Histocompatibility of Acellular Matrix Bone with Osteoblast and Vascular Endothelial Cells

The culture of stem cells is an essential element in tissue engineering.The base for the repairing of bone defect in tissue engineering is still its vascularization. Co-culture of the osteoblast and the vascular endothelial cell (VEC) can not only promote the proliferative activity of the osteoblast but can also accelerate the revascularization of the regenerating bone. Thus it can help to increase the ability of regenerating bone in the repairing of bone defect. The bone source and the site for supplying bone were limited in autografting, meanwhile it is difficult in the modeling of self-tissue, therefore its clinical application is limited. There is certain immunologic rejection in the isotype heterogenous bone grafting and the tissue engineering bone provides a new way for solving this problem. In this experiment, the osteoblast and the vascular endothelial cell were compounded by using the acellular rib of pig. Then the tissue engineering bone was constructed and implanted into the animal. Observe and study the biological compatibility, try to find out the pathological process of in vivo ossification, bone defect repairing and revascularizarion for the heterogenous bone grafting. Thus it will provide the theological base for the clinical application of tissue engineering bone. This chapter is divided into 3 parts: Part I: The culture of the seed cell in tissue engineering and the study of cellular compatibility, Part II: Study the effect of heterogenous bone acellular matrix (HBACM) on the biological compatibility between the osteoblasts and the vascular endothelial cells, Part III The experimental research of implanting the compound cell of heterogenous bone acellular matrix into the animal body.


Introduction
The culture of stem cells is an essential element in tissue engineering.The base for the repairing of bone defect in tissue engineering is still its vascularization.Co-culture of the osteoblast and the vascular endothelial cell (VEC) can not only promote the proliferative activity of the osteoblast but can also accelerate the revascularization of the regenerating bone.Thus it can help to increase the ability of regenerating bone in the repairing of bone defect.The bone source and the site for supplying bone were limited in autografting, meanwhile it is difficult in the modeling of self-tissue, therefore its clinical application is limited.There is certain immunologic rejection in the isotype heterogenous bone grafting and the tissue engineering bone provides a new way for solving this problem.In this experiment, the osteoblast and the vascular endothelial cell were compounded by using the acellular rib of pig.Then the tissue engineering bone was constructed and implanted into the animal.Observe and study the biological compatibility, try to find out the pathological process of in vivo ossification, bone defect repairing and revascularizarion for the heterogenous bone grafting.Thus it will provide the theological base for the clinical application of tissue engineering bone.This chapter is divided into 3 parts: Part I: The culture of the seed cell in tissue engineering and the study of cellular compatibility, Part II: Study the effect of heterogenous bone acellular matrix (HBACM) on the biological compatibility between the osteoblasts and the vascular endothelial cells, Part III The experimental research of implanting the compound cell of heterogenous bone acellular matrix into the animal body.

General methods (Parts I -III) 2.1 Part I: The culture of the stem cells in tissue engineering and the study of cellular compatibility
The osteoblast (A group) and vascular endothelial cell (B group) of the rabbit was cultured, and then both of the cells were co-cultured (C group).The morphology and the growth of the cells in the 3 groups were observed under the inverted phase contrast microscope.Immunocytochemical stain of typeⅠcollagen was used to identify the osteoblast.Immunocytochemical stain of the vascular factor VIII was used to identify the VEC.The sections were made by the co-cultured cells.Three days later, observe the mixed growth of 2. The heterogenous bone acellular matrix had good biological compatibility, with low antigenicity, no cytotoxity or neoplastia；The co-cultured cells had strong potency of proliferation in the extracellular matrix of the heterogenous bone acellular matrix.3. The major ossification of the tissue engineering bone in the body was the cells cultured in vitro.There was synergistic action between the osteoblast and the vascular endothelial cell whether in vivo or in vitro.Fracture healing could be accelerated and the repairing of bone defect could be promoted by using the co-cultured cell as seed cell.

Part I:
The culture of the seed cell in tissue engineering and the study of cellular compatibility

Introduction
The culture of seed cell is an essential element in tissue engineering.Osteoblast is the seed cell in tissue engineering, but the base for the repairing of bone defect in tissue engineering is still its vascularization.Co-culture of the osteoblast and VEC can not only promote the proliferative activity of the osteoblast but it they can also accelerate the revascularization of the regenerating bone.Thus, it can help to increase the ability of regenerating bone in the repairing of bone defect.To this end, we conducted related studies and reported as follows.

The culture of osteoblast (group A)
The calvaria cultures were taken from two 2week-old newborn rabbits and the soft tissue was removed.After several times of rinsing in PBS, the soft tissue was cut into blocks of one mm 3 in size, which were then digested in 1:1 of 0.25% trypsin and 0.1% collagenase at 37 ℃ for 10 min.After the digestion terminated, the solution was centrifuged at 1200 r/min for 5 min and the supernatant was discarded.Then, the cells was suspended in 20% of fetal calf serum DMEM and inoculated in culture flasks, which were kept in the cell incubator at 37 ℃ with 5% of CO 2 and saturated humidity.The tissue blocks were digested using 1:1 of 0.25% trypsin and 0.1% collagenase at 37 ℃ for 20 min.The cells were collected via centrifugation and were inoculated in culture flasks.The above steps were repeated twice.Then, the culture medium was replaced every two to three days.The cells that became monolayer were used for subculture.

The culture of VEC (group B)
The bilateral kidney cortex of the above two 2-weekold newborn rabbits were cut into fragments, which were then digested with 0.25% of trypsin at 37 ℃ for 20 min.After the digestion terminated, the solution was centrifugal at 1200 r/min for 5 min and the supernatant was discard.Then, the cells was suspended in 20% of fetal calf serum DMEM and inoculated in culture flasks, which were kept in the cell incubator at 37 ℃ with 5% of CO 2 and saturated humidity.Then, the culture medium was replaced every two to three days.The cells that became monolayer were used for subculture.

Co-culture of osteoblast and VEC (group C)
Primary cultured osteoblast, which was in logarithmic growth phase, was inoculated by the ratio of 1:1 in the culture flasks.20% of fetal calf serum DME were added in the flasks and kept in cell incubator at 37 ℃ with 5% of CO 2 and saturated humidity.Then, the culture medium was replaced every two to three days.The cells that became monolayer were used for subculture.

4.2.2
The index of observation 4.2.2.1 Observation under the inverted phase contrast microscope Observe morphology and the growth of the cells in the three groups under the inverted phase contrast microscope.

Immunocytochemical stain
The cells of group A and group B were inoculated with the density of 2 × 10 5 cells per well into the six-pore board with a built-in glass slide for making cell climbing slices.Three days later, immunocytochemical stain of typeⅠcollagen to identify the osteoblast for group A. Immunocytochemical stain of the vascular factor Ⅷ to identify the VEC for group B.

Histological observation
The cells of group C were inoculated with the density of 2 × 10 5 cells per well into the six-pore board with a built-in glass slide for making cell climbing slices.Three days later, observe the mixed growth of the two kinds of cells after HE stain and Masson trichrome stain.

Test of the activity of alkaline phosphatase (ALP)
The cells of groups A, B and C were respectively inoculated with the density of 3 × 10 3 cells per well into 96-pore board (12 pores each group).Test the activity of ALP by using the method of paranitrobenzene phosphate via a machine of UV / fluorescence / visible high efficiency analyzer (Perkin Elmer, USA).Observe whether the VEC would affect the ALP activity produced by the osteoblast.

Test of the cellular activity by the method of methythiazolyl tetrazolium bromide (MTT)
The cells of groups A, B and C were digested and inoculated with the density of 4 × 10 3 cells per well into 96-pore board.12 pores were taken on the 1 st , 3 rd , 5 th and the 7 th day, respectively.Added 5 mg ml -1 of MTT and reacted for four hours.After the liquid was discarded, dimethyl sulfoxide was added and oscillated for 10 min.An enzyme mark instrument (Perkin Elmer, USA) was used to obtain the adsorption at the wavelength of 570 nm and to analyze the growth and proliferation of the cells in the three groups.

Statistical methods
The result of the experiment was expressed by mean±standard deviation.The analysis of variance and q test of the measurement data among groups were done by using SPSS 10.0 software packet.This difference had statistic meanings (P<0.01).

Observation under the inverted phase contrast microscope
Primary cultured osteoblast and VEC began to adhere to the wall four hours later and was adherent completely in 48 hours.Osteoblast displayed various shapes, some were spindle, and some were long triangles, with massive sharp horn like (Fig. 1a).VEC showed ovoidal and changed to typical 'flagstone' appearance when it fussed to monolayer (Fig. 1b).When VEC and osteoblast were co-cultured, they grew closely.There was no contact inhibition, rejection and phagocytosis.The volume of osteoblast was large and the volume of VEC was relatively small (Fig. 1c).

Immunocytochemical stain
The immunocytochemical stain of osteoblast typeⅠcollagen and VEC factor VIII were both positive (Figs. 2 and 3).The cytoplasm revealed yellow staining and the cell nuclear blue staining.Results show that the primary cultured cells were osteoblast and VEC.

Histochemistry stain
The HE staining and Masson staining of the cells of group C showed the mixed growth of two types of cells with good cellular compatibility.The volume of osteoblast was large and the volume of VEC was relatively small (Fig. 4).VEC appeared red by Masson trichrome stain (Fig. 5).

Test of the activity of ALP
After three days of growth, the activity of ALP in each group was as follows.Group A (0.1568 ± 0.0132) U/L, group B (0.1148 ± 0.0145) U/L, group C (0.5018 ± 0.0873) U/L and the F value was 202.811 (P <0.01).The ALP activities from high to low in order were group C, group A and group B. The ALP activity of group A was higher than that of group B (P<0.05).The ALP activity of group C was obviously higher than that of group A and group B (P<0.01).

Test of the cellular proliferation activity by MTT method
The activity of the cells is listed below (Table 1, Fig. 1).On the first day, group B> group A > group C, the difference was statistically significant (P <0.01, P <0.05).On the third day, the activity of group A and B was higher than that of group C, the difference was statistically significant (P <0.01) and there was no significant difference between group A and group B (P> 0.05).On the fifth day, results show that there was no significant difference among group A, B and C.However, on the seventh day, the activity of group A was higher than that of group B (P <0.01), and the activity of group C was higher than that of group B (P <0.01), the difference was statistically significant and there was no significant difference between group A and group C (P> 0.05).

Discussion
Osteoblast is the progenitor cell in tissue engineering; while VEC is rarely used as seed cell in tissue engineering.The three basic processes of bone graft are the vascularization of the graft, bone regeneration and bone-terminal fusion.The ultimate goal is to repair bone defects.The vascularization of the graft is the key step, which affects the total process of transplantation and repair, and it play a decisive role on the mode and effect of bone regeneration and fusion.Zhu, et al indirectly cultured osteoblast and VEC using nest dish culture method, in which the mediums of the two kinds of cells contact with each other, while the two kinds of cells do not contact.Results show that the medium of VEC can enhance the proliferation of osteoblast.However, in this paper, VEC was directly cocultured with osteoblast.We observed whether the cellular compatibility of the two kinds of cells was good or not; what's the impact on the osteogenic activity of the osteoblast.
From the experimental results, immunocytochemical stain of typeⅠcollagen and the vascular factor VIII identified the osteoblast for group and the VEC for group B. Observation under the inverted phase contrast microscope, HE staining and Masson trichrome staining showed that the two kinds of cells mixed grew well, and the cells were well distributed.There was no contact inhibition, rejection and phagocytosis, explaining that osteoblast and VEC are with good cellular compatibility.
Brighton et al and Katagiri et al find in the experiments that the epidermal cells, endothelial cells and osteoblast may be the bone progenitor cells, which can behavior the characteristics of osteoblast after culture.For example, they can synthesize ALP, osteocalcin and type I collagen.The internal environment of body is maintained by the synergy of a variety of cells.And in vitro environment, osteoblast and VEC might have effects of mutual synergies.
Lu et al find that the TGF-β release system can significantly promote bone marrow stromal cell proliferation and differentiation to osteoblast.Moreover, the number of surface cells, the activity of ALP and the osteocalcin production was significantly higher than that of the control group that is without the system.The bone morphogenetic protein, vascular endothelial growth factor (VEGF) and tetracycline have similar regulation with TGF-β to osteoblast.VEC can synthesize and secrete VEGF, thus significantly promote the activity of ALP in osteoblast.
Seen from the test of ALP activity by paranitrobenzene phosphate method, the ALP activity of group A was obviously higher than that of group B (P <0.05); while the ALP activity of group C was remarkable higher than that of groups A and B (P <0.01).Results show that VEC can significantly enhance the ALP activity in osteoblast, it further explained that VEC could increase the function of osteoblast, including the proliferation of osteoblast, and could promote osteoblast to transform to bone cell.The results were consistent with that of Ref.. On the other hand, osteoblast can secrete VEGF, FGF and other pro-angiogenic factors, which affect VEC to promote angiogenesis.In the bone marrow hemopoietic microenvironment, osteoblast plays a central role by producing colony-stimulating factor to promote the regeneration of hematopoietic stem cells and VEC.
Results of the MTT test show that activity of VEC on the 1 st day and the 3 rd day was strong.It began to decline on the 5 th day.However, osteoblast began to proliferate fast on the 5 th days.The proliferation activity of co-culture cells was lower than osteoblast and VEC on the 1 st and the 3 rd day.From the 3 rd day, the two types of cells have good cellular compatibility and started into the logarithmic growth phase, thus demonstrating strong proliferation activity.On the 5 th days, the cell proliferation activity obviously accelerated, and has no significant difference with osteoblast and VEC.On the 7 th day, the proliferation activity of osteoblast and co-culture cell was remarkably higher than that of VEC.Whether these were due to the reason that the two kinds of cell competed for living sites on the board from the 1 st to the 3 rd day after co-culture, which affected the cell proliferation activity.It needs to further study.The experiment in this paper showed that the co-culture cells proliferated slowly in the early stage and faster in the later stage.Osteoblast and the co-culture cells are with strong proliferation, the proliferation of VEC is comparatively weak, indicating strong potency of proliferation of co-culture cells.
The experimental results show that, there was good cellular compatibility between the osteoblast and VEC.VEC could increase the function of osteoblast, and promote the proliferation of osteoblast.The VEC cultured in vitro may help the revascularization of the tissue engineering bone.Thus, it can provide normal nutritional, and the regulation of nerves and body fluid.Therefore, the co-cultured cells between osteoblast and VEC are expected to become the seed cells of the tissue engineering bone.

Conclusion
The conclusion section should be an independent paragraph.A reader who choose to read onlt the conclusion must be able to reasonably understand the topic.The conclusion should start with a sentence or two that describe the experiment before concluding results.
1.There was good cellular compatibility between the osteoblast and the VEC.
2. The ossification activity of the osteoblast was significantly increased by the VEC.
3. The co-cultured cells had strong potency of proliferation.

Introduction
Tissue engineering is a science that uses cell biology and engineering principles to research and develop biological alternatives for the repair and improvement of wounded tissue and function.The histocyte that is cultured in vitro and with high concentration is amplified and is absorbed in a biocompatible extracellular matrix (ECM) that is degradable and can be absorbed by human body.The seeded cells continue to grow and reproduce during the degradation and the absorption process in the scaffold, and develop to corresponding new tissue and organ with original special function and morphology, then the expected purpose of wounds reparation and reconstruction function was attained.In this experiment, the osteoblast and the vascular endothelial cell (VEC) were compounded by using the acellular rib of pig.Observe and study the biological compatibility.Thus it will provide the theological base for the clinical application of tissue engineering bone.

Preparation for the framework material for HBACM
Fresh pork ribs were selected and the soft tissue around was removed.Then the soft tissue was made into skeletons in size of 2.0cm × 0.5cm × 0.5cm.(7) the skeletons were freeze-dried and sterilized with ethylene oxide, for use, and then observe the degree of acellular under scanning electron microscope.

Cell culture
The co-culture osteoblast and VEC, the identification of osteoblast, VEC and the co-cultured cells were the same as the former.

Compound of the cell with HBACM
Experimental groups were as follows: Group A: osteoblast compound with HBACM; Group B: VEC compound with HBACM; Group C: osteoblast + VEC compound with HBACM.

Observation under the inverted phase contrast microscope
Observe the adhesion of the cells with the material, the distribution of the cells in the pores of the material, and the growth, differentiation and proliferation of the cell.

Histological observation
The three groups of cells were compounded and cultured with the materials for 5 days.After being fixed with alcohol and stained directly by Masson trichrome, they were observed under optical microscope.Three of the materials in each group were taken and embedded in paraffin after decalcification to determine on histological sections.Then observe the biological compatibility after HE stain, toluidine blue and Masson trichrome stain.

Observation under the scanning electron microscope
The three groups of cells were compounded and cultured with the materials for 5 days.One of the compound materials in each group were taken and rinsed in PBS for thrice, fixed with 2.5% glutaraldehyde, dehydrated with alcohol gradiently, and then fixed in iso-amyl acetate.After dried and coated with gold, the samples were observed by using a scanning electron microscope (JSM-5900) to test the adhesion, growth, proliferation and the matrix secretion of the cells on the HBACM.

Test by flow cytometer
The three groups of cells were compounded and cultured on the culture plate with HBACM.Three compounds of cellular material were selected individually from each group on the 1d,3d,5d and 7d.Then they were digested.Collect and count the cells.The cells were staining in PI dye and were detected using a flow cytometer (Coulter Corporation, U.S.) to analyze the effect of HBACM on on cell cycle and DNA content.Then the DNA index (DI values), DI = 1.0 ± 0.15 was calculated as normal diploid cells.Analyze the cell cycle and the ploidy by flow cytometer to test the toxicity of the material to the cell.

Statistical methods
The result of the experiment was expressed by mean±standard deviation.The analysis of variance and q test of the measurement data among groups were done by using SPSS 10.0 software packet.

Observation of HBACM under scanning electron microscope
The structure of the bone trabecular of HBACM was integrated, the bone lacunas were empty, and no residual cellular components were found (Fig. 1).

Observation under the inverted phase contrast microscope
When the three groups of cells were co-cultured with HBACM for 12 hours, the cells could adhere and grow in the pores and on the surface of HBACM.With the culture time gone, grew, differentiate and proliferate along the edge of HBACM pore gradually.The cells gradually extended, stretched across the pores, and the pseudopodium tended to contact with each other from the 5 th day co-cultured with HBACM (Fig. 2).

Histological observation
The HBACM of compound cell was directly Masson stained.Results show that the cells in each group tightly adhered with materials.They gathered into a group, connected into a network, and there were a large number of proliferations of the cell on the surface and in the pores of HBACM.The number of cells adhesion on HBACM in osteoblast + VEC group was the maximum (Fig. 3).Stained histological sections showed that the cells were closely attached along the edge of materials, and the histocompatibility of cells and HBACM was good.

Scanning electron microscope results
After cultured with HBACM for five days, the cells were tightly adhered on the surface of HBACM.Osteoblast appeared spindle-shaped or polygonal, the adjacent cells connected to each other with processes (Fig. 4).VEC was oval-shaped, with angular protrusions, and was closely attached to the materials (Fig. 5).The co-cultured cells were showing their own form, and the two kinds of cells tightly combined with each other.The cell compatibility and compatibility between the cells and HBACM were good, with network collagen attached around (Fig. 6).A plenty of cells adhered on the surface of the materials of osteoblast + VEC group, and much collagen formed.However, less collagen formed on the surface of VEC group.

Test by flow cytometer
The three groups of cells were compounded and cultured with the material for one, three, five, seven days, respectively.The testing results of cell cycle and ploidy level are listed Table 1.The S phase cells of the co-cultured group on the 1 st and the 3 rd day was higher than that of the single cell group, no heteroploid cells were found.The effect of HBACM on the cell cycle and the ploidy of the three groups of cells are listed on Table 1.  1.The influence of the HBACM to the cell and DNA index of three group cells

Discussion
As a kind of artificial extracellular matrix (ECM), scaffolds provide three-dimensional space for the growth, reproduction and metabolism of the cells.The biocompatible of scaffold directly affects the adhesion, growth and proliferation of the cell.Different kinds of ECM are widely used nowadays, they are: biodegradable polymer; ceramic material; composite materials and natural extracellular matrix material (NECM).
Heterogeneous bones, which are obtained from animals such as pigs and cattle, have a rich resource and lower cost, and are easy to access and process.Moreover, the bone tissue retains the dense pore structure naturally and is with good effect of scaffold, which provides three-dimensional space for the proliferation, differentiation and the osteogenesis of the cells.At the same time, the bone tissue is with high bone induction activity and degradation rate matched well with the growth rate of new bone.Thus, it has great potential for developing as bone substitute material.However, if the untreated heterogeneous bones were implanted into human body, there will be a strong immune rejection.Hence, a variety of physical, chemical treatments are used to eliminate the antigen as far as possible, and simultaneously retain the useful biologically active substances and its bone induction activity, as well as its mechanical strength.Those are also the research focus of the heterogeneous bones tissue engineering transplantation.This study focused on the transplantation of the heterogeneous bones and the elimination of the cells antigen, and then used the heterogeneous bones as a superior scaffold for bone tissue engineering.

Biological compatibility of HBACM
Bone extracellular matrix is the intercellular substance of calcified bone tissue, and is consist by organic and inorganic ingredients.The organic ingredients are mainly: collagen fibers and some amorphous material, which are both the secretion of bone cells.Inorganic components are mainly calcium salt, which exist mostly as hydroxyapatite crystals.Lowpermeability method was selected to produce HBACM in this experiment.The cells were burst and the membrane structure was damaged.Other methods were also used to extract the cell membrane, cytoplasm, organelles and other ingredients.Only the cell was extracted, and the inorganic and organic ingredients were retained.Results show that the cells of the three groups could be attached to the HBACM and grew.The reasons maybe as follows: the natural pore system was not significantly damaged; the original trabecular bone, the trabecular bone gap and the lumen system was retained; the three-dimensional structure of the bone salt still existed.This natural structure was conducive to cell adhesion, growth and provide generous interior space and surface for the secretion of ECM.Therefore, the acellular bone matrix showed excellent biocompatibility.

Antigenicity of HBACM
It is generally believed that transplantation antigen of heterogeneous bones present in bone cells, hematopoietic cells, white blood cells, red blood cells, plasma, blood vessels and bone matrix.To transplant heterogeneous bones, we should reduce or eliminate the antigenicity of transplanted bones.After a series of physical and chemical treatment, the rib of pig was made as HBACM, which did not contain cells under the scanning electronic microscopy.Thus, it was of low antigenicity.Observation from the inverted phase contrast microscope, the three groups of cells grew along the edge of the pores of the material.The cells gradually extended, stretched across the pores, and the pseudopodium tended to contact with each other, they gradually grew, differentiated and proliferated.Stained histological sections and the result of scanning electron microscopy showed that the cells were closely attached, gathered into a group, and connected into a network.These indicated that the compatibility between cells and the compatibility between cells and HBACM were good, without any mutual exclusiveness.These further explained that there were none or extremely low antigenicity when using this method to prepare acellular bone matrix.

Cytotoxicity of HBACM
Flow cytometry could be used to determine DNA content and proliferation activity of the cells, and could analyse the cell cycle and the ploidy level.When the body suffers from cancer or precancerous lesions with malignant tendency, the DNA content of the cells changes abnormally, leading DNA aneuploid.Flow cytometry can detect aneuploid cells by measuring DNA content, which has been an effective tool making cancer for the diagnosis of tumor cytology.In this study, flow cytometry was used to determine the ploidy level and the DNA content of the three groups of cells that co-cultrued with acellular matrix scaffold in vitro.No aneuploid cells were found, indicating the scaffolds were without cytotoxicity and tumorigenesis.We could see from the result of cell cycle analysis that the cells of each group had normal growth cycle in the acellular matrix scaffolds.The S phase of the co-cultured group on the 1 st and the 3 rd day was higher than that of the single cell group, indicating high proliferation of the cells on scaffold materials when osteoblast was co-cultured with VEC.
With the research and development of biodegradable scaffolds with three-dimensional and multi-space structure, the bioactive bone transplantation materials formed by the three kinds of cells and acellular matrix bone shows a broad application prospects.

Conclusion
It is preferred not to use abreviations in the conclusionsor at least use the fuul description once, at the first conclusion.
molecules, living cells, and blood supply in the grafted tissue.However, the bone source and the site for supplying bone were limited in autografting, meanwhile it is difficult in the modeling of self-tissue, and therefore its clinical application is limited.There is certain immunologic rejection in the isotype heterogenous bone grafting, which worried many people.At the same time, the bone source is limited.Since the 1980s, with the rapid development of cell culture technology, tissue engineering bone provides a new way for solving this problem.In this study, heterogenic acellular extracellular matrix was compound with cell to construct tissue engineering bone, which was implanted into the animal.Then the bone defect repairing was observed.

Mark the cell with BrdU (5 -bromodeoxyuridine)-labeled cells
400 μL of BrdU was added into 200 mL of serum-free F 12 culture medium.35 bottles of 3 generations and 5 generations of rabbit osteoblast, VEC and the co-cultured cells were taken with the culture medium being discarded, and then, the serum-free F 12 culture medium marked by BrdU were added (6mL per bottle).The bottles were kept in the cell incubator at 37 ℃ with 5% of CO 2 and saturated humidity for one hour.After that, the supernatant was discarded and the cells were digested and collected, which was washed for thrice with culture medium to remove BrdU that had not incorporated cells.

The compound cell of heterogenous bone acellular matrix
30 blocks compound cell of heterogenous bone acellular matrix (pork ribs) with the size of 2.0cm × 0.5cm × 0.5cm were divided into three groups (10 blocks each group).Group A was compound with osteoblast, Group B was compound with VEC, and the Group C was compound with co-cultured cells.The three groups of cells marked with BrdU were inoculated with the density of 6 × 10 5 cells per block into HBACM, respectively.One block of each group was taken for in vitro testing, and the remaining 27 blocks were used for implanting into the animal body.

Preparation for animal model of bone defect
Resect the 1.5cm long radius of the double forelimbs of the rabbit and prepare animal model of bone defect.The tissue engineering bone which had been compounded and cultured for one week by the heterogenous bone acellular matrix and the cells of each group was implanted to the left side.The heterogenous bone acellular matrix was implanted to the right side as a control.
Select the material at 3 weeks, 6 weeks and 12 weeks to observe bone fracture.

6.2.4
The index of observation 6.2.4.1 Observation of gross morphology Three, six and 12 weeks after the operation, three animals in each group were sacrificed and the radius were dissected.Observe the development and the prognosis of the tissue engineering bone at various phases by naked eye.

X-ray examination of bone defect
After three, six, and 12 weeks of the operation, samples were taken, respectively.The X-ray (CR) radiography was used to observe the healing of bone defects, and in order to understand the conjugation of the implanted tissue engineering bone and the peripheral normal radius, also to understand the formation, change and moulding of the callus at the site with bone defect.

Trace and test the BrdU marked cells
The paraffin sections was prepared in three and six weeks, respectively, for BrdU immunohistochemistry stain, to detect BrdU marked cells, then to understand the survival of seed cell in the experimental animal.After HBACM and BrdU marked cells was co-cultured in vitro for one week, one tissue engineering bone in each group was taken for the production of paraffin section, in order to detect the compound cell of heterogenous bone acellular matrix in vitro.

6.2.4.4
Routine histological examination About 1.5 cm of the samples was selected from three experimental animals at various phases.The samples were taken with the implanted bone tissue engineering as the center and some normal bone on both ends.After fixed, decalcified, dehydrated and embedded, the samples were made into paraffin sections for HE, toluidine blue, Masson staining.
Pathological changes (such as growth and differentiation of the cells in vivo, inflammatory cell infiltration, degradation of the scaffold, rebuilding and remodeling of callus, recanalization of canal, and the revascularization) of HBACM compound with the cells in each group were observed under optical microscope.

Immunohistochemistry of type I collagen
The paraffin sections of the sixth weeks was taken for type I collagen immunohistochemistry stain, to understand the secretion of type I collagen by each group of cells at various phase.

Image analysis
Masson stains at the 3 rd and the 6 th week were selected and the border between the tissue engineering bone and the normal bone was detected by an image analyzer to measure the area of the vessel in each unit.The revascularization of tissue engineering bone in each group was observed.

Statistics method
The result of the experiment was expressed by mean±standard deviation.The analysis of variance and q test of the measurement data among groups were done by using SPSS 10.0 software packet.

Observation of gross morphology
The samples were taken and observed at the 3rd, 6th, and 12th week, respectively.Results show that the speed and degree of repairing the bone defect by tissue engineering bone from high to low in order were: that of the co-cultured cell group, that of the osteoblast group, that of the VEC group and that of the simple framework material group (Figs.1a, 1b, 1c, and 1d).

X-ray examination of bone defects
We could see from the X-ray examination that the speed and degree of repairing the bone defect by tissue engineering bone from high to low in order were: that of the co-cultured cell group, that of the osteoblast group, that of the VEC group and that of the simple framework material group (Figs.2a, 2b, 2c, and 2d).

Trace and test the BrdU marked cells
One week after compound in vitro, we could see from the immunohistochemical staining result that cells compound and grew well with HBACM, the cells attached to the pores of HBACM, closely adhere to the edge of HBACM and grew (Fig. 3).When the bone was implanted for three weeks and six weeks, the marked cells could still be detected in HBACM in vitro, and the new cartilage could also be observed (Figure 4).

Routine histological examination
In the 3 rd week, a large number of cartilages formed in tissue engineering bone.On the juncture with the normal bone, the formation of cartilage zone could be seen.osteoblast and osteoclasts coexisted in the pores of HBACM, in which the capillary grew (Fig. 5).In the 6 th weeks, the pores in HBACM gradually grew confluent, the blood vessel increased in the pores, and the cartilage cells gradually transformed to bone cells (Fig. 6).In the 12 th weeks, the marrow between tissue engineering bone and normal bone recanalized, and the fracture healed (Fig. 7).Fig. 4. BrdU immunohistochemical staining (400×).When the bone was implanted for three weeks, marked cells could still be detected in HBACM, and the new cartilage could also be observed.
Fig. 5. Toluidine blue in the 3rd week (200×) On the juncture with the normal bone, the grewformation of cartilage zone could be seen in the pores.osteoblast and osteoclasts coexisted in the pores.

Immunohistochemistry of type I collagen
After the three groups of cells compound with HBACM were implanted in vivo, type I collagen was expressed (Fig. 8), while VEC group was lower expressed.
Fig. 6.HE stain in the 6 th week (100×).In the 6 th weeks, the pores in HBACM gradually confluent, the blood vessel increased of HBACM.

Discussion
As the research development of cell biology, molecular biology, and biological engineering and materials science, tissue engineering was born as a new discipline."Tissue engineering" was put forward in 1987 by the National Science Foundation (USA), which refers to the science that uses cell biology and engineering principles to research and develop biological alternatives for the repair and improvement of wounded tissue and function.The basic premise of modern tissue engineering is to guide the tissue repair and regeneration by the application and the control of cell tissue and the micro-environment.It is a thoroughly new therapeutic mode to repair bone defects by using the methods and tools of tissue engineering, which has broad application prospects.With the rapid development of the current research of bone tissue engineering, tissue engineering bone has become one of the most promising tissue engineering achievements that will be come into use in clinical applications, which will lead a revolutionary change in the treatment of the fracture healing and the bone defects caused by trauma, tumor resection and malformation correction.

Trace and test the Brdu marked cells
The structure of BrdU is similar with thymidine, it is characteristic is that the methyl, which connects the fifth carbon atom and pyrimidine ring of phase Ⅲ in thymine, is substituted by bromide atom.During the synthesis process, BrdU can specifically incorporate into DNA at S phase, which is the same as thymine.Simultaneously, there is no cross-reaction between BrdU and thymine, and it is without radioactive material contamination.Therefore, BrdU has been used to mark cells in both in vivo and in vitro, and has been widely applied into the research of cell kinetics, as its high accuracy, rapidity and safety for marking cells.Generally speaking, it can be detected in vivo within eight weeks, and will not be longer than 12 weeks.In this study, BrdU was used to mark cells that cultured in vitro.After compound the marked cells with HBACM, in vitro test and in vivo test for implantation at the 3 rd week and at the 6 th week were done.Results show that cells compound and grew well with HBACM, the cells attached to the pores of HBACM, closely adhere to the edge of HBACM and grew.When the bone was implanted for three weeks and six weeks, the marked cells cultured in vitro could still be detected in HBACM, and the new cartilage could also be observed.These indicated that the histocompatibility between HBACM and cells was good; and it further indicated that the major ossification of the tissue engineering bone in the body was indeed due to the cells cultured in vitro.

Pathological process of bone tissue engineering repairing bone defects
Fracture healing is a complex process, which is continuous and is the result of the mutual synergistic effect between osteoblast and osteoclasts.The research results in this experiment showed the pathological process after the tissue engineering bone was implanted into animal body.The endochondral bone formation is the main effect during the fracture healing process.Under the effect of osteoblast, HBACM gradually osteoblasted outward; gradually transformed the fibrous tissue among the fracture ends and the pores of HBACM into cartilage.Then the pores grew confluent.At the same time, the surface of bone trabecula in HBACM began to change into small cavities, which gradually interconnected to form larger cavities.Vascular formed, the chondrocyte gradually proliferated, calcified and ossified in the larger cavities.The fracture healing process completed when the larger cavities grew confluent and recanalized with autogenous marrow cavity.

Comparison of the osteogenic ability of bone tissue engineering in vivo
Type I collagen is the major extracellular matrix secreted by osteoblast.It is expressed from the beginning of cell proliferation, and achieved to the maximum at synthesis stage of matrix.The results showed that after the three groups of cells compound with HBACM were implanted in vivo, type I collagen was expressed (Fig. 8), while VEC group was lower expressed.From the gross specimen and X-ray results, we could see that the speed and degree of repairing the bone defect by tissue engineering bone from high to low in order were: that of the co-cultured cell group, that of the osteoblast group, that of the VEC group and that of the simple framework material group.These indicated that fracture healing could be accelerated and the repairing of bone defect could be promoted by using the cocultured cell as seed cell.Simultaneously, the simple framework material group could also achieve fracture healing, which indicated that the HBACM might contain bone morphogenetic protein, with a strong cross-species induction of osteogenic activity; namely, induced undifferentiated mesenchymal cells differentiate into cartilage or bone, its effect was non-species-specific and could repair bone defects.

Comparison of revascularization ability of bone tissue engineering in vivo
There has been a significant progress in the research of bone tissue engineering.However, with the experiment object becoming large-scale, the research of bone tissue engineering bone is facing the key problem --rapid vascularization.Like other types of transplanted bone, adequate blood supply is the decisive factors to ensure tissue engineering bone to survive in vivo.Current research on the reconstruction of the blood supply in the new tissue engineering bone is still at the initial stage.The main methods are as follow: (1) combined transplantation of VEC and osteoblast (2) the use of VEGF to promote the growth of blood vessel (3) the application of microsurgical techniques in the revascularization of tissue engineering bone, including osteoblast + biomaterials + tissue flap with vascular pedicle embedded; osteoblast + biomaterials + vascular bundle implantation.Zhengfu Fan, et al prepared a deep facial flap animal model with a nameless vascular pedicle in forelimb.When the facial flap was applied to cover the biomaterials of compound cells to repair the 1.5 cm of radial bone defect, the speed of revascularization was obviously higher than that of the control group without facial flap after the repairing of bone defect was finished.Casabona, et al further designed a bio-engineered prefabricated flap.Human bone marrow stromal cells which were cultured for two weeks were delivered into the latissimus dorsi of athymic mice by a porous hydroxyapatite ceramic model.Eight weeks after the implantation, histologic examination revealed the presence of spongious bone tissue with rich blood supply.A simple myocutaneous flap was thus transformed into a composite osteomyocutaneous flap.Although the flap can cover any area or any known blood vessels, and had the possibility of preshaping the graft to the exact characteristics of the defect.However, two operations should be taken in vivo.The image analysis showed that the number of revascularization of tissue engineering in co-cultured cells group in the body in the 3 rd week was higher than that of the osteoblast group, the difference was statistically significant (P <0.05).In the 6 th week, the number of revascularization from high to low in order was: that of the co-cultured cells group, that of the VEC group, that of the osteoblast group, and the difference was statistically significant (P <0.01).The combined transplantation of VEC and osteoblast were used in this experiment and the same result was obtained, which made the new bone tissue engineered bone to entirely replace the autogenous bone graft entirely possible, and provide a excellent experience for tissue engineered bone to be implanted in vivo.
It can be seen, the activity of osteoblast could be enhanced, the fracture healing could be accelerated, the repairing of bone defect could be promoted and the revascularization of tissue engineering bone could be improved by using the co-cultured cell osteoblast and VEC as seed cell.There was mutual synergistic action in vitro and mutual promoted effects in vivo between the osteoblast and the VEC

Conclusion
This in vivo animal study suggests that: 1.The osteogenesis process of the regenerating bone was the formation of endochondral bone regeneration.2. The major ossification of the regenerationg bone occurred to the cells cultured in vitro.3.There was synergistic action between the osteoblast and the VEC whether in vivo or in vitro.4. Fracture healing could be accelerated and the repairing of bone defect could be promoted by using the co-cultured cell as seed cell.

Fig. 1 .
Fig. 1.Curves of cells viability in different groups

Fig. 2 .
Fig. 2. The immunocytochemical stain of osteoblast type I collagen (100×).The result was positive.The cytoplasm yellow staining and the cell nuclear blue staining.

Fig. 3 .
Fig. 3.The immunocytochemical stain of VEC factor VIII(100×).The result was positive.The cytoplasm revealed yellow staining and the cell nuclear blue staining.

Fig. 4 .
Fig. 4. HE staining of the cells of group C (100×).The HE staining showed the mixed growth of two types of cells with good cellular compatibility.

Fig. 5 .
Fig. 5. Masson staining of the cells of group C (100×).The Masson staining showed the mixed growth of two types of cells with good cellular compatibility.

Fig. 1 .Fig. 2 .
Fig. 1.Observation under the inverted phase contrast microscope (100×) when the osteoblast was co-cultured with materials for 5 days.The cells gradually extended, stretched across the pores.

Fig. 7 .
Fig. 7.In the 12 th weeks, medullary cavity formed in the tissue engineering bone.

Fig. 8 .
Fig. 8. Immunohistochemical of type I collagen in the 6 th week

Table 1 .
Comparison of different cells viability

6.2.3 Animal experiments 6.2.3.1 Animal grouping 27
New Zealand big-ear rabbits weighted 2.5 kilogram were selected and they were grouped into three groups with nine in each group.Groups of A, B, C were HBACM compound with osteoblast, VEC and co-cultured cells marked by BrdU.Animal grouping is listed as follows.

Table 1 .
Animal grouping of the New Zealand rabbits www.intechopen.com