Cell Tissue Bank (2010) 11:233240

DOI 10.1007/s10561-009-9151-2

Multi-variety bone bank in China

Dan Li Long Bi Guo-Lin Meng Min Liu
Jing Jin Yin Liu Zhen Wang Jian Liu
Yun-Yu Hu

Received: 3 December 2008 / Accepted: 17 August 2009 / Published online: 19 September 2009
Springer Science+Business Media B.V. 2009

Abstract This is a descriptive report of the establishment and operation of a Chinese bone bank,
though not a typical one. While being engaged in
collection, processing and storage of allogeneic
tissues, the bone bank to which the author belongs
concurrently develops and produces new, non-human
derived, graft materials. Among others is reconstituted bone xenograft (RBX) which possesses strong
osteoinductive capability without evoking immune
rejection. Hence, its appellation multi-variety bone
bank, which was established by Dr. Hu Yunyu in
1990, the first of its kind in China. There are several
salient features discriminating this bone bank from
others. At this hospital-based non-profit institution,
allograft hemi-joints are freshly prepared and distributed deep-frozen, instead of being freeze-dried on an
industrialized basis for convenient transportation.
The former has much more superior biological and
mechanical properties as compared with the latter.
However, allogeneic tissues are sometimes in short
supply due to limited number of donors and the risk
of some potential donors carrying viruses such as
human immunodeficiency virus (HIV), hepatitis B
virus (HBV), or hepatitis C virus (HCV). New graft

D. Li (&)
L. Bi
G.-L. Meng
M. Liu

J. Jin
Y. Liu
Z. Wang
J. Liu
Y.-Y. Hu
Institute of Orthopaedics & Traumatology, Xijing
Hospital, The Fourth Military Medical University,
Xian, Peoples Republic of China
e-mail: bonebank@fmmu.edu.cn

materials, including reconstituted bone xenograft

(RBX), were developed that serve as a supplement
to allografts. RBX has been successfully used in
clinical practice for the management of old fractures,
nonunions and bone defects, most notably of contaminated, infected open fractures and osteomyelitis
with the use of anti-infective reconstituted bone
xenograft (ARBX). Additionally the multi-variety
bone bank serves as a training base for educating
professional personnel and researchers (postgraduates) in theories and technologies of tissue banking.
Up to now, eighteen special technical staff members
and approximately sixty senior researchers have been
trained at this institution.
Keywords Bone banking
Bone allograft

Bone xenograft
Quality control

Introduction
The earliest attempts to use frozen bone grafts date
back to 1910 when Bauer experimented with frozen
dog bone grafts. However, it was not until 1942 when
Inclan first described a protocol to create a bank of
frozen human bone grafts (Inclan 1942). In 1949 the
first full service tissue bank, named the United State
Navy Tissue Bank, was established in Bethesda,
Maryland by Dr. George W. Hyatt based on the
experiences of treating the wounded during the

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Second World War. During the Korean War, the US

Navy Tissue Bank provided a center for the procurement, processing, storage, shipment, and distribution
of frozen or freeze-dried tissue allografts (especially
bone and skin) in order to allow tissue transplantation
during wartime. Moreover, many patients were also
treated in their medical facility during peace time
(Strong 2000). For the next 50 years, tissue banks
gradually developed in other countries and bone
banks of various sizes were established in Japan,
Britain, Canada, Russia, France, Poland and China. In
order to set the standards for tissue banking procedures and techniques, many organizations such as the
American Association of Tissue Banks (AATB), the
European Association of Tissue Banks (EATB) and
the Asia Pacific Association of Surgical Tissue Banks
(APASTB) were founded. These institutions also
provided professional training and organized the
procedure for requesting tissue donations.
Bone allograft transplantation in China was developed at the same time as that in Europe and the US,
however, the length of time elapsed between actually
storing the tissues and creating a bone bank was
relatively brief. The first tissue bank of China was
established in Taiyuan City in the Shanxi province in
1988 with the funding and support of the International Atomic Energy Agency (IAEA) by Radiation
Protection Research of China. Shanxi Medical Tissue
Bank, the first of its kind in China, adopted many
advanced technical and administrative ideas that have
been popular in Western countries, thus setting an
example in the development and operation of tissue
banks in this country. It was at this time when China
was advancing the policy of reform and opening to
the world. Chinese scientists and scholars, who were
focusing on research in this field, marched outside of
the country to pursue new knowledge and widen their
vision of their specialty.
At the same time, research on bone allograft
transplantation and bone banking had also begun in
the Institute of Orthopedics & Traumatology in
Xijing hospital, the Fourth Military Medical University of China. Under the leadership of Professor Hu
Yunyu, a well-known orthopedic specialist, we
carried out not only the procurement, processing,
and storage of bone allografts, but also advanced
basic research on bone xenografts (Hu et al. 1993).
After 2 years of preparation, the first multi-variety
Bone Bank of China was established as a basic

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Cell Tissue Bank (2010) 11:233240

allograft bone bank in our institution (Hu 1995).

From the time of establishment, the functions of this
Multi-variety Bone Bank have been determined to be
the following: (1) the procurement, processing, and
storage of bone allografts, (2) the processing and
production of bone xenografts, (3) the development
of new type of bone transplantation materials (such as
artificial bone substitute), (4) basic research on tissue
engineering, and (5) professional training of technical
staff and advanced research fellows.
Professor Hu YunYu, the founder of the bone bank
and inventor of RBX, visited several institutions
in Hong Kong, the US, Japan and France, giving
lectures and making academic exchanges on RBX
and related research. The Technical Manuals and the
Standards for Tissue Banking published by the
American Association of Tissue Banks and Standards
of the American Red Cross are held as guidelines for
operation at this institution. Soon afterwords, BeiJing
Jishuitan Hospital, Shanghai 9th hospital and Huaxi
Hospital at Sichuan University successively set up
their own bone banks, and Hubei Joint Bone Bank
was established in Wuhan City. At the same time,
several laws and regulations with respect to organ
donation and tissue transplantation were laid down
and put into practice by the central and local
governments in China, with all transplantationrelated procedures being put under strict surveillance
of the Ministry of Health and State Food and Drug
Administration.
As tissue banks began to develop in China, quality
control was instituted and strictly enforced as a
prerequisite for all procedures from procurement and
processing to storage. Now most of the tissue banks
in China adhere to the guidelines formulated by
AATB and EATB, though each bank has set a quality
control system of its own. Quality control in a tissue
bank begins with screening of donors, during which
time the personnel of the tissue bank are not allowed
to contact the potential donor. There are two legal
ways to obtain the information about a potential
donor, one is from the local red cross association, the
other being from the donor center of some medical
universities. The first course of action is to obtain
consent from the donor or the donors family. Once
the donors give their consent, they will be allocated a
specific donor number for identification. The ages of
male donors should be younger than 55 years, while
females should be younger than 50. The donors

Cell Tissue Bank (2010) 11:233240

medical and social histories are carefully examined,

and those who are afflicted with the following
diseases should be excluded: septicemia, systemic
viral disease (HIV infection and viral hepatitis),
untreated syphilis, clinically active tuberculosis,
leprosy, systemic mycosis, rheumatoid arthritis, systemic lupus erythematosis, polyarteritis nodosa, sarcoidosis and clinically significant metabolic bone
disease. At the time of tissue procurement, a serum
sample of sufficient quantity is obtained and used to
perform routine serological tests. Major tests commonly identify: HIV 1 and 2 antibodies, hepatitis B
surface antigen, hepatitis C antibody, and syphilis.
Samples taken from the harvested tissue are also
tested for aerobic and anaerobic bacteria contamination. Additional tests for HIV such as p24 antigen and
polymerase chain reaction (PCR) are also quite
commonly employed. For deceased donors, tissue
harvesting must be executed in a sterile operating room
as soon as possible after the death of the donor, within
24 h of death when the body has been refrigerated or
within 12 h when it is not refrigerated. As is the case
with most tissue banks in China, the bone bank of ours
adopts a multi-step approach to reducing the bioburden
of the graft materials in order to ensure their safety and
utility when used. With that objective in mind, we
practice strict aseptic manipulations while processing
the product, including environment monitoring, regular washing and disinfection of the manipulation room,
use of aseptic technique, and packaging in a laminar air
flow room. The graft materials are freeze-dried or
deep-frozen at -80C. All the end products are
terminally sterilized by exposing them to a dose of
25 kGy of gamma radiation.
Quality control is an important responsibility of
the bone bank (Kostiak 2000; Nather 2004). Although
all the larger bone banks in China have set up criteria
for quality control according to the standards of
AATB or EATB (Li and Sun 2004), there is currently
no state standard for bone banks in China. Establishing this requires advanced understanding of the
proper technique, environment, and equipment as
well as management of a multi-variety bone bank. As
one of the main centers, our bone bank is entrusted to
draft the basic technical standards for bone banks in
China. This standard has often referred to the
standards of AATB and EATB. After passing the
evaluations by the related specialists, the drafted
standard should be published soon as a state standard.

235

Allografts
The procurement of allografts
Since 1998, sixty-nine cadaver donors (56 male and
13 female) with an average age of 36 were tested
according to the standards of AATB. Six donors were
excluded for HBV (2 donors were HBsAg positive, 1
donor was HBsAg and HBcAb positive, 1 donor was
HBsAg, HBcAb and HBeAg positive), HCV (1 donor
was HBsAg and HCV-Ab positive) and syphilis (1
donor was syphilis-Ab positive) infections, leading to
an exclusion rate of 8.7%. There were no cases of
positive HIV detection. Ligaments, tendons and long
bones with articular facets from the donors that met
the preset standards were procured in a sterile
manner. Except for the cranial bone, other bones
including the rib, pelvis and centrum were procured
cleanly.
The femoral heads obtained from hip replacement
operations were the main sources of bone from living
donors. Six of 88 living donors were excluded for
HBV infection (HBsAg positive), giving an exclusion
rate of 6.8% from this donor pool.
A total of 480 osteoarticular allografts (hemijoint), 2,632 packages of cancellous bone granules,
237 packages of bone plates, and 51 packages of bone
blocks were procured or produced from the 145
screened donors according to the AATB standards.
The hemi-joints were cryopreserved and the other
types of allografts were stored after being freezedried and irradiated for sterilization.
The application of allografts
The osteoarticular allografts stored at -86C were
used primarily in limb salvage procedures for malignant bone tumors. Cases from 1998 to 2007 are
shown in Fig. 1. The number of cases from 1998 to
2001 was higher than those for later years.
The implanted positions of the hemi-joint allografts are shown in Fig. 2: distal femur (DF) 24.07%,
proximal femur (PF) 20.37%, proximal tibia (PT)
21.30%, distal tibia (DT) 6.48%, proximal humerus
(PH) 16.67%, ulnar and radial (UA) 5.1%, and others
(TO) 5.38%.
Allografts applied in different diseases are shown
in Fig. 3. Most numbers (43%) of the allografts were
used during bone tumor operations.

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Fig. 1 Cases of hemi-joint allografts applied during the

operations from 1998 to 2007

Fig. 2 The ratio of different hemi-joint allografts

Fig. 3 The ratio of allografts applied in different diseases

The knee joint is the most common site of

malignant bone tumors, and the proximal femur is
the most common site of transferred cancers (Pardasaney et al. 2006). When these tumors occur, either
a segmental bone allograft or a segment bone
allograft with artificial prosthesis is an effective
alternative (Canadell et al. 1994; Finn and Simon
1991). The preparation, storage and application of

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Cell Tissue Bank (2010) 11:233240

hemi-joint allograft were the characteristics of our

multi-variety bone bank and the institute. All the
bone allografts were procured in a germ-free environment. The periosteum, soft tissue and bone
marrow were removed in a class 100 clean room.
All the hemi-joint allografts were soaked and washed
with normal saline supplemented with antibiotics
before they were triple vacuum packaged and cryopreserved. A total 480 hemi-joint allografts have been
applied and used in limb salvage procedures for
malignant bone tumors over the years. For the hemijoint allograft, the knee joint, proximal femur and
proximal humerus, which are the most common sites
for bone tumors, were used for 83% of the allografts.
Liu JiZhong et al. reviewed 90 patients with malignant bone tumors who had undergone segmental
resection followed by transplantation with deepfrozen allograft from May 1992 through January
1999 (follow-up period 1889 months, mean
37.5 months). The results showed that skin necrosis
(11 patients, 12.2%), early infection (6 patients,
6.67%), fracture of allograft (3 patients, 3.35%) and
nonunion (10 patients, 11.1%) were the main complications that prevented functional recovery of the
affected limb. 73.3% of the 90 patients had excellent
or good limb reanimation, with the function in hip
and knee joints being better than those in shoulder
joint, and the patients who had received interpositional allograft had more satisfactory results than
those who underwent hemi-joint allografts (Wang
et al. 2006; Guo et al. 2005; Liu et al. 2001a, b).
The population in China has a large incidence of
serum hepatitis, and it has been reported that about
10% of Chinese are hepatitis or HBV carriers (Sang
et al. 2000). All donors positive for hepatitis were
excluded from the current study. Although no HIV
positive patients have been detected up to now in this
bone bank, there have been positive markers for
syphilis and hepatitis C. Thus, proper examination of
the donors blood is critical for their selection (Pink
et al. 2006).

Serial products of xenograft

The processing of xenografts
The processing and production of each bone xenograft, such as reconstituted bone xenograft (RBX),

Cell Tissue Bank (2010) 11:233240

antibiotic reconstituted bone xenograft (ARBX) and

the injectable BMP xenografts (IBX), were preformed according to the standard of each product.
Native bone morphogenetic protein (BMP) extracted
from bovine cortical bone (Urist 1965) was the
essential component of the above three products.
RBX is composed of BMP and bovine cancellous
bone and is prepared in the following way: cancellous
bone from calf femoral head is cut into 2 mm 9
2 mm 9 2 mm granules, which are washed with
copious amounts of heated water (5055C) to clear
them of blood cells and tissue debris; subsequently,
the bony granules are defatted with 1:1 chloroform/
methanol for 4 h, immersed in 30% hydrogen peroxide for 48 h, followed by partial decalcification in
0.6 mol/l HCL and, finally, they are repeatedly rinsed
with distilled water to reach a pH value of approximately 7. The granules are freeze-dried, then a
certain amount of BMP is taken to make a suspension
and some bony granules processed as above are
mixed with the suspension in a ratio of bone to BMP
(2 g:30 mg). The mixture is vigorously stirred and
vacuum-suctioned leading to thorough infiltration of
BMP into the milliporous carrier. The composite is
freeze-dried again, followed by sterilization with
ethylene oxide to make the end-product of RBX.
ARBX is composed of RBX and an antibiotic release
system. When preparing ARBX, gelatin, glycerol and
distilled water are mixed in the ratio of 2:1:5
(g:ml:ml), and the mixture is dissolved in water bath
at 80C, then cooled to 55C. Next, gentamicin is
added to gelatin solution according to the ratio of 1
part gentamicin (g) to 10 parts gelatin solution (ml)
(1:10). The mixture is thoroughly stirred and is used
to encapsulate RBX granules using a specially
designed apparatus. Each RBX granule contains
approximately 0.2 ml of gelatin/gentamicin mixture.
The gelatin/gentamicin/RBX is dried, sterilized with
ethylene oxide and stored until used. IBX is another
form and is composed of BMP and polyvinylpyrrolidone (PVP), an excipient.
The application of xenografts
A total of 2,495 cases were treated with RBX from
1993 until now, and 238 of the cases cured with
ARBX. A total of 572 cases were treated using IBX
from 2001 until now. RBX was indicated for use
primarily in the treatment of old fractures, bone non-

237

unions and defects. ARBX was applied to open or

contaminated bone injuries and osteomyelitis. IBX
was utilized in minimal injury operations and closed
fractures.
Autogenous bone is by no means the most ideal
graft substitute, but it has been regarded as the gold
standard of bone grafts. However, due to the limited
supply of the bone and potential for secondary
damage during the operation, the application of
autogenous bone is quite limited. With the establishment, development, and operation of the bone bank,
bone allografts began to play a more important role in
the area of bone grafting. However, the limited donor
supply and potential risks of infection from HBV and
HIV are still the main reasons for limiting the use of
allogeneic bones in bone grafting. Although the
earliest records of bone graft were those of bone
xenografts (Galvan et al. 2006; Sanan and Haines
1997), the xenografts could not be used clinically due
to the risk for extensive immunological rejection.
Professor Hu Yunyu, the creator of the multi-variety
bone bank, developed a biological composite termed
the reconstituted bone xenograft (RBX) by chemically treating fresh calf cancellous bones to make
them antigen-free and combining them with an
extract of calf cortical bones that is from derived
bovine bone morphogenetic protein (bBMP), a strong
osteoinductive growth factor. Reconstitution of the
two components resulted in a composite with strong
osteoinductive activity without evoking an immune
rejection. This composite demonstrated little immunogenicity and exhibited excellent mechanical property. The intense immune rejection incurred through
the use of xenogeneic bone in the past is the main
reason why xenograft has not been extensively used
in clinical practice. Probably separate processing of
the two components of bone xenograft may hold the
key to its successful use, i.e. bovine cancellous bone
is subjected to serial chemical procedures including
defatting, deproteinization and partial decalcification
to eliminate the antigenicity while retaining the
collagenous carrier with part of the calcium salt
therein; in the meantime, the growth factor BMP that
has strong osteoinductive capacity is extracted from
bovine cortical bone using the technique described by
Urist. Then, the two components are combined
together under favorable conditions. RBX possesses
strong osteoinductive capacity without evoking
immune rejection, additionally it retains porous

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structure of native bone and some mechanical

strength. Therefore we would not be caught in the
dilemma of obtaining a nonantigenic carrier at the
expense of osteoinductivity of xenograft or using a
graft with residual antigenicity and consequent
immune rejection, as occurs when bone xenograft is
being treated in the traditional way. The composite is
also especially well-suited for the treatment of bone
non-unions and defects (Hu et al. 1993). Moreover,
the function of our multi-variety bone bank is not
limited to the processing and storage of bone
allografts, but can also be used in the production,
development and storage of bone xenografts. The
combined use of autograft, allograft and xenograft is
convenient for both patients and surgeons.
Although the amount of bone xenografts used in our
institute was much lower than that of allografts, the
development of a variety of xenografts is still another
characteristic of our multi-variety bone bank. With
good osteoconductive and osteoinductive abilities,
appropriate mechanical strength and perfect spacing
rate, RBX has been used to treat old fractures, bone
non-unions and defects with a cure rate of 93% (Liu
et al. 2003). This product was the first bone repairing
material, in conjunction with bone growth factors, to
be approved for marketing by the State Food and Drug
Administration of China (SFDA). Currently, more
than 100,000 packages have been sold by the Chinese
mainland marketing companies. Based on research on
RBX, we generated ARBX, which exhibits both antiinfective and bone repairing abilities, and has been
shown to release antibiotics in an infected bone
location for more than 30 days. This product could
be used in the one-stage treatment of open and
contaminated bone injuries and may be particularly
useful for the treatment of osteomyelitis (Li and Hu
2001; Jang et al. 2005). Due to its handling characteristics, IBX can be used in an office setting.

Cell Tissue Bank (2010) 11:233240

Case 1 Male, 10 years old, with osteogenic sarcoma of right distal femur, had allografts applied at
the limb for the operation (Fig. 5).
Case 2 Male, 11 years old, with chronic osteomyelitis and was treated with ARBX (Fig. 6).

Professional training
Two types of professional training has been carried
out in this multi-variety bone bank. One training
regime was targeted to those who planned to work as
technical staff at bone banks. About 18 people were
trained with this paradigm from 1990 to 2001. The
longest training time was 3 months and the shortest
training period was 1 month. Another training session
was prepared for postgraduates who wanted to obtain
a masters or doctoral degree and pursue research on
bone grafts or bone graft substitutes. More than 60
people have been trained with this paradigm according to standardized protocols covering half a year to
3 years.
The third characteristic of this bone bank is the
professional training of the technical staff and senior
researchers. Eighteen special technical staff members
have been trained in our bone bank. Although the
number is small, it is consistent with the current
situation in China. Seldom do bone banks in China
have a training program. On the other hand, we have
trained a sizeable number of senior researchers
(approximately 60 master degree graduates and

The application of different bone grafts

of multi-variety bone bank and the typical cases
A total of 6,705 units of different bone grafts were
processed and produced in our multi-variety bone
bank from 1998 to 2007. As shown in Fig. 4, bone
allografts were the major products and the number of
cases increased. The number of cases with bone
xenografts also grew, but less so than allograft cases.

123

Fig. 4 Cases with different bone grafts of multi-variety bone

bank

Cell Tissue Bank (2010) 11:233240

239

Fig. 5 Male, 10 years old, with osteogenic sarcoma of right

distal femur (a), had allograft applied at the limb for the
operation. Three months after operation, the patient got
satisfying function and the bone of him begun to link with

the allograft bone (b). Four years post-operation, the function

was still acceptable and the allograft bone had been completely
integrated with host bones (c)

Fig. 6 Male, 11 years old, with chronic osteomyelitis of the

tibia (a), had been applied with ARBX and external fixation.
Six months later, the wound of skin was healed and chronic

osteomyelitis was controlled (b). One year later, after the

remove of external fixation, good function was recovery and
chronic osteomyelitis was cured (c)

doctors) who have contributed to the development of

our multi-variety bone bank. Although most of them
have previously been trained as orthopedic surgeons
and very few have been dedicated to basic science
research, they all recognized and understood the
technology and the management styles of modern
bone banks. Overall, proper scientific experiment and
recognition of allografts and xenografts will play
important roles in spreading public awareness about
the options for bone grafts.

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