT are shown by TP (transverse processes), NB (new bone) and VB (vertebral
body). The juncture of fused bone with transverse processes at higher magnification
was shown (middle and right panels). C. Morphometric analyses of bone volume (BV)
and structure mode index (SMI) of newly formed bone were compared to transverse
processes and the vertebral body was analyzed by one-way ANOVA with Tukey
post-hoc test. *P < 0.05 vs. vertebral body, **P < 0.05 vs. transverse process. D.
Histology photograph of the new bone (NB) incorporated (black arrow) into the
transverse processes (TP) on undecalcified bone sections 4.5 cm long and 5 µm
thick with Masson Tri-Chrome staining at magnification indicated with the scale bar
(10 mm). In the right panels, TP and NB are magnified (10×) to indicate different
bone marrow contents.
3.3. Biomechanical testing
To examine the biomechanical strength, specimens of fused bones from the PLF
study were randomly selected and the three-point bending test was performed to
determine the maximum force, work-to-break and elasticity. Specimens were
selected to those with and without instrumentation and then divided into unilateral
and bilateral fusion samples. Unilateral failure in two animals was associated with a
broken ABC coagulum in the middle part which occurred during inappropriate
manipulation prior to implantation, but the surgical team’s decision was to proceed
with testing the performance of such implants. The bone volume was 50% higher in
the contralateral implants of those sheep than in sheep with the symmetrical bilateral
implants. The bone volume was adjusted due to mechanical loading through the axial
spine weight bearing transfer (Figure 6). The mechanical strength (as examined by
Force and Work) of the newly formed bone without instrumentation was higher than
the native transverse processes bone. However, with instrumentation the strength of
new bone was considerably lower. These biomechanical parameters were supported
by the micro-CT findings (Figures 6A-B). When spinal fusion was achieved
unilaterally, both maximum force and work-to-break of the newly formed bone were
higher when compared to bilateral specimens (Figures 6D-E).
Figure 6. Biomechanical parameters (three-point bending test) of the samples from
PLF sheep treatment groups. A-C. Comparison of dissected newly formed bone
samples obtained from INS+ and INS- groups with the transverse processes alone
(TP). D-F. Fused bone obtained from bilateral implants as compared with unilateral
implants. Force, work and elasticity have been individually determined and analyzed.
Results are shown as box and whisker plots (n=3-6). * P < 0.05 INS- vs INS+: and
bilateral vs unilateral (Kruskal Wallis test with post-hoc Mann Whitney U test).
We demonstrated for the first time that Autologous Bone Graft Substitute
(ABGS) containing recombinant human BMP6 (rhBMP6) dispersed within autologous
blood coagulum (ABC) achieved a successful lumbar fusion when applied as an
implant inside and surrounding the implanted cage between L4 and L5 vertebra as in
Anterior Lumbar Interbody Fusion (ALIF) procedure. In addition, when placed as a
cylinder shaped implant posteriorly, ABGS resulted in a successful fusion at ectopic
bilateral sites in-between L4-L5 transverse processes as in Posterolateral Lumber
Fusion (PLF) procedure. In parallel, the sheep that received ABC alone failed to
achieve lumbar fusion in both ALIF and PLF models as examined at 16 weeks and
27 weeks, respectively, following the surgery. The newly formed bone and the quality
of lumbar fusion was qualified by radiographs and histology and quantified by micro
CT and biomechanical parameters . In the PLF model, evaluation of ABGS
combined with sheep ALLO particulates used as compression resistant matrix
improved biocompatibility as well as handling properties and resulted in fusion
comparable to ABGS without ALLO. The use of instrumentation to stabilize the
lumbar segments as it is used routinely in the PLF procedures also resulted in fusion
but the amount of bone volume as quantified by micro CT was reduced when
compared to the procedure without instrumentation. The stiffness and lack of motion
with instrumentation may have contributed to this reduced bone volume . It needs
to be noted that we have not yet evaluated the ABGS without ALLO with
instrumentation. It remains to be examined whether the mechanical rigidity has a
negative influence on bone formation particularly in four-legged animals.
BMP, as an injectable drug, is not efficient for local bone formation, so
originally it was delivered with an appropriate collagenous carrier to stimulate
osteogenesis in preclinical studies . Although autologous and/or allogenic
collagenous matrices are preferred to minimize immune insults in humans, the
clinically approved rhBMP2 or rhBMP7/OP-1 based bone graft substitute have
employed animal (bovine) derived collagens. For spine PLF indications, these
collagen scaffolds are combined with highly mineralized Ca-P and/or carboxymethyl
cellulose to achieve acceptable handling properties. These composite implants
invariably result in immunological and foreign-body responses at the local implant
sites. To overcome this unwanted biology, high doses of BMPs have been employed
[19,31]. As BMP2 is known to bind avidly to Noggin, a BMP antagonist predominant
in the bone, and to exhibit a weak affinity to collagen/mineral composites, high doses
of BMP2 (12-40 mg/site) have been employed in preclinical and clinical studies.
BMP2 dissociates at the implant sites resulting in unwanted local and systemic safety
issues and in inconsistent lumbar fusion in humans.
Autologous blood coagulum (ABC) in ABGS serves as a native physiological
carrier for BMP6 and its advantages, as compared to commercially used rhBMP2 on
bovine collagen molecule are: 1) provides circulating blood-borne osteoprogenitors,
2) promotes rhBMP6 binding with plasma proteins tightly within the fibrin mesh-work
and slow release of the intact protein, 3) decreases inflammation and when used with
highly mineralized devitalized ALLO suppresses the formation of multinucleated giant
cells , 4) reduces immune responses and avoids generation of antibodies to
rhBMP6 in rabbits  and humans , and 5) provides a permissive environment
for bone differentiation by its buffering capacity. Additionally, BMP6 binds reversibly
to Noggin, a natural BMP antagonist present in bone, has affinity for most of the type
I and II BMP receptors and has a high specific alkaline phosphatase activity in
osteoblastic cell cultures as compared to BMP2 or BMP7 [55,66,67].
ABGS examined at 100 µg rhBMP6/mL ABC in ALIF and at 62.5 µg
rhBMP6/mL ABC and 187.5 µg rhBMP6 /mL ABC in PLF resulted in successful
fusion. It appears 100 µg rhBMP6/mL ABC is an optimal dose as there is no
significant difference in the quantity of bone formed and the quality of fusion with a
higher dose as seen in the PLF model. Studies using rhBMP2 soaked in bovine
tendon derived absorbable collagen sponge (ACS) and applied within a cortical
dowel allograft or a threaded titanium interbody cage were shown to promote anterior
interbody L7-S1 lumbar fusion at a dose of 1.5 mg/mL concentration in a nonhuman
primate and in sheep ALIF models [57,68,69]. The addition of HA/TCP synthetic
ceramic granules or allograft bone chips with absorbable collagen sponge (ACS) also
was shown to promote fusion with rhBMP2 in the posterolateral lumbar fusion
preclinical models albeit at high doses .
RhBMP2/ACS (INFUSE) has been approved as an autograft substitute for
treating DDD at one level vertebra-disc-vertebra from L2 to S1 using an anterior
approach in skeletally mature patients. Each device contains 12 mg of rhBMP2 (in
total) and includes a sheet of collagen soaked with 6 mg and filled separately in 2-LT
cages that are inserted into the intervertebral disc space. The efficacy of the device
has also been the subject of a number of studies [22,25]. However, the off-label use
to promote spinal fusion with posterior approaches has produced unwanted safety
concerns [25,46]. RhBMP7/Collagen/CM-Cellulose (OP-1 Putty) has also not been
approved for PLF; however, both rhBMP2 (INFUSE) and rhBMP7 (OP-1 Putty) have
been allowed for humanitarian device exemption (HDE) use in PLF where the
autograft is not feasible.
The dose that we found in the current ALIF and PLF studies in sheep is
comparable to the reported dose in our recently published rabbit PLF study  and
in our rabbit ulna study , suggesting the optimal dose of 100 µg/mL ABC is
translatable from small (rabbits) to large animals (sheep). These observations
supported our clinical study design to evaluate ABGS/ALLO with instrumentation in
posterolateral lumbar interbody fusion (PLF) in humans (https://osteoprospine.eu).
The prepared cylinder-shaped ABGS/ALLO implants exhibit a uniform distribution of
ALLO particles across the ABC providing biocompatibility, good handling properties,
and a sustainable release of rhBMP6 over 7-10 days as examined in vitro . This
rhBMP6 release is likely to follow in accordance with the dissolution of blood
coagulum in vivo. A randomized, double-blinded controlled Phase II study on
posterolateral lumbar interbody fusion (PLIF) utilizing ABGS with human devitalized
allograft particulates and is being conducted at the Department of Orthopedics and
Traumatology, AKH University Hospital in Vienna.
We provide evidence that ABGS in which ABC served as a native physiological
carrier for a small dose of BMP6 in ALIF and PLF sheep models. In the ALIF model,
implanted ABGS containing rhBMP6 in ABC/ cervical human cage fused significantly
when placed in between the denuded L4-L5 vertebrae as compared to control
implants for a 16-week period. In the PLF model, ABGS implants either with or
without ALLO showed a complete fusion at a success rate of 88% without
instrumentation and at 80% with instrumentation, as examined by histology,
radiographs, micro CT analyses and biomechanical testing at 27 weeks following
surgery. In the implants that contained ALLO, the mineralized bone particles were
substituted by the newly formed remodeling bone via creeping substitution. We
believe the novel ABGS will provide an acceptable approach to treat spine disorders
For animal studies, we thank to Mirjana Marija Renic and Djurdjica Car for their
excellent technical assistance.
Funding: This program was funded by the European Community’s Seventh
Framework Program [FP7/2007-2013, grant agreement HEALTH-F4-2011-279239
(Osteogrow)], Horizon 2020 [GA No 779340 (OSTEOproSPINE)] and the Scientific
Center of Excellence for Reproductive and Regenerative Medicine [project
"Reproductive and regenerative medicine - exploration of new platforms and
potentials", GA KK01.1.1.01.0008 funded by the EU through the ERDF].
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CRediT author statement
Lovorka Grgurevic: Conceptualization, Methodology, Validation, Formal analysis,
Investigation, Resources, Data Curation, Writing - Original Draft, Writing - Review &
Editing, Visualization, Supervision, Project administration, Funding acquisition.
Igor Erjavec : Methodology, Validation, Investigation, Resources, Data Curation, Writing -
Original Draft, Writing - Review & Editing, Visualization, Supervision, Project
Munish Gupta: Conceptualization, Methodology, Validation, Investigation.
Marko Pecin: Methodology, Validation, Investigation.
Tatjana Bordukalo-Niksic: Methodology, Validation, Formal analysis, Investigation,
Resources, Writing - Original Draft, Writing - Review & Editing.
Nikola Stokovic: Methodology, Validation, Investigation, Resources, Writing - Original
Draft, Writing - Review & Editing, Visualization.
Drazen Vnuk: Methodology, Validation, Formal analysis, Investigation.
Vladimir Farkas: Methodology, Validation, Investigation.
Hrvoje Capak: Methodology, Validation, Investigation.
Milan Milosevic: Methodology, Validation, Formal analysis, Investigation.
Jadranka Bubic Spoljar: Methodology, Validation, Investigation.
Mihaela Peric: Methodology, Validation, Formal analysis, Investigation, Resources, Data
Curation, Writing - Review & Editing, Supervision, Project administration.
Mirta Vuckovic: Methodology, Validation, Investigation.
Drazen Maticic: Conceptualization, Methodology, Validation, Investigation, Supervision.
Reinhard Windhager: Methodology, Validation, Formal analysis, Investigation.
Hermann Oppermann: Conceptualization, Methodology, Validation, Investigation,
Resources, Writing - Original Draft, Writing - Review & Editing, Supervision.
T. Kuber Sampath: Conceptualization, Methodology, Validation, Formal analysis,
Investigation, Writing - Original Draft, Writing - Review & Editing, Supervision.
Slobodan Vukicevic: Conceptualization, Methodology, Validation, Formal analysis,
Investigation, Resources, Data Curation, Writing - Original Draft, Writing - Review &
Editing, Supervision, Project administration, Funding acquisition.
Autologous bone graft substitute composed of autologous blood coagulum as a native
carrier for rhBMP6 induces new bone formation in anterior lumbar interbody fusion and
posterolateral lumbar fusion in sheep spine
In ALIF sheep model rhBMP6/ABC/cage implants fused at 16 weeks following
In PLF sheep model rhBMP6/ABC/allograft implants showed an 88% fusion without
instrumentation and 80% with instrumentation at 27 weeks after surgery
An optimum rhBMP6 dose of 100 µg/ml autologous blood coagulum with and without
allograft was needed for successful PLF in sheep.