Download Bone transplantation and immune response

Journal of Orthopaedic Surgery 2009;17(2):206-11
Review article:
Bone transplantation and immune response
Hamid Shegarfi,1 Olav Reikeras2
1
2
Institute of Basic Medical Sciences, Department of Anatomy, University of Oslo, Norway
Department of Orthopedics, Rikshospitalet University Clinic, University of Oslo, Norway
BASIC SCIENCE
ABSTRACT
Bone is the second most common transplant tissue
after blood, with the iliac crest autologous graft being
most used. Bone transplantation induces osteogenesis
to repair bone defects. Despite being the most
efficient, autogenous bone requires an additional
incision and its supply may be inadequate. Deepfrozen allogeneic bone can be an alternative, but is at
risk of microbiological contamination, transmission
of unrecognised germs, delayed incorporation, and
cellular and humoral immune reactions. Synthetic
graft substitutes combine scaffolding properties with
biological elements to stimulate cell proliferation and
differentiation and eventually osteogenesis. However,
they generally lack osteoinductive or osteogenic
properties and have various effects on bone healing.
We present an overview of bone grafts and graft
substitutes in clinical use, and the immune responses
to allogeneic bone.
Key words: bone substitutes; bone transplantation; immune
system; major histocompatibility complex
Bone cell types
Bone formation (osteogenesis) entails cellular
activity by osteoblasts, osteocytes and osteoclasts.
Osteoblasts possess signalling molecules and act as
mechanosensory cells. Mechanical stress enhances
osteoblastic activity.1,2 Osteoclasts resorb bones and
are multinucleated and formed by the fusion of cells
of the monocyte/macrophage linage. Osteocytes are
mature bone cells accounting for over 90% of adult
bone cells.
Bone structure
The matrix (made by osteoblasts) is the major
constituent of bone that surrounds the bone cells. Bone
matrix has inorganic and organic constituents. The
inorganic components are mainly crystalline mineral
salts and calcium in the form of hydroxyapatite,2,3
initially laid down as unmineralised osteoid.
Mineralisation then occurs through the action of
osteoblasts secreting vesicles containing alkaline
phosphatase. This enzyme cleaves the phosphate
Address correspondence and reprint requests to: Dr Olav Reikeras, Rikshospitalet-Radiumhospitalet Medical Care, University
of Oslo, Norway. E-mail: [email protected]
Vol. 17 No. 2, August 2009
groups from monophosphate ester substrates, which
act as foci for calcium and phosphate deposition. The
vesicles then rupture and act as a nucleus for crystals
to grow on. Transforming growth factor β (TGF-β),
vitamin D metabolites, and parathormone (PTH) are
important mediators of calcium regulation. The organic
constituent is mainly composed of type-I collagen,
which is synthesised intracellularly as tropocollagen
and then exported out of the cell. Tropocollagen then
associates into fibrils. Other components include
glycosaminoglycans, osteocalcin, osteonectin, bone
sialo protein, and cell attachment factor.3
Bone transplantation and immune response 207
la
A
ll
B D
K
-
lll
lb
C/E
N
A/E
D/L/Q
T
D
B/C
E
M
RT1
(Chrom20)
H2
(Chrom17)
HLA
HLA-92
A,G,F
-
(Chrom6)
Figure 1 The major histocompatibility complex gene region
of the rat (RT1), mouse (H2), and human (human leukocyte
antigens, HLA)
Bone and immune system
Bone provides solid support for body structure and is
an organ that acts as a reservoir for critical ions such
as calcium and phosphate. Immune cells (T cells, B
cells, and natural killer [NK] cells) are produced in the
bone marrow. NK and B cells are matured in the bone
marrow. Memory-B and -T cells migrate back into the
bone marrow. The interaction between immune cells
and bone is classified as osteoimmunology.3
Major histocompatibility complex
Major histocompatibility complex (MHC) is a genetic
region important for the immune system and a key
mediator for allogenic transplantation. MHC molecules
are called human leukocyte antigens in humans, RT1
in rats, and H2 in mice (Fig. 1). The MHC gene complex
is divided into 3 sub-regions, the class I, II, and III.
Only class I and II are involved in allotransplatation.
MHC molecules are heterodimeric transmembrane
glycoproteins that belong to the immunoglobulin
superfamily. They are able to present endogenous and
foreign peptides on the surface of antigen-presenting
cells (APC) and communicate with T cells. Acute
rejection is generally mediated by T cell responses
to donor MHC antigens that differ from those in the
recipient (MHC-mismatch). Chronic rejection is due to
a chronic alloreactive immune response. Rejection may
also be initiated by non-MHC genetic factors such as
minor histocompatibility antigens, which are peptides
derived from proteins encoded by polymorphic genes
outside the MHC locus and inherited independently
from MHC molecules.4
Bone transplantation
Transplantation of organ or tissue within oneself is an
autograft, from others is an allograft, from a genetically
identical recipient (such as an identical twin) is an
isograft, from another species is a xenograft, and from
one animal to another within the same inbred strain is a
syngeneic graft.5,6 Allograft rejection (e.g. as occurs in 60
to 75% of first kidney transplants and 50 to 60% of liver
transplants) is a major concern. Bone transplantation
stimulates skeletal repair and regeneration. Bone grafts
have biological and mechanical functions. The biological
activity of a graft includes osteogenesis (inherent
activity), osteoinduction (activity of surrounding host
tissues), and osteoconduction (ingrowth of host tissues).
The mechanical environment affects bone remodelling.
Skeletal graft incorporation is a multifaceted process in
which multiple variables determine success or failure.
The sequence includes haemorrhage, inflammation,
revascularisation, substitution, and remodelling.7,8
Successful graft incorporation is defined as the ability
of the graft and surrounding tissue to function and
maintain mechanical integrity.4 Autologous bone is the
most efficient material and usually harvested from the
iliac crest, but has disadvantages (donor-site morbidity,
inadequate supply, size and shape problems). Allografts
from cadavers are increasingly common, but risk disease
transmission and immune reactions.9,10 Autologous
bone is also superior to allogeneic bone.9,11,12
Immunoreactions to bone grafts
Frozen or cryo bone allo-transplantation is less likely
to result in complications and rejection. Even deepfrozen allogeneic bone (containing no living cells) can
cause complications in 25 to 35% of patients.9 Bone can
be stored frozen for indefinite periods. Sterilisation
and disinfection can reduce the immunologic defence
reactions and the risk of infection without damaging
the bone matrix. Nonetheless, the causes of rejection
remain poorly understood.
NK cells do not participate in the rejection of
solid organ transplants. Cardiac and skin allograft
transplanted into mice with intact NK cells but absent
T and/or B cells do not get rejected and survive
Journal of Orthopaedic Surgery
208 H Shegarfi and O Reikeras
indefinitely.6 However, NK cells are able to recognise
and reject intact allogeneic MHC class I molecules on
foreign cells by their receptors. Unlike T cells, which
can recognise foreign or mismatch MHC molecules
directly or indirectly, NK cells predominately recognise
foreign molecules directly. In bone cryografting, intact
cells are unlikely to survive and thus NK cells do not
have a role in cryoallograft rejection.9,12
T cells express receptors on the surface that are
able to recognise either the whole MHC molecules
(direct recognition) or MHC molecules presenting
non-self peptide on their peptide-binding group
(indirect recognition). MHC is highly polymorphic
and varies between individuals, and therefore
the host T cells that usually recognise the foreign
MHC lead to variably powerful immune responses.
Identical twins and cloned tissue are MHC-matched,
and therefore not subject to T cell–mediated rejection.
On their peptide-binding grooves, MHC class I and II
present peptides of 8 to 9 and 15 to 24 amino acids in
length, respectively. Foreign peptides presented on
the MHC-I and -II molecules can activate cytotoxic
T cells (CD8+ T cells) and T-helper cells (CD4+ T
cells), respectively. They have no cytotoxic activity
and cannot kill infected host cells. They activate
and direct other immune cells such as macrophages
via interferon-γ production, or other cytokines, and
dramatically inhibit bone generation (Fig. 2).13
BONE GRAFT PROCESSING
To reduce immune sensitisation and disease
transmission, superfluous proteins, cells, and tissue
should be removed as much as possible. The most
common methods for preservation are freezing to below
-70º (which has minimal effect on bone properties)
and freeze-drying (which has substantial effect).
Autogenous, cancellous bone is robustly osteogenic
as it contains living osteogenic cells that can induce
differentiation of host mesenchymal cells into osteoblasts
and its structure can serve as an osteoconductive
support for the ingrowth of sprouting capillaries,
perivascular tissue, and osteoprogenitor cells from the
recipient bed. However, it does not provide structural
support. Autogenous, cortical bone provides structural
support and is somewhat osteogenic, osteoinductive,
and osteoconductive. However, revascularisation is
slow and primarily the result of peripheral osteoclast
resorption and vascular invasion of haversian canals.
Therefore, the graft becomes weaker than normal
bone, and large portions of dead cortical autograft may
remain for long periods.
Deep frozen bone retains its material properties
MHC-match
MHCmismatch
MHC-match
presenting foreign
peptide
CD8+
T cell
CD8+
T cell
CD8+
T cell
APC
APC
APC
CD4+
T cell
CD4+
T cell
CD4+
T cell
T-cell
receptor
Peptide
MHC-l
MHC-ll
Peptide
T-cell
receptor
Figure 2 Antigen-presenting cells (APC) to both CD4+ and
CD8+ T cells in major histocompatibility complex (MHC)match, MHC-mismatch, and MHC-match presenting peptide
of a MHC-mismatch molecule.
and can be used after thawing. Freeze-drying alters its
material properties, and thus even after rehydration it
is friable and remains weak in torsion and on bending.14
Cortical allografts provide structural support and
are osteoconductive to a limited degree, but are
not osteogenic or osteoinductive. Vascularisation is
lacking and such grafts sustain fatigue micro-damage
by cyclic loading over time. As necrotic bone cannot
repair itself in response to damage, up to 50% of these
grafts may fracture.
Morcellised and cancellous allogeneic bone grafts
provide limited mechanical support in compression
and are osteogenic only. However, they have an open,
porous lattice-like structure that provides the same
stages of incorporation as in cancellous autografts.
EXPERIMENTS IN RATS
A rat model of tibial allotransplantation was developed
to investigate biometric and histological changes of
fresh versus prefrozen graft and the corresponding
humoral immune responses. Necrosis of the fresh
allogeneic grafts was confirmed by mechanical
testing and radiological signs of resorption, and
Vol. 17 No. 2, August 2009
an alloantibody response was detected. Although
frozen bone allografts impede the antibody response
against MHC antigens and improve incorporation,
they nevertheless perform more poorly than frozen
syngeneic grafts.9
Even though the bone marrow was drained after
2 hours of freezing at -80ºC, it is possible that MHC
peptide proteins from the donor cells (such as osteocytes
deep inside the bone) survive the freezing and can be
targeted by APCs after transplantation. APCs present
these peptides on their MHC-II proteins to CD4+ cells
and lead to a gradual, long-term immune response.
No measurable antibody response was detected after
transplantation of frozen allogeneic bone. If immune
responses against MHC antigens contribute to the
poorer healing of frozen allogeneic (compared with
syngeneic bone), it is likely the mediated cells rather
than humoral responses are responsible.
SUBSTITUTES TO BONE GRAFTING
Dematerialised bone matrix
This is produced through decalcification of cortical
bone. It retains the trabecular collagenous structure
of the original tissue and can serve as a biologic
scaffold without structural strength.15 It possesses
osteoconductive properties, but its clinical results
are not uniformly good,16 owing to the biological
properties of the graft, the host environment, and the
methods for graft preparation.
Bone transplantation and immune response 209
from a variety of materials (including natural and
synthetic polymers, ceramics, and composites)
designed to mimic the 3-dimensional characteristics
of bone tissue.19 Matrices also act as delivery vehicles
for growth factors, antibiotics, and chemotherapeutic
agents, depending on the nature of the injury to be
repaired. This intersection of matrices, cells, and
therapeutic molecules is known as tissue engineering.
The composition of these materials can be adjusted
depending on the specific application of the matrix.
They are osteoconductive but not osteoinductive and
vary in structural support. They are usually supplied
as ceramics or injectable materials that harden in situ.
Scaffold ceramics are mostly made of calcium
phosphate.20 They are osteoconductive but not
osteoinductive, unless growth factors or BMPs are
added to create a composite graft. They are brittle
and have little tensile strength.21 They provide
an osteoconductive matrix for host osteogenic
cells to create bone under the influence of host
osteoinductive factors. After incorporation with the
bone, they gradually acquire mechanical strength
similar to cancellous bone.22 Injectable calcium
phosphate combines cement properties with those
of a void-filler.23 The initial compressive strength
when hardened is similar to that of cancellous bone.
Eventually it undergoes long-term remodelling and
is replaced by new bone.
CLINICAL APPLICATION
Traumatic complications
Bone morphogenetic proteins
Bone morphogenetic proteins (BMP) have
tremendous potential in bone formation at fracture
and non-union sites, and are effective alternatives
or enhancers of autologous bone grafts for bone
regeneration in animal studies.17 Two recombinant
BMPs—rhBMP-2 and rhBMP-7 (also known as
osteogenic protein-1) are available for clinical use.
Each has been evaluated in randomised controlled
trials involving trauma patients.18 The action of
BMP is mediated through receptor kinases and
transcription factors called Smads that regulate
the expression of target genes. More clinical and
long-term experience is necessary to confirm that
treatment with BMPs is beneficial.
Osteoconductive bone graft substitutes
Synthetic graft substitutes, or matrices, are formed
Bone loss and delayed or non-unions are difficult to
treat. Autogenous corticocancellous bone grafting
provides the most effective standard of care.
Implantation of osteogenic protein-1 in combination
with a collagen matrix results in the repair of critical
size defects in long bones.24–26 After debridement and
stabilisation, intramedullary application of BMPs in
combination with bone grafts or bone substitutes has
given good results.27 BMPs appear effective for the
treatment of traumatic complications although the
number of patients is small.
Spinal fusion procedures
Autogenous bone graft from the iliac crest remains the
gold standard because it has all 3 properties essential
for adequate fusion. Demineralised bone matrix
products and synthetic bone grafts (ceramics) were
developed to minimise the invasive harvesting of
Journal of Orthopaedic Surgery
210 H Shegarfi and O Reikeras
autografts28 and to provide scaffolds similar to those
of autologous bone.29–31 They are effective in filling
bone defects, but synthetic bone grafts do not have
osteoinductive properties. Thus, they require the host
bone to be well vascularised to enable availability
of appropriate cellular and osteogenic factors. For
anterior lumbar fusions, the use of rhBMP-2 and
a collagen sponge versus autografts resulted in
equivalent fusion rates and clinical outcomes.32 A
human pilot study using rhBMP-2 with biphasic
calcium phosphate also demonstrated similar results
for posterolateral fusions, with significantly reduced
surgical time and blood loss.33
Revision of endoprostheses
In order to select the appropriate bone graft material,
bone loss around prostheses is classified as cavitary,
segmental, contained, and uncontained.34,35 In typeI revisions, no graft is required. In type-II revisions,
bone graft is required for filling cavitary defects or
reconstructing a segmental bone loss. In type-III
revisions, a large quantity of bone graft is required for
implant stability. Cavitary or contained bone defects
(type II) can usually be treated with morselised
autogenous or allogeneic cancellous bone. Mixing
demineralised bone matrix with the morselised
material may enhance the grafting. Significant
segmental bone deficiency (types II and III) requires
structural or bulk allografts for reconstruction.
Corticocancellous allografts can also be used to
reconstruct large cavitary defects. Segmental cortical
defects of the femur are usually reconstructed with
cortical allogeneic onlay grafts. The cortical grafts
must be securely fixed to the host and cover at
least half the surface of the recipient bone. Major
cavitary defects (types II or III) require an impaction
allografting technique using morselised grafts to
restore bone stock,36,37 but there are no long-term
studies to support this strategy.38
FUTURE
Bone preservation techniques39 (including freezing,
freeze-drying, thimerosal, alcohol, demineralisation,
boiling, and autoclaving) have an impact on the host’s
immune response to the graft. Improved preservation
techniques for donor bones are needed, especially
to exclude any immunogenic peptide longer than 5
amino acids that can evoke immune responses in the
host. Proteases such as trypsin and pepsin are useful
to degrade foreign peptides,40 but may destroy useful
proteins such as collagen. Trypsin and pepsin need
acidic conditions (pH 2–3) to be fully active and this
may be harmful for bone mass. Extensive panel testing
and critical analysis of the experimental system are
needed to determine the right conditions (pH, duration
of exposure, choice of protease and buffer) for successful
destruction of allopeptides during bone grafting.
Collaboration between immunologists, biochemists,
and material scientists is necessary for the selection and
modification of biological and biomechanical parameters
so as to optimise the outcome of allografting.
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