Bone 140 (2020) 115548
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Bone
journal homepage: www.elsevier.com/locate/bone
Review Article
Discovery of bone morphogenetic proteins – A historical perspective
a,⁎
T
b
T. Kuber Sampath , A. Hari Reddi
a
perForm biologics Inc., Holliston, MA, United States of America
Lawrence Ellison Center for Musculoskeletal Regeneration, Department of Orthopedic Surgery, School of Medicine, University of California at Davis, Sacramento, CA,
United States of America
b
A R T I C LE I N FO
A B S T R A C T
Keywords:
BMP
Bone repair
Review
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.
1. Early history
Bone has natural capacity for healing upon fractures. The use of
antiseptic decalcified bone has long been realized [1,2] for the construction of bone following osteomyelitis and applied it to repair bone
deformities. Huggins [3,4] demonstrated for the first time an experimental means of inducing new bone at ectopic non-bony sites and
showed that urinary bladder epithelium when implanted under abdominal fascia in dogs resulted in osteoblast differentiation locally as
evidenced by the presence of high alkaline phosphatase activity and
accumulation of hydroxyapatite containing mineralized nodules. He
described this phenomenon as “Epithelial Osteogenesis”. This finding
showed that “live” epithelium provided stimulus (signal) for the adjacent connective tissue fibroblast-like cells (cells) to respond under a
permissive tissue-matrix environment (scaffold) to form bone. It has
now been realized that three biological principles required for tissue
engineering are: signals, cells and scaffold.
2. Bone induction by demineralized bone matrix
Urist discovered that demineralized bone is capable of inducing new
bone when implanted ectopically in the skeletal muscle [5] and he
described this phenomenon as “Bone Formation by Autoinduction”. His
observation was the first proof that acellular demineralized bone matrix
has a bone morphogenic activity as it is capable of inducing new bone
at ectopic sites. Urist and Huggins showed that demineralized dentin
matrix is also capable of inducing new bone in vivo [6]. Urist and Strates
⁎
later named the bone- and dentin- derived substances as “Bone Morphogenetic Proteins” [7]. Independently, it was shown that allogenic
demineralized bone matrix when implanted in a rat subcutaneous site
initiates a cascade of cellular events mimicking the biological processes
of embryonic bone formation [8,9]. This matrix-induced new bone
formation involves migration, proliferation of fibroblast-like mesenchymal stem cells and their differentiation into chondrocytes to form
cartilage as a transient tissue which upon calcification and concurrently
with vascular ingrowth formed new bone and bone marrow at predictable time intervals in a reproducible manner. As a result, it was
believed that bone matrix likely contained a set of morphogenic proteins responsible for the cascade of cellular events associated with
matrix-induced new bone formation.
3. Discovery of bone morphogenetic proteins
The progress in identifying bone morphogenetic protein from the
demineralized bone matrix has been slow due to the difficulty in isolating protein from the insoluble bone matrix and not having a defined
bioassay for in vivo bone formation. However, it was eventually demonstrated that proteins responsible for bone formation could be solubilized from the demineralized bone matrix by dissociative extraction
using 4 M guanidium chloride or 8 M urea containing 1 M NaCl or 1%
SDS at neutral pH over 16 h. The extracted proteins could be reconstituted with an insoluble residual inactive collagenous matrix and assayed their bone forming ability in a rat subcutaneous site [10] (Fig. 1).
In this dissociative extraction and reconstitution assay, the
Corresponding author.
E-mail address: kuber.sampath@performbiologics.com (T.K. Sampath).
https://doi.org/10.1016/j.bone.2020.115548
Received 24 June 2020; Received in revised form 3 July 2020; Accepted 6 July 2020
Available online 27 July 2020
8756-3282/ © 2020 Elsevier Inc. All rights reserved.
Bone 140 (2020) 115548
T.K. Sampath and A.H. Reddi
Fig. 1. A copy of the discussion note describing the steps to perform dissociative extraction and reconstitution and the data obtained from the rat subcutaneous
implants on Day 12. Sampath TK, Reddi AH. (1981) Proc Natl Acad Sci USA 78:7599–7602.
solubilized bone extract provided the signals and the inactive residual
collagenous matrix served as scaffold and the subcutaneous ectopic site
recruited the responding mesenchymal stem cells to undergo proliferation and differentiation into endochondral bone formation. This
advance for the first time provided not only a reproducible bioassay for
bone morphogenetic protein but also helped to qualify that the bone
forming activity is homologous among the mammals [11]. These findings permitted the isolation, characterization and identification of
several bone morphogenetic proteins from bone matrix extract.
By utilizing the amino acid sequences obtained from enriched bone
inductive protein fractions isolated from bovine bone [12], several
genes encoding “putative” bone morphogenetic proteins (BMP1, −2,
−3 and −4) were identified by molecular cloning techniques. Of them,
BMP2, −3 and −4 were members of the TGF-beta family of proteins.
BMP1 was a mammalian tolloid proteinase responsible for processing
extracellular matrix proteins like collagens and certain member of the
TGF-beta family of proteins including TGF-beta and GDFs, and was later
found to be a contaminant. Independently, the bovine osteogenic protein was highly purified and shown to be composed of homodimers of
an 18 kDa protein and a 16 kDa protein [13] (Fig. 2).
The purified bovine osteogenic protein by SDS-PAGE gel and 125Ilabeled, was divided in half and electrophoresed either untreated
(oxidized) or after reduction (reduced & pyridylathylated).
Based on amino acid sequences obtained from these highly purified
bovine osteogenic proteins, a relationship to Drosophila DPP and
Xenopus Vg-1 was established. As a result, a consensus gene construct
was assembled and used to obtain the OP-1 gene for the first time [14].
The OP-1 gene was later renamed as BMP7 [15]. Subsequently, the 18
and 16 kDa proteins were identified as homodimers of OP-1 and BMP2,
respectively [13]. Later, several additional BMP-related genes were
cloned [16] from human cDNA and genomic libraries using oligonucleotide probes whose constructions were based on known BMP gene
sequences. These genes were called Growth and Differentiation Factors
(GDFs). Independently, other researchers have identified a set of morphogenetic proteins called cartilage-derived morphogenetic proteins,
CDMPs (also called CDMP1/GDF-5/BMP11; CDMP-2/GDF-6/BMP12;
CDMP-3/GDF-7/BMP13) as they were shown to be expressed predominantly in cartilage [17].
Fig. 2. Purification of highly purified Bovine Osteogenic Protein.
4. BMP structure and function
Like for all members of the TGF-beta superfamily, BMPs are synthesized as a large precursor and then processed as a mature disulfide
linked dimer. For a BMP to be active dimerization is a requirement
[18,19]. The products approved for clinical use employ mature disulfide-linked BMP homodimer applied locally in combination with a
collagenous scaffold. BMP exerts its function by binding to a specific
Ser/Thr kinase receptor heterodimeric complex composed of one type I
receptor and one type II receptor [20–22]. The binding of BMP ligand to
2
Bone 140 (2020) 115548
T.K. Sampath and A.H. Reddi
A
B
C
D
Fig. 3. Photomicrographs of the OP-1/BMP7 induced endochondral bone formation in the rat subcutaneous implants (stained with toluidine blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
they are expressed in tissues other than bone [31,32]. BMPs serve as
inductive signals for a number of tissues during organogenesis, suggesting that they may have therapeutic utility in tissues other than
bone. For example, BMP2 has a developmental role in cardiac tissue
[33], BMP4 (a structurally close member to BMP2) in lung development
[34] and BMP7 in kidney morphogenesis [35,36]. The loss of BMP6
exhibits hemochromatosis [37] and gain of BMP6 function results in
anemia through disturbance of the iron-hemojuvelin-hepcidin loop. The
loss of GDF-8 results in enhanced skeletal myogenesis with high metabolic activity exhibiting a lean phenotype [38], whereas GDF-11
(closely related to GDF-8) appears to have a positive role in the ageing
process [39].
its receptor complex may also be facilitated by extracellular matrix
proteins like Type IV collagen and Heparan Sulfate proteoglycan and co
receptors that include Endoglin and Hemojuvelin. The ligand-receptor
complex subsequently induces the phosphorylation of intracellular effector proteins SMAD1/5/8 to mediate downstream signaling. The
binding of BMP to its receptor complex is tightly controlled in the extracellular milieu by endogenous antagonists [23] (e.g., Noggin).
Recombinant human BMP when implanted with allogeneic collagenous matrix is capable of inducing new bone at ectopic sites in a
dose-dependent manner [24]. BMP2 [25,26], OP-1/BMP7 [27,28] and
BMP6 [29,30] all have been shown to induce new bone formation in the
rat subcutaneous implants, restore large segmental defects in rabbits
when implanted with a collagenous scaffold. The doses employed vary
based on the specific BMP and the substratum used as carriers. The
efficacy of BMP2, −6 and −7 has been shown to exhibit a comparable
bone forming activity in the rat subcutaneous implant assay and this
activity is dose-dependent. A single BMP is sufficient to induce the
cascade of cellular events leading to the formation of new bone at ectopic sites, an example was shown for OP-1/BMP7 in Fig. 3, as well as it
elicits this response in diaphyseal segmental defect models of small and
large animals [27,28].
A, negative control (Day12), rat bone collagenous matrix (25 mg) as
carrier. B, rat collagenous carrier consituted with 125 ng of rhBMP7
(Day 7), evidence of chondrogeneis is seen. C, rat collageneous carrier
with rhBMP7 (Day12), note the extensive bone formation and remodeling. D, rat collagenous carrier plus rhBMP7 (Day21), note hematopoietic bone marrow differentiaitn in the newly formed ossicles.
While BMPs are capable of forming bone at ectopic and bony sites,
5. BMP clinical studies
Several clinical trials have been conducted to assess the safety and
efficacy of recombinant BMP containing devices for the treatment of
acute diaphyseal bone fractures, delayed union, tibial nonunion and
spinal fusion. Two BMP products, rhBMP2 (InFUSE® [40] and Amplify®) [41] and rhBMP7 (OP-1 Implant® [42] and OP-1 Putty® [43])
are licensed under a PMA and HDE, respectively, for marketing and
clinical use in the U.S.
BMP, as an injectable drug, is not efficient for local bone formation.
It therefore required delivery using an appropriate carrier to induce
new bone formation at local implant sites. Collagenous matrix not only
serves as a carrier for BMPs but also acts as a substratum to facilitate
mesenchymal cell recruitment and proliferation and subsequently differentiation into endochondral bone formation. While it is preferred to
3
Bone 140 (2020) 115548
T.K. Sampath and A.H. Reddi
use autologous or allogenic collagenous matrix as carrier/substratum to
minimize immune responses in humans, thus far the clinically approved
BMP-based osteogenic devices have routinely employed xenogeneic
(bovine-sourced) collagenous matrices and/or bovine collagen-mineral
composites. The collagen-mineral composite carrier exhibits a weak
affinity for rhBMPs and invariably provokes an inflammatory and foreign body giant cell responsiveness at the implant site, due to the high
mineral content. In order to overcome these insults, high doses of
rhBMPs were employed. For example, rhBMP2 is applied in 12–40 mg
doses for single-level posterolateral lumbar fusion [41,44] resulting in
local and systemic safety issues and an unwanted clinical outcome.
A preferred carrier for BMP would be an autologous physiological
carrier which does not provoke an inflammatory and immune responses
and exhibits a high affinity for BMPs. Hence low doses of BMPs could be
employed to induce bone formation without causing any unwanted
safety issues. Recent studies have shown autologous blood coagulum
(ABC) can serve as a physiological carrier for BMPs. An autologous
bone graft substitute (ABGS) that contains rhBMP6 and ABC was capable of inducing new bone formation in a rat subcutaneous site [29],
repairing diaphyseal segmental defect in rabbits [29] and promoting
posterolateral lumbar fusion in rabbit [30] and sheep [45] models.
Furthermore, it was shown ABC overcomes an inflammatory and foreign body responses when it was used with high mineral content allograft and synthetic ceramics in vivo [45,46]. These findings led to the
evaluation of ABGS for safety and efficacy in a Phase I study in patients
with Distal Radius Fractures [47] and a Phase I/II study in patients with
High Tibial Osteotomy [48].
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6. Conclusion
BMPs were purified from bovine bone extracts employing a subcutaneous implant assay for bone induction. By utilizing the primary
amino acid sequences obtained from purified bovine bone inductive
proteins, numerous BMP genes were identified from human cDNA and
genomic libraries. They are called BMPs, CDMPs and GDFs and constitute a large member of the TGF-beta superfamily of proteins. BMP
proteins are highly conserved from fly to humans. Furthermore, they
are expressed in many organs during embryogenesis and in part can be
recapitulated during adult tissue repair. BMPs signal through a set of
specific Ser-Thr kinase receptors and act under the influence of a concentration gradient, which is governed by extracellular matrix proteins
and a family of BMP antagonists. Though recombinant BMP protein
containing osteogenic devices are approved for therapeutic use in orthopedic medicine, there are numerous challenges due to the high doses
employed suggesting need for a physiologically acceptable scaffold. The
demonstration that autologous blood coagulum can serve as a physiological carrier for BMPs suggests that some of these challenges could be
overcome in the future.
Declaration of competing interest
TKS is Co-Founder and CEO of perForm biologics Inc., and has
ownership in the company, and AHR has no conflict of interest.
Acknowledgement
TKS thanks David C. Rueger for research collaboration in the purification of bovine osteogenic proteins when both worked at Creative
BioMolecules, Inc.,
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