ECM Repair of rabbit radial bone defects in rabbits
【Abstract】 The home-made rabbit auricular cartilage acellular matrix
(extracellular cartilage matrix, ECM) to repair bone defects of the characteristics and mechanism. [Methods] A total of 40 New Zealand white rabbits were randomly divided
into A, B groups in its backbone on both sides of the middle of production of 5 mm of radial bone defect model. On the right as the experimental side of the defect area A group of acellular cartilage matrix composites implanted autologous bone marrow stem
cells, B group only implantation of acellular cartilage matrix, the left side of the blank control. 2,4,6,10 weeks, respectively, animals were killed, specimens radiological and histological examination. [Results] X-ray and biopsy confirmed complex in 6 weeks has
been the formation of dense bone tissue, 10 weeks has been generating new marrow. [Conclusion] showed that rabbit auricular cartilage acellular matrix and autologous bone marrow stem cells in complex with good osteogenic ability, guided tissue
regeneration, to prevent nonunion role.
Key words Bone graft Bone defects acellular cartilage matrix cartilage
Abstract: [Objective] The characteristics and mechanism of the bone defect repair with the using allogenic extracellular cartilage matrix (ECM) were studied. [Method] Forty New Zealand white rubbits were divided into three groups at random: A and B on the right side , the defect was covered with the complex of ECM and autologous mesenchymal stem cells (MSCs), the ECM in the groups A and B, respectively.The defects on the left side served as controls correspondingly.The rabbits were killed at the 2nd, 4th , 6th, 10th week.Samples were taken for radiological and histological studies. [Result] In the experimental sides the bone defects were healed.New bone appeared in the way of intramembranous ossification and entochondrostosis. [Conclusion] It is suggested that the complex of ECM and autologous MSCs has the effects on guiding bone regeneration and preventing from nonunion.
Key words: bone defect; bone grafting; extracellular cartilage matrix (ECM); chondrocyte
Clinically, long bone defects caused by nonunion are more common. A lot of bone defect repair materials, autogenous bone graft results although ideal, but its source is
limited. How to choose a material to enhance the effect of bone defect, which is the focus of the people being studied. Authors used by collagenase - detergent treatment to
remove the antigenic composition of rabbit auricular cartilage composite autologous
chondrocyte implantation in rabbit radial defects in the backbone, respectively, 2,4,6,10-week observation of bone repair capacity. Specifically reported as follows.
1 Materials and methods
New Zealand white rabbits (purchased from Tongji Medical College, Huazhong University of Science and Technology animal testing center), a total of 40, both male, months of age ranged from 2 to 3 months, weighing 2.0 ~ 2.5 kg.
1.2 Preparation of rabbit auricular ECM
Cut the sides to take 2-month-old rabbit ear, removing the skin and cartilage
membrane, fresh cartilage washed three times with PBS solution, using detergent
-step 〔1〕 right cartilage acellular processing: (1) fresh hypotonic Tris buffer cartilage placed in buffer containing protease inhibitor, 4 ? constant
cted 48 h after the
successive washing with distilled water 24 h ; (3) DNase ? and RNase ? mixture at
ion at 4 ?
thoroughly cleaned 24 ~ 48 h. Completed the removal of the cartilage cells, processed, placed in sterile PBS solution to save backup.
1.3 bone marrow stem cells isolation and culture
By Kadiyala et al 〔2〕 described method, to remove the healthy 2-month-old New
Zealand white rabbits, and promethazine intramuscular injection of ketamine anesthesia, bilateral iliac iodophor disinfection, after shearing, in sterile conditions, with 16 bone puncture needle In the iliac wing wear Ru Gusui lateral cavity, syringes,
needle tube containing 0.1 ml heparin pre-saline solution 6 ml of bone marrow
aspiration medium Mc5A moved to 5 ml centrifuge tube mixing, 1 500 r / min centrifugation 5 min , the upper fat and supernatant discarded. Adding proper amount
of suspension cells in serum-free culture medium, slowly moved into containing
lymphocyte separation medium, accounting for 1.077 in the centrifuge tube (cell culture medium and cell separation medium, the ratio of 1:1), to 2 000r/min centrifuge 20 min,
carefully draw interface, creamy white layer of nucleated cells by adding Mc5A culture medium 10 ml, made from cell suspension moved into 25 cm2 culture bottle, placed in 37 ? 5% CO2 and saturated humidity incubator, inverted microscope observation showed the same pattern homogeneous distribution of uniform cells, MSCs can be used for the collection and transplantation of 〔4,5〕, counting cells in a bottle containing
1.5 × 105A/ ml, the collection of low-temperature storage of bone marrow MSCs.
1.4 Preparation of animal model
2 ~ 3 months old New Zealand rabbits 40, both male, weighing 2.0 ~ 2.5 kg, were randomly divided into two groups of 20, bilateral radial bone in his middle bite to 5 mm radial bone defect model created the right as the experimental side, the left side of
the blank control, A group, the right side of the defect implanted acellular cartilaginous
matrix composite autologous cultured chondrocytes, B group only implantation of acellular cartilage matrix, respectively, 2,4,6, 10 weeks, animals were killed, specimens
radiological and histological examination.
4,6,10 weeks after operation, under the same conditions, respectively, making X-ray
observation of repair of bone defects. Because ECM is not developing, if there is high
density shadow means that there are new bone formation. To 12 weeks of X-ray films
according to the endogenous bone area of bone defect line of X-class efficacy
assessment 〔3〕. 100% 5 points, 75% ~ 99% of the 4 points, 50% ~ 74% of 3 points,
25% ~ 49% for 2 points, 1% ~ 24% for 1 minute, 0 for 0 minutes.
1.6 Histological observation
2,4,6,10 weeks, respectively, all animals were killed first, take bilateral repair, will foot radius separation, formalin-fixed and 1 week, and later isolated from the radial
bone in the original defect area as the center were cut radius 10 mm, As a histological biopsy, HE stain.
1.7 Data statistical analysis
Data with the x-? s, said, using t test, P <0.05 as statistically significant.
A set of experiments side (composite side of the body): The first 4 weeks when punctate shadows, portion of ground-glass shadow; 6 weeks, when the shadow density,
there are large areas of calcification, bone mass increased; 10 weeks has been
completely bony connections, most of the appears irregular bone marrow cavity, we can see a small amount of bone marrow cavity similarities (Figure 1,2). B set of experiments side (ECM implant side): After 4 weeks, no significant calcification; 6
weeks also see a small part of the bone connected to calcification widened; 10 weeks when the bone defect have more calcification, bone mass increased , are all part of the bony connection, part of the marrow cavity appeared irregular (Figure 3,4). C control
group, 10 weeks still no new bone formation defect (Figure 5,6) (Table 1). Table 1 rabbit radial bone defects in the backbone of 10 weeks of treatment efficacy rates when the results of X-ray films A set of experiments side (composite side of the body): After 2 weeks can be seen woven into the group of bone and cartilage cells, inflammatory cells less, and can be seen wire Mission-shaped fibrin as well as the proliferation of small
arteries, fibrous callus formation, the new trabecular bone began to appear; 4 weeks, woven bone mass increased significantly and the kitchen was like; six weeks when a large number of new dense bone tissue, weaving the gradual absorption of bone degradation, a very small number of cartilage cells and fibrous tissue filling bone
defects, 10 weeks after the formation of cortical bone and mature bone marrow, accompanied by callus growth, we can see similarities bone marrow cavity (Figure 7). B set of experiments side (ECM implant side): 2 weeks to have more cartilage cells and
fibrous tissue and a small amount of inflammatory cells; 4 weeks when a small amount of new bone, no marrow cavity appeared; 6,10 weeks when woven bone , there is a little irregular medullary cavity, but still see the cartilage cells (Figure 8). C group and the
control group was still visible after 10 weeks most of the defect, a small amount to fill granulation tissue (Figure 9). Reposted elsewhere in the paper for free download http://
2.3 Analysis of trabecular bone histomorphometry
A set of experiments side (composite side of the body) new trabecular bone area (17.43 ? 4.58) mm2, higher than the B group of the experimental side (ECM implanted side) new trabecular bone area (11.92 ? 3.19) mm2, there are significant differences,
statistically significant (t = 5.59, P <0.05); while the A set of experiments side and B set of experiments side with the C group and the control side (8.25 ? 5.33) of the new trabecular bone area compared with a significant difference, with statistically
significance (t = 6.68, P <0.001; t = 9.56, P <0.05).
There are many ways of repairing bone defects, with autogenous bone graft, allograft, artificial bone transplantation, the ideal fix is to autogenous bone graft, but
subject to donor restrictions. In recent years, study of long bone defects, more and more used for cell cultivation of bone graft material can be divided into two kinds of artificial and natural, artificial material has good biocompatibility, degradability and
absorbability but they are costly, the degradation rate of plasticity is low, in particular its acidic metabolites will reduce the pH value of the surrounding polymer, thereby affecting the growth of nuclear organization 〔4〕, but also can cause fibrosis and the
possible immune response in surrounding tissue 〔5〕 ; natural materials allograft
bone, xenograft bone, etc., by the use of acellular allogenic cartilage after treatment by production of extracellular cartilaginous matrix (extracellular cartilage matrix, ECM) to repair rabbit radial bone defects of 5 mm to observe the effect of its restoration . The results showed that: ECM composite bone marrow stromal stem cells (A group) best, followed by B group, less for the C group, indicating ECM can be used as bone defect repair tissue material may be used as cell culture scaffolds.
3.2 ECM organizational structure of
ECM only as support the protection of cells and the maintenance of osmotic pressure effect. In recent years, extracellular matrix research and development is very rapid. Now studies suggest that ECM is a "messenger proteins" rich environment, and the interaction of chondrocytes with the ECM regulate many biological processes, including the cell adhesion, growth, differentiation and survival 〔6〕.
ECM biological material has the following advantages: (1) After detergent enzyme
-step processing production, in addition to the cells of cartilage components, which would exist in addition to the nuclear membrane of the major histocompatibility antigens, avoiding the organizational rejection. (2) ECM mainly composed of collagen, elastin, proteoglycans and glycosaminoglycans, these ingredients are good hydrophilic. (3) ECM has a certain biological activity, to promote bone marrow stromal stem cell
proliferation, differentiation and so on. This experiment allogeneic ECM vesting in the body are two kinds of outcomes: (1) allogeneic ECM absorb a portion of narrowing in the composite autologous bone marrow stem cells in repairing tissue, bone marrow
stem cells in the material supports the migration, differentiation and proliferation; (2) bone marrow stem cells themselves secrete a number of active substances, arouse their cartilage cells and bone cells to transformation, to form new bone tissue.
3.3 the direction of research and development of bone defects
Bone marrow stromal stem cells into bone cells are able to transform into bone with a clear ability. But to single out the implantation of cells and can not effectively form a
new organization, the main reason lies in liquid form into the body's cells under the influence of local micro-environment can not maintain the local effective concentration, leading to decline in the number of new organizations. In tissue engineering of the
occurrence of new bone tissue process, the growth factor can not only play a role in inducing osteogenesis, but also can promote the proliferation and differentiation of the seed cells, directly promoting the formation of bone tissue 〔7〕. ECM as a stent graft
bone defect, the bone comes from the surrounding host bone periosteum 〔8〕,
osteogenic activities in order to soft tissue invasion and formation of bone resorption, osteoclast lacuna within the cells and osteoblasts derived from the transplanted bone
the surrounding bone marrow stem cells 〔9〕, absorption and reconstruction at the
same time. Lacunae gradually formed tubular structures, surrounded by new bone formation can be observed in active bone osteoinduction phenomenon. 4 weeks after
transplantation may be a relatively strong bone callus, the healing of the surface of the main junction, especially in the early stage of bone graft healing as the main method.
Figure 1A set of experiments side (composite side of the body): 10 weeks after the
specimen showed bone defect has been completely connected, form a smooth, relatively hard bone Figure 2A set of experiments side (composite side of the body): 10 weeks, the shadow density, there are large areas of calcification, bone mass increased bone has
been completely connected to most of the bone marrow cavity appeared irregular, showing a small amount of bone marrow cavity similarities Figure 3B set of experiments side (ECM implant side): 10 weeks of bone defect to have more
calcification , bone mass increased, both are part of the bony connection, there is still a small part of the surrounding fibrous granulation tissue Figure 4B set of experiments
side (ECM implant side): 10 weeks of bone defect have more calcification, bone mass increased, both are part of the bony connection, part of the marrow cavity appeared irregular Figure 5 the control group: 10 week defect mostly fibrous granulation tissue,
no obvious new bone formation Figure 6C the control group: 10 weeks still no bone defects showed new bone formation Figure 7A set of experiments side (composite side of the body): 10 weeks after the formation of cortical bone and mature bone marrow,
accompanied by callus growth, showing that bone marrow cavity similarities Figure 8B set of experiments side (ECM implant side): 10 weeks when woven bone, there is a little irregular medullary cavity, but still see the cartilage cells in Figure 9C and control
groups: 10 weeks later most of the defect is still visible, a small amount of granulation tissue to fill the bone defect in the currently accepted model of radial defect of the middle stage is greater than 1.0 cm. Johnson et al 〔10〕 experimental bone defects
that require bone growth in a number of unfavorable environment for the conduct, if the bone defect of more than 3 ~ 4 times the diameter of the backbone, that is not self-
repair, because the partial muscle coverage of small bones, red bone marrow content of
less and so on. In the short term to restore bone supporting role, but also the integration over time, and then be achieved with the host bone plastic the same shape and function.
The test results showed that the rabbit ear ECM and autologous bone marrow stem
cells, ECM complex or simple to repair bone defect compared with the control group compared to the good osteogenic ability to explain ECM can be used as an ideal bone defect repair materials, Its advantages: Adapted easy to produce simple, organizational
compatibility is good and if this material further in-depth study and further
improvements in the production, will greatly enhance the value.
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