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Abstract
We describe a fifty-year old woman with
nonunion of humerus who had undergone two unsuccessful
operations. Union was obtained by the use of autologous
platelet-rich gel (PRG). Platelet concentration rich in growth
factors is a novel osteoinductive therapy that could achieve
this goal in the treatment of disturbances of bone healing
processes. This article reports the efficiency of percutaneous
autogenic PRG injection into the humeral diaphyseal nonunion gap
as a minimally invasive treatment method offering the advantage
of decreased morbidity associated with the classic grafting
techniques. According to our knowledge, this is the first report
where PRG was used in nonunion treatment.
J.Orthopaedics 2006;3(3)e15
Introduction:
Most diaphyseal fractures of the humerus heal
without secondary surgical intervention. The two most common
methods for fixation of a humeral diaphyseal fracture are plate
osteosynthesis and intramedullary nail fixation [1]. Recently,
rigid humeral intramedullary nails are the most commonly used,
they can be with or without interlocking. When nonunion does
occur, it is likely to be related to the severity of the initial
injury, the transverse pattern of the fracture, distraction of
the fracture, soft tissue interposition or inadequate
immobilization. Despite continued advances in the treatment of
long bone fractures, disturbances of healing processes remain a
difficult challenge [2]. Surgical methods are various and
include open reduction and internal fixation with plates and
screws, reamed intramedullary nailing and external fixation [1].
Supplementing fixation bone graft reliably achieves union.
Autogenous cancellous bone harvested from the iliac crest has
been reported as the most successful material for bone grafting
and alternative substances must be equally successful in
achieving union, as well as providing some increase in benefit,
to justify their use [3].
Platelet concentration rich in growth factors
is a novel osteoinductive therapy that could achieve this goal
in the treatment of disturbances of bone healing processes [4].
Platelet a-granules contain over 30
growth factors. The most important are platelet derived growth
factor (PDGF), transforming growth factor-b
(TGF-b), vascular endothelial growth
factor (VEGF), insulin-like growth factor (IGF) and epithelial
growth factor (EGF) [5]. By concentrating platelets a higher
level of growth factors might be reached which could stimulate
the halted healing processes in bone. The activator for
platelet-rich plasma (PRP) is a mixture of thrombin and calcium
chloride. After connecting these substances platelet-rich gel (PRG)
is formed [6].
This article reports the efficiency of
percutaneous autogenic PRG injection into the humeral diaphyseal
nonunion gap as a minimally invasive treatment method offering
the advantage of decreased morbidity associated with the classic
grafting techniques.
Case Report
A fifty-year old woman after a fall
experienced humeral fracture in the beginning of 2002. The
fracture had been treated using a rigid humeral intramedullary
nail without interlocking and probably with cause an inadequate
immobilization nonunion occurred (fig. 1). The patient started
to attend our ambulatory outpatient clinic in February 2003 and
was qualified to operation. In March the nail was removed and
fixation performed using a rigid intramedullary nail with
interlocking. Unfortunately the nail without interlocking had
been in position to long and was displaced and leaned at the
clavicle inducing a considerable reduction of the range of
motion. Despite this treatment the union did not occur. In
February 11 months after the last operation there was no
radiological evidence about bone healing processes occurring in
the fracture site (fig. 2.) and on 22.02.2004 percutaneous
autologous platelet-rich gel (PRG) grafting into the nonunion
site was performed.
PRG preparation procedure
108 ml of whole blood with 12 ml of
anticoagulant (sodium citrate) was drawn into sterile tubes and
centrifuged. Whole blood was drawn at a rate of 12 minutes, with
a centrifuged speed of 3200 RPM (GPS system, Biomet). The blood
was centrifuged into its three basic components: red blood
cells, platelet-rich plasma (PRP) sometimes referred to as
“buffy coat”, and platelet-poor plasma (PPP). Because of
differential densities, the red blood cell layer forms at the
lowest level, the PRP layer in the middle and the PPP layer at
the top. The cell separator separates each layer from the less
dense to the more dense; therefore it separates PPP first and
PRP second, leaving the residual red blood cells. Then the top
yellow serum component PPP was removed into a 30 ml syringe.
Next the tubes were shaken vigorously for 30 seconds to suspend
platelets and then two 10 ml syringes were connected to the
tubes to extract 12 ml PRP. The PRP application requires
initiating the coagulation process with a mixture of 3 ml 10%
calcium chloride mixed with 4800 units of bovine thrombin. The
PRP was stored at room temperature. After connecting these
substances platelet-rich gel (PRG) was formed.
Surgical procedure
In general anaesthesia an 18-gauge needle was
introduced immediately into the gap of nonunion under
fluoroscopic guidance. By injecting PRG the area was literally
peppered. The second injection into the gap or operation using
bone grafts was not performed.
Following the outpatient procedure, the
participant was followed up on a regular basis with clinical
examinations, roentgenograms, dual-energy x-ray absorptiometry (DEXA)
examinations and functional evaluations. All examinations were
taken at 3 days and 3, 5, 8, 12, 18 and 24 weeks after
percutaneous injection. X-ray films were taken in two views:
anteroposterior and lateral. DEXA examination was performed at
LUNAR DPX once per one visit. The University Ethic Committee
refused its performance more than once in one visit in the
patient. Union was determined on strict roentgenographic
criteria: the patient was considered healed when 75% of the
circumference of the bone at the defect site had resolved.
During the first visit at the 3rd week and
subsequent periods the healing process was noted on
roentgenograms. At the 8th week over 75% of the circumference of
the bone at the defect site had resolved and during later visits
remodeling of the union was observed on X-ray films (fig. 3).
The patient also exhibited an increase in bone mineral density
in DEXA examinations (table 1). A maximum value in all cases was
reached at the 18th week. 12 months after PRG injection
the intramedullary nail was removed. Despite carry on
active rehabilitation the range of motion is lower than normal.
Discussion
The use of growth factors in combination with
tissue engineering seems to be the most promising method in the
future for the treatment of bone and cartilage defect [7].
Growth factors are cytokines with regulatory functions for
healing in tissues of the musculoskeletal system. These small
peptides are synthesised by resident cells at the site of injury
such as mesenchymal stem cells and chondrocytes, or by the
infiltrating inflammatory process. During clotting, platelets
aggregate and release numerous regulatory molecules to the
fracture site such as PDGF, TGF-b, VEGF,
IGF and EGF. They play an important role in the initial phase of
the healing process [8]. It is generally accepted that once
cells enter the wound, their proliferation and differentiation
are most likely to be determined by the type and level of growth
factor presented in the site of the fracture [7,8].
Table 1. Bone mineral density
(BMD) increasing.
|
|
Values
(%) |
|
BMD increasing
– 3th week |
14 |
|
BMD increasing
– 5th week |
28 |
|
BMD increasing
– 8th week |
50 |
|
BMD increasing
– 12th week |
83 |
|
BMD increasing
– 18th week |
112 |
|
BMD increasing
– 24th week |
98 |
Autologous bone graft potentially contributes
three vital local components: osteoconduction, osteoinduction
and osteogenic cells. According to some authors to reach
successful in the treatment of disturbances of bone healing
process, an alternative technique must also provide these
osteogenic components. However, surgical procedures requiring
harvesting of the iliac crest bone graft introduce a variety of
potential risks to the patient, including increased surgical
time and stay in hospital, increased blood loss and
postoperative pain, infection, scar, etc [3]. For this reason
many surgeons perform a secondary operation of nonunion with
using bone marrow as a low-invasive method. Connolly [9] and
Haley et al. [10] have demonstrated that percutaneous injections
of autologous bone marrow can successfully treat between 78% and
95% of nonunions. In more bone defects or persistent nonunions
to increase the possibility of the rate of success many authors
connect osteoconductive biomaterials, such as demineralized bone
matrix (DBM) which possesses also osteoinductive properties,
with bone marrow which dominates osteoinductive and osteogenic
properties. This mix can substitute autograft with success [3].
However, the level of the osteoprogenitor cells in aspirated
bone marrow is totally different. It ranges between 12 and 1224
progenitors/cm2 [11]. That is why nowadays, application of the
concentration of active substances begin to be in common use.
Hernigou et al. injected bone marrow concentration into a gap of
noninfectious atrophic nonunion. They achieved union in all
cases where obtained concentrate with high density of
osteoprogenitors cells were >1500/cm3. They mentioned that the
fibrous tissue interposed between the bone ends ossified after
the injection of bone marrow. However, they could not explain
the exact mechanism that allows the transformation of fibrous
tissue into callus [11].
The autogenous percutaneous bone marrow
injection offers the advantage of decreased morbidity associated
with the classic open grafting techniques. Additional advantages
are decreased cost and hospitalization. Although autologous bone
marrow harvesting is thought to be a relatively simple
procedure, it has not been without complications. Donor site
problems include bleeding, hematoma, chronic pain and infections
[9]. The time of operation including harvesting and application
is too long to use general short-term intravenous anaesthesia.
The use of platelet-rich gel (PRG) to enhance
bone regeneration and soft tissue maturation has increased in
the fields of maxillofacial surgery over the last decade
[12,13,14]. Autologous platelets as a source of healing factors
have been shown to promote tissue repair in several clinical
situations in orthopaedic surgery. Lowery et al. used PRG with
allogenic grafts in lumbar spinal fusion with good results. They
observed no radiological or clinical evidence of pseudoarthrosis
in all patients [15]. Kitoh reviewed clinical results of
distraction osteogenesis with transplantation of marrow-derived
mesenchymal stem cells (MSCs) and PRG in 3 patients. A mixture
of PRP and osteoblast-like cells were injected into the callus.
In one case they had to increase the distraction to 1,5 mm/day
between 34th and 47th because callus formation was likely to
consolidate prematurely. Enhanced callus formation was observed
radiographically after the first transplantation of MSCs and PRG
in all three cases. However, they connected 2 osteoinductive
biomaterials and we do not know exactly the influences of PRP on
bone formation in vivo [16].
To estimate bone healing processes we used
X-ray and DEXA examination. During the first visit at the 3rd
week and subsequent periods healing processes were noted on
roentgenograms. At the 8th week over 75% of the circumference of
the bone at the defect site had resolved and during later visits
on X-ray films remodeling of the union was observed. Many
authors have reported that DEXA is an accurate, reproducible and
non-invasive technique for measuring mineral density [17,18].
The patient also exhibited an increase in bone mineral density
in DEXA examinations. At the 18th and 24th week the BMD in union
site was higher than above and below its. Unfortunately we did
not receive agreement from the University Ethic Committee for
performing more than one DEXA examination during one visit, so
we can not estimate a precision error and repeatability of
examination.
Platelet-rich gel dominates only
osteoinductive properties [19,20], but our investigation showed
that percutaneous injection in this case is a sufficient method
to obtain union, which is less invasive procedure than bone
marrow injection. By using special dual-caniule applicator PRP
and thrombin with calcium ions are injected into the delayed
union site. PRG comes into being by connecting PRP and thrombin
during 5-25 seconds and the liquid can reach even a small gap
before gelatinous mass will be formed [6].
Our results confirm that percutaneous
autologous platelet-rich gel grafting could be an effective and
safe method for the treatment of nonunion. In contrast to PRG,
recombinant growth factor such as BMP-7 is expensive and
cartilage-derived morphogenetic protein is not available for
clinical use [21]. We believe that it will be in common use in
orthopedics in persistent bone disorders [22]. According to our
knowledge, this is the first report where PRG was used in
nonunion treatment and further trials are needed to determine
whether platelet gel will be able to induce healing processes in
nonunion.
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