Calcium sulfate induced versus PMMA-induced membrane in a critical-sized femoral defect in a rat model
Scientific REPORtS |
Calcium sulfate induced versus PMMA-induced membrane in a critical-sized femoral defect in a rat model
OPEN Aimed to investigate the characteristics of CS-induced membrane in comparison with the PMMAinduced membrane. Cellular components, histological changes, growth factor expressions of IL-6, VEGF, BMP-2, and TGF-?1 in the two induced membranes were compared at 2, 4, 6 and 8 weeks, respectively. We also compared the histological changes at the bone defects between CS and PMMA groups. The structural characteristics of induced membrane were similar between CS and PMMA. Endochondral ossification took place in the CS-induced membrane at 8 week. Levels of VEGF, BMP-2 and TGF-?1 in CS-induced membrane were insignificantly higher than those in PMMA-induced membrane at different time points. The expression of IL-6 was significantly higher in PMMA-induced membranes at 2nd week. In addition, osteogenic and neovascular activities of induced membranes increased with time and peaked at 6 weeks. CS promoted endochondral ossification at the broken ends of the bone defect than PMMA did. CS-induced membrane has a better capacity of generating VEGF, BMP-2 and TGF-?1.osteogenic and neovascular activities achieve highest level at 6 week. CS may have the potential to replace PMMA as a novel spacer in Masquelet technique.
Clinical management of critical-sized segmental long-bone defects resulting from severe trauma, surgical
excision of tumour and debridement after posttraumatic chronic osteomyelitis or infected non-union poses a major
problem. Segmental bone defects can be managed by autologous bone graft (ABG) as well as by adequate soft
tissue envelope. Vascularized fibular graft and bone transportation with Ilizarov external fixation are two widely
used techniques in the treatment of defects exceeding 5 cm1. Vascularized bone graft needs specialized
microvascular anastomosis, and is prone to stress fracture, complications at the donor site, incomplete bone healing and
risk of resorption1,2. Ilizarov bone transportation also has such disadvantages as pin tract infection, joint stiffness,
neurological injury, irregular lines and so on3.
A more novel method, developed by Masquelet et al.4, utilizes the body?s natural foreign body reaction and
fresh autologous cancellous bone graft to successfully repair massive diaphyseal defects in human patients.
Masquelet technique involves a two-step procedure5,6. During the first step, a bone defect is filled with PMMA
to induce formation of an encapsulation membrane. The second step starts after 8 weeks or so to reconstruct the
defect. The PMMA is removed and the cavity is filled with autologous morselized cancellous bone. Of all the
roles played by PMMA in Masquelet technique, like adding stability and hindering fibrous growth, acting as a
foreign body to induce formation of a vascularized membrane is perhaps the most significant. This is the reason
why Masquelet technique is known as the induced membrane technique. The induced membrane can prevent the
graft from resorption and create a favorable micro-environment for vascularity and corticalization, promoting
bone formation. Since 1986, the induced membrane technique has been widely used to manage bone defects at
the tibia, femur, humerus, hand, ulna, mandibular and elsewhere1,7?11.
PMMA cement, introduced by Buchholz and Engelbrecht in 197012 for localized antibiotic delivery, is a
widely used spacer because of its benefits of being able to bear weight and variable antibiotic elution rates. It also
bears such shortcomings as limited antibiotic release, incompatibility with many antimicrobial agents, and a
need for surgical removal of the non-biodegradable cement before surgical reconstruction of the bone defect13,14.
Therefore, extensive research pursuits are targeting alternative, biodegradable materials to replace PMMA,
including calcium sulfate (CS)14,15.
CS has benefits of good biological compatibility, osteogenic property and osteoconduction16. Recently it has
been found to be potentially osteoinductive17, inducing differentiation of bone marrow mesenchymal stem cells
into osteoblasts. Due to its complete biodegradability, an additional significant advantage over PMMA, the use of
CS as a delivery vehicle has been investigated18. It has also been widely used in clinic as a local antibiotic carrier
for the treatment of chronic osteomyelitis, because it is totally absorbed over a period of several weeks, releasing
the entire antibiotic load19. We found that it also induced formation of membrane-like structure in clinical cases20,
but the histological and biochemical characteristics of the CS-induced membrane were unclarified. There has
been no research addressing these problems as well as the differences between the PMMA-induced versus the
CS-induced membranes in repair of large segmental bone defects.
Therefore, we hypothesized that PMMA might be replaced by CS since they can both accomplish the
important role of inducing membrane and the major disadvantage of PMMA might be overcome and made up for by
the major benefit of CS. The degradability of CS may allow one-stage reconstruction of large segmental bone
defects, sparing the necessity of surgical removal of the spacer.
As the first attempt to testify our hypothesis, the present study was aimed to characterize CS-induced
membrane and make comparisons between the CS-induced and PMMA-induced membranes in repair of large
segmental femoral defects in a rat model.
Materials and Methods
Animal care. Male Sprague-Dawley rats (n = 60; Guangzhou, Guangdong, China), weighing
approximately 260?280 g were randomized into two experimental groups and one empty control group (n = 20, each).
Critical-sized femoral defect was created in all rats. In experimental groups filled with CS (Stimulan Rapid Cure,
Biocomposites, Keele, U.K.) (CS group), and PMMA (Simplex P, Stryker, Kalamazoo, MI) (PMMA group),
respectively. Nothing in the control group. The general anesthetic used was sodium pentobarbital. Iidocaine was
used for additional regional anesthesia before osteotomy. The research was conducted in accordance with the
Declaration of Helsinki and with the Guide for Care and Use of Laboratory Animals. All animal experimental
procedures and the animal use and care protocols were approved by the Review Committee on Ethical for the use
of Human or Animal subjects of NanFang Hospital (Project SYXK No. 2015-0056, Southern Medical University,
Guangzhou, Guangdong, China).
Surgical procedure. After intramuscular injection of sodium pentobarbital (3%, 1 ml/kg body weight), all
rats were shaved and placed on a heating blanket during surgery. After the superficial fascia and muscles of the
right hind limb were incised, exposing the lateral aspect of the femoral bone. A six-hole, 1.0 mm stainless steel
mini-plate (BIORTHO, Jiangsu Province, China) was applied to the femur shaft and secured in place with two
proximal and two distal 1.1 mm cortical screws (BIORTHO, Jiangsu Province, China). A critical-sized defect
(CSD) measuring 10 mm was created in the femur bone shaft, beneath the plate, using a high-speed power drill.
The bone defects were filled with CS or PMMA cylinders (2.8mm in diameter, 10 mm in length) respectively21.
The wounds were closed with interrupted 4-0 Vicryl sutures. Analgesia was given for 3 days postoperatively.
Penicillin 20,000 IU/kg was administered intramuscularly immediately, 24 and 48 hours after operation.
Five animals in each group were sacrificed each time at 2, 4, 6 and 8 weeks to harvest the membranes
surrounding the CS/PMMA spacers for analysis. Subsequently the animals were euthanized with an overdose
anesthetics administered intraperitoneally.
The membranes were divided into four fractions which were respectively fixed in 10% formalin solution for
later paraffin embedding, shock frozen (?80 ?C) for later PCR analysis, shock frozen for later Western-Blotting
analysis, or fixed in 10% formalin solution for further immunohistochemistry (IHC) analysis.
Digital radiographs. Digital radiographs were taken immediately, 2, 4, 6 and 8 weeks post-operation under
anesthesia using an Oralix AC Densomat X-ray machine (Gendex Dental System, Milan, Italy). The femur
samples were harvested at 2, 4, 6 and 8 weeks postoperatively for histological examination and micro-CT scanning.
Micro-computed tomography (Micro-CT). The morphology of the newly formed and reconstructed
femur bone were using an animal micro-CT scanner (Skyscan1176, Bruker Micro-CT, Belgium). The Scanning
parameters with an 80 kVp energy setting, intensity of 313 uA with 280 ms acquisition time and a voxel resolution
of 18 ? m. The defective region was identified by a contour as a traced region of interest (ROI), the relative
measurements of bone mineral densities (BMDs) and bone volume/total volume (BV/TV) were calculated.
Histological observation of bone defects. After micro-CT scanning, the femur samples were decalcifed
in 10% Ethylene Diamine Tetraacetic Acid (EDTA, pH = 7.4) and dehydrated in graded ethanols (70?100%).
Finally, they were embedded in paraffin and cut in 5? m thick sections for staining. Masson?s Trichrome and
Safranin O-fast green staining were utilized according to the manufacturer?s protocols.
General histological observation of induced membrane. The harvested samples were embedded in
paraffin and cut in 5?m thick sections for histological staining. Stained with Hematoxylin & Eosin and used
to an Optiphot-2 (Olymbus@) photomicroscope to distinguish cells. The Safranin O staining, Collagen II and
Aggrecan immunohistochemistry staining were carried out for further determination.
Real-time polymerase chain reaction (Real-time PCR). The membranes were stored in liquid
nitrogen before RNA analysis. RNA was isolated according to the manufacturer?s instructions. The amount of
RNA was measured as OD values on UV-1750 UV spectrophotometer by Shimadzu (Japan). Amplifications
were performed with the ABI 7300 Real-Time PCR System (ABI, Carlsbad, CA, USA) with different
primers. The primers used were as follows: Interleukin-6 (IL-6) forward AGACTTCCAGCCAGTTGCCTT
and reverse TTGTGGGTGGTATCCTCTGTGA; Vascular endothelial growth factor (VEGF) forward
TCCTGTGTGCCCCTAATGC and reverse ACGCACTCCAGGGCTTCAT; Transforming growth factor-?1
(TGF-?1) for ward GACGGAATACAGGGCT T TCG and reverse CCTCGACGT T TGGGACTGAT;
B one morphogenetic protein-2 (BMP-2) for ward AGAGCT T TGATGTCACCCCG and reverse
GGAGACACCTGGCTTCTCCTC. The expression of each compound was related to compare the housekeeping
gene GAPDH with one selected control sample.
The content measurements of TGF-? 1 and BMP-2. The membrane tissues were homogenized in
lysis buffer at 4 ?C, followed by centrifugation at 20000 ? g for 30 min at 4 ?C. TGF-?1 and BMP-2 were detected
using monoclonal goat anti-rabbit TGF-?1 antibody (BA0290, Boster) and monoclonal goat anti-mouse BMP-2
(Ab6285, Abcam), respectively. Determination of GAPDH with mouse anti-GAPDH antibody (Abcam) served
as a loading control. The blots were blocked (10% non-fat dried milk in 1m M Tris, 150 m M NaCl, pH 7.4) for
1 h, incubated for 1 h at RT with primary antibody in blocking buffer with 0.5% Tween20 and 0.5% bovine serum
albumin (BSA) and then incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (Santa
Cruz Biotechnology) diluted 1:1000 in blocking buffer with 0.5% Tween20 and 0.5% BSA at RT. The density of
individual bands was determined using Fusion V software and normalized to the density of the corresponding
Statistical analysis. All data collected were reported as means with standard deviations and P values ? 0.05
were defined as statistical difference. For normally distributed data, Student t test or one-way analysis of variance
(ANOVA) was used to compare differences between 2 different groups or among more than 2 groups. All
statistical analyses were performed using SPSS 13.0 software (SPSS Inc, Chicago, IL, USA).
The tracked X-ray radiographs of the typical models. At the immediately after surgery, as presented
in Fig.?1A,D, both CS and PMMA were radiopaque so that the bone substitute and the broken ends were clearly
At 4th week, much of the CS appeared fuzzy, dotted and flocculent, indicating partial degradation and
absorption. A membrane-like structure appeared around the bone defect while the PMMA material remained relatively
clearly visible (Fig.?1B,E).
At 8th week, CS was completely degraded and absorbed, membrane-like structure also appeared. The PMMA
material showed no significant change compared with previous (Fig.?1C,F).
Nothing grew into the critical-sized bone defects in the empty control group at different time points
Micro-CT measurement. The micro-CT cross sections showed more obvious new bone formation in CS
group than in PMMA group. Further compared with the empty control group, CS and PMMA groups exhibited
significantly more new bone formation but there were gaps still existing in the bone defect (Fig.?2A?L).
The quantity of the newly formed bone was calculated by morphometric analysis (Fig.?2M and N). The data
demonstrated significantly greater BMDs in CS group than in PMMA group at 4, 6 and 8 weeks postoperatively
(P < 0.05). Compared with the control group, there were significant differences in 2 experimental groups at 4, 6
and 8 weeks postoperatively (P < 0.05). However, there was no significant difference among the 3 groups at the
2nd week postoperatively (P = 0.135). The BV/TV results exhibited a similar trend. The mean BV/TV percentage
in CS group was significantly higher than that in PMMA group at 4, 6 and 8 weeks postoperatively (P < 0.05), but
there was no significant difference at the 2nd week among the 3 groups (P = 0.59).
Histological analysis of bone defects. Through Masson?s Trichrome staining, we can find the following
At the 2nd week, we found lots of blue-staining fibrous tissues, lymphocytes, macrophages and micro-vascular
sections in both CS and PMMA groups. The broken ends presented even and no new bone tissue formed (Fig.?3A and E).
At the 4th week, there were more blue-staining fibrous tissues and micro-vascular sections in both CS and
PMMA groups. The lymphocytes and macrophages reduced significantly in number compared with those at the
2nd week. The diameter of vascular section increased obviously compared with previous. A small amount of new
bone formed at the broken ends (Fig.?3B and F).
At the 6th and 8th weeks, more fibrous tissues and vascular sections were seen. A few lymphocytes and
macrophages were also apparent. Obvious endochondral ossification was seen at the broken ends. Much more new
bone tissue formed near the ends (Fig.?3C,D and G,H).
At the same time, the histological sections of the end of bone defect stained with Safranin O and fast green
demonstrating the similar results to those of Masson?s Trichrome staining (Fig.?4).
Gross observation and radiographic analysis of the induced membrane. Gross observation
showed the following macroscopic appearances of the induced membranes at the bone defects filled with CS/
PMMA cement for 2, 4, 6 and 8 weeks (Fig.?5).
CS group. At 2nd weeks, a thin soft membrane-like structure formed and adhesive to the surrounding muscle
tissues. The outer layer of membrane was dark red fibrous tissue-like structure, and the inner layer was smooth
and light green. Slight degradation of the CS was observed in the bone defect. The smooth light green inner
layer did not adhere to the CS. At 4th weeks, the induced membrane was gradually thickening. The outer layer
of membrane was still dark red, but a clear border appeared between the membrane and surrounding muscle
tissues. The CS partly degraded in the bone defect. Some clear yellow-green liquid was found in the bone defect.
The inner layer of the membrane became yellow-green. A small amount of bone callus formed around the broken
ends of the bone defect. At 8th weeks, the membrane thickened more and became elastic (Fig.?5C). Most of the CS
degraded in the bone defect. There was an amount of clear yellow-green liquid in the bone defect. A lot of bone
callus generated around the plate and the both broken ends, but the bone callus was not yet fully mature, with an
obvious boundary with surrounding bone tissue.
PMMA group. At 2nd weeks, around the PMMA a thin and dark red induced membrane structure formed
which was similar to the periosteum, but thicker and tougher than the periosteum. The induced membrane
adhered to the PMMA and its surrounding muscle tissue. No degradation of PMMA or any liquid was found
in the induced membrane. At 4th weeks, the induced membrane was generally thickening, and tightly adhesive
to the PMMA. Some bone callus formed around the broken ends. The PMMA around the bone defect showed
no significant change compared with before. At 8th weeks, the induced membrane around the bone defect partly
adhered to the surrounding tissues (Fig.?5D). Its thickness increased significantly. The PMMA still showed no
obvious change compared with before, but some bone callus generated and surrounded the plate and both ends
of the bone defect.
Control group. At 2nd weeks, no membranous-like tissue was generated. The bone marrow cavity was closed,
and osteosclerosis showed at both broken ends. A small amount of fibrous tissues grew gradually into the bone
defect. At 4th weeks, significant osteosclerosis showed at the broken ends. Lots of fibrous tissues grew into bone
defect. At 8th weeks, the bone defects were already filled with white fibrous tissue.
As the CS-/PMMA-induced membranes matured, their thickness and fibrosity increased significantly. The
CS-induced membranes were generally thicker than the PMMA-induced ones. Specifically, the thickness of
CS-induced membrane (1095 ?m) was significantly greater than that of PMMA-induced membrane (842 ?m) at
8th week (p < 0.05) (Fig.?6).
Morphological structure of different induced membranes. CS group. As shown in Fig.?7A, at the
2nd weeks, local and mild acute inflammatory reaction and diffuse edema were noticed, capillaries as well as
neutrophils, monocytes, fibroblasts, myofibroblasts, and collagen were seen on the outer surface of the membrane,
and synovial-like epitheliums were observed in the inner part of the membrane. At the 4th week, the membrane
was similar to that described above, and myofibroblasts and collagen fibers mostly arranged parallel to the spacer
surface, but less edematous reaction appeared (Fig.?7B). At the 6th week, the thickness and fibrosity increased
significantly; only a handful of monocytes, neutrophils and small capillaries were observed; larger vessels developed
in the outer part of the membrane (Fig.?7C). At the 8th week, the edema was almost completely absorbed and
multinucleated giant cells decreased in number; endochondral ossification, newly formed bone tissue and even
mature lamellar bone appeared in the induced membranes (Fig.?7D).
PMMA group. At different time points postoperatively, the morphological characteristics of induced
membranes around PMMA were generally similar to those around CS. The extent of inflammatory and edema reaction
was more severe than in CS group at 2th week. But endochondral ossification or newly formed bone tissue was not
observed at the 8th week (Fig.?7E?H).
Endochondral ossification of the induced membranes in CS group at 8 weeks
postoperatively. The 8th week-induced membranes in CS group were further identified. H&E and Safranin-O staining
confirmed endochondral ossification and newly formed bone tissue (Fig.?8A,B). IHC staining also showed
multiple sites of fixation for antibodies against Collagen-II and Aggrecan, demonstrating the endochondral ossification
presented in Fig.?8C and D.
The contents of TGF-?1 and BMP-2 in the induced membranes. The TGF-?1 and BMP-2 protein
contents went up gradually at 2, 4 and 6 weeks but declined after 6 weeks (Fig.?9). The expressions of VEGF,
TGF-?1 and BMP-2 were insignificantly higher in CS groups than in PMMA ones (Fig.?10A?C). The expression
of IL-6 showed a gradually decreasing tendency in both groups; it was significantly higher in PMMA group than
in CS group at 2nd week but insignificantly higher at 4, 6 and 8 weeks (Fig.?10D).
Our present work has made the following four main findings. Firstly, CS as a spacer can also induce formation of
a membrane which may be thicker than that induced by PMMA. Secondly, endochondral ossification may take
place in the CS-induced membranes but not in the PMMA-induced membranes, suggesting that CS-induced
membranes may have a real osteogenic potential to repair bone defect. Thirdly, the expressions of factors (VEGF,
TGF-?1 and BMP-2) may be insignificantly higher in CS-induced membranes than in PMMA-induced ones at
different time points. The contents of the factors may increase with time and peak at 4 to 6 weeks and decline
gradually afterwards. Fourthly, CS may promote endochondral ossification at the broken ends at different time
points better than PMMA.
Through histological staining we found that a membrane-like fibrotic capsule formed around the bone defect
in CS. The inner layer of the CS-induced membrane there were synovial-like epithelial cells and a large number of
VEGF could induce angiogenesis, which played an important role in bone repair and bone regeneration.
Moreover, blood vessels can provide nutrients and oxygen and play an important role in regulation of bone
remodeling by attracting endothelial cells and osteoclasts and by stimulating osteoblasts differentiation.
BMP-2 can promote chemical trends, gather, differentiation of BMSCs, which can directly mediate osteogenic
differentiation of BMSCs and immature osteoblasts. In our present work, the increased BMP-2 production in the
membranes might have played a role in the osteogenic differentiation of BMSCs.
TGF-?1 can also promote proliferation and differentiation of BMSCs, help synthesis of extracellular matrix
protein and regulate the immune function29. TGF-?1 has been shown to play a central role in foreign body
encapsulation. Therefore, TGF-?1 has been considered as a putative regulator of osteoclastic-osteoblastic interaction
and is continuously synthesized in induced membranes21,30.
The expression of IL-6 showed a gradually decreasing tendency. It was significantly higher in PMMA group
than in CS ones at 2nd week. This might have been related to the PMMA exothermic inflammation reactions.
The induced membrane plays an important role in bone repair and healing. Despite the significant differences
we measured between CS and PMMA membranes induced around the bone defects, it is interesting to note that
both of the induced membranes can promote bone healing.
We observed a significant increase in the BMDs and BV/TV of the bone defect from 2 to 8 weeks in the two
groups. We also found more obvious new bone formation in CS group than in PMMA group through the
structural characteristics. Compared with previous studies on PMMA-induced membrane, CS-induced membranes
also contained well-vascularized fibrous tissue that was mostly preserved during follow-up. And most
importantly, we found some intramembranous ossification in the CS group specimens. We speculate that CS might
provide a source of calcium for new bone tissue, and promote proliferation and differentiation of osteoblasts to
form new bone tissues.
Being aware that our observations in a rat model cannot be extrapolated directly to human conditions, we
believe that CS may replace PMMA in that it promotes bone growth but does not require a secondary surgery in
Masquelet technique. Therefore, the healing process would capitalize on all this angiogenic and osteogenic
activity and thus could result in a much quicker healing than the PMMA does.
Our present study also has a major limitation. We compared the histological and biochemical characteristics
of the membranes induced by CS and PMMA. It is only a preliminary proof that CS can induce membranes, but
further experiments of molecular biology are required to verify and compare the differences between CS- and
PMMA-induced membranes. Further animal experiments are necessary to verify our hypothesis that CS might be
used in one-stage reconstruction of segmental bone defects, sparing surgical removal of the spacer.
The membranes induced by CS are similar to those of PMMA in many respects. The osteoinductivity of CS is
significantly better than that of PMMA. Thus, to maximize the bone-healing effect, we propose that PMMA can
be replaced by CS early (6 weeks) when the osteogenic activity of CS is at the peak. Since CS has benefits of good
biological compatibility, osteogenic property, osteoconduction, potential osteoinductivity, and a capacity of being
totally absorbed, it has been widely used in clinic as a local antibiotic carrier for treatment of chronic
osteomyelitis in our hospital. So we hypothesized that one-stage surgery may be achieved to reconstruct small bone defects
with CS as a spacer.
The authors thank Pro.Liang and all the staff in the Key Laboratory of Bone and Cartilage Regenerative Medicine,
Nanfang Hospital of Southern Medical University. The work was supported by Guangdong Provincial Science and
Technology Plan Projects
(No.2016B090913004, No.201508020035 and NO.2015A020212022)
Yun-fei Ma and Bin Yu designed the research. Lei Wang and Xiang Zhang performed the animal experiments.
Cheng-he Qin, Bo-wei Wang, Bo-wei Wang and Nan Jiang analyzed the data. Yan-jun Hu and Qing-rong Lin
performed all other experiments and prepared all figures. Yun-fei Ma and Nan Jiang wrote the main manuscript
text, and all authors reviewed and commented on the manuscript. Bin Yu and Yan-jun Hu are co-corresponding
Competing Interests: The authors declare that they have no competing interests.
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