Repair of alveolar cleft bone defects by bone collagen particles combined with hUC-MSCs in rabbit

Background: Alveolar cleft is a kind of cleft lip and palate, which seriously affects the physical and mental health of patients. In this study, a similar model of human alveolar cleft phenotype was established in rabbits to evaluate the effect of bone collagen particles combined with human umbilical cord mesenchymal stem cells (hUC-MSCs) on the repair of alveolar cleft bone defects. Methods: In this study, 24 adult Japanese white rabbits (JWRs) were selected and randomly divided into 4 groups. Including normal group, control group, materials group and MSCs group. The model of alveolar clefts was established by removing the incisors on the left side of the upper jaw. The normal group did nothing. In the control group, the incisors were removed and sutured directly. In the material group, the incisor were removed, then filled with bone collagen particles, and finally sutured. In the MSCs group, the incisors were first removed, then filled with bone collagen particles incubated by hUC-MSCs, and then stitched. Blood biochemical analysis was performed 3 months after the operation. Skull tissues were collected for gross observation, and micro-focus computerized tomography (micro-CT) analysis. Paraffin sections were prepared for histological and immunohistochemical staining. Results: The bone collagen particles and hUC-MSCs are not biotoxic and can promote alvenlus regeneration. Bone collagen particles combined with hUC-MSCs were much better than those used alone in inducing bone repair and regeneration. Conclusions: HUC-MSCs can be used as a bone generation inducer combined with bone collagen materials for bone regeneration and repair.

and the development of jaws, but also affects the physical and mental health of patients. Therefore, it is very important to establish an animal model of alveolar clefts that is similar to human alveolar cleft disease and can be duplicated. This method can provide a theoretical basis of the occurrence and development of the disease, and also provide a good scientific research foundation for the repair of alveolar clefts.
The most commonly used model animals are primates [1,2], sheep [3],canine animals [4], felids [5], rodents and rabbits [6,7]. Rabbits have the advantages of short growth cycle, easy feeding and low cost. Moreover, compared with other animals, rabbits are of moderate size, gentle temperament and easier to operate. Studies in our laboratory have also proved that rabbits can be used to establish animal models of alveolar cleft [8].
Therefore, rabbits were selected as experimental animals to establish the animal model of alveolar clefts. Alveolar cleft caused only by facial muscles can be corrected or repaired by surgical suture, but there is still no effective treatment for alveolar clefts caused by bone defect.
At present, the treatment methods of alveolar clefts can be divided into distraction osteogenesis and bone grafting.
Traction osteogenesis refers to the technology of bone correction or repair by applying physical traction force in a specific direction and frequency to partially or completely detached biological tissue so that the gap is gradually replaced by new bone. Binqer et al.
used traction osteogenesis to correct alveolar cleft [9]. Although this method avoids the immunogenicity of the foreign implanted tissue, the procedure is complicated, requires a long treatment period, and requires two operations to place and remove the retractor. Still not a good treatment.
Bone materials commonly used in bone grafting to include autogenous bone, allogeneic bone, allogeneic bone and tissue-engineered bone. Boyne et al. have repaired oronasal fistulas by inserting a small amount of cancellous bone from the autogenous ilium into the fractures [10]. This method is considered to be the gold standard for the clinical repair of alveolar clefts [11,12]. Nevertheless, autologous bone grafts are bound to cause donor trauma and deformity, which an ideal repair should avoid. Nique et al. once used allograft bones to treat alveolar clefts, and postoperative imaging showed that the tooth successfully erupted and grew into the graft bone, but this process required a long time [13]. El Deeb et al. fill hydroxyapatite in the crack of the alveolar clefts and found no tooth eruption [14]. Allogeneic bone allograft or artificial bone, although can avoid donor area deformity, but there is a risk of immune rejection and transmission of disease. With the development of tissue engineering technology, the application of tissue engineering bones to repair alveolar fractures are no longer a problem. Currently, the most widely used scaffold materials are collagen, hydroxyapatite and so on [15,16]. Because of the complex structure and function as bone tissue, it is difficult for a single material to meet the demand. It is commonly used to repair alveolar clefts by combining scaffold materials with factors or stem cells that induce bone regeneration. Compared with factors, stem cells have the advantages of low cost and easy access. Common stem cells are bone marrow mesenchymal stem cells [17], umbilical cord mesenchymal stem cells [18.19], embryonic stem cells and so on. Compared with stem cells derived from bone marrow, stem cells derived from umbilical cord have the advantages of low immunogenicity, rapid proliferation, wide availability and no ethical concerns [19,20]. Chronic spinal cord injury to dogs has been treated with hUC-MSCs combined with collagen scaffolds [21].
In this study, bone collagen granules prepared by decellularization and degreasing of bovine cancellous bone were used as scaffold materials. The main ingredients are collagen and hydroxyapatite. It not only preserves its natural three-dimensional porous structure, but also reduces its immunogenicity. On this basis, composite human umbilical cord derived mesenchymal stem cells were used to repair alveolar fissures.
The purpose of this study was to investigate the feasibility and effectiveness of bone collagen particles inoculated with hUC-MSCs in the rabbit model of alveolar clefts. The results indicate that hUC-MSCs combined with bone collagen particles may be a reliable alternative therapy for the repair of alveolar bone defects.

Blood analysis
Blood routine (Table 1&4), liver function (Table 2&5), renal function (Table 3&6) and BGP of each group were measured at 3 and 6 months after the surgery, and the values of each group were compared with those of the normal group. The blood routine results showed that NEUT content increased and LYM content decreased in the control group at 3 months, and they all tended to be normal at 6 months. The content of CRP, EO and NEUT in the material group increased at 3 months, while the content of LYM decreased, and all of them tended to be normal at 6 months. CRP contents in MSCs group decreased from 6 months, and other indicators showed no significant abnormality. Liver function results showed that ALT and AST levels in the control group were higher than those in the normal group at 3 and 6 months. The content of ALT and AST in the material group was higher than that in the normal group at 3 months and normal at 6 months. No significant abnormalities were found in the MSCs group. Renal function results showed that CR levels in the control group, the materials group and the MSCs group increased significantly at 3 months and tended to normal at 6 months. BUN content of the control group and MSCs group increased significantly at 3 months and tended to be normal at 6 months. BGP results showed that at 3 months, the control group was lower than the normal group, while the materials group and the MSCs group were not significantly different from the normal group ( Figure 2a). At 6 months, the content of BGP in the material group decreased, while that in the MSCs group increased even higher than that in the normal group (Figure 2b). To sum up, there was a certain inflammatory response to both the control group and the material group at 3 months, which increased the load on the liver and kidney. The MSCs group only increased the load of the kidney, but had no significant effect on the liver. At 6 months, the indicators of each group were basically normal. Serum BGP was continuously secret in MSCs group and was higher than normal group. We hypothesized that hUC-MSCs could prolong bone repair time. This indicated that neither bone collagen granule material nor hUC-MSCs were toxic, and that hUC-MSCs could reduce inflammatory reactivity.  Sirius red stainings: The Sirius red staining results showed that the orange color were darker at 6 months after the surgery than at 3 months after the surgery ( Figure 6A). The statistical results showed that the percentage of type 1 collagen in the normal group was about 60%, that in the BMP group was about 40%, and that in the control group and the material group was extremely low. At 6 months after the operation, the percentage of type 1 collagen in the normal group and the BMP group remained basically unchanged, while the percentage of type 1 collagen in the material group increased to 40%, while the percentage of the control group remained extremely low. Compared with the control group and the material group, the BMP group had the highest bone trabecular content and almost all of them were bright orange. The results showed that the effect of collagen particles combined with hUC-MSCs on inducing collagen types 1 were stronger than that of collagen particles alone.  8c&d). The black areas indicate the presence of osteoblasts. Therefore, it can be speculated that the number of osteoblasts at 3 months after collagen granule implantation is significantly higher than that at 6 months after implantation.

IHC (BMP2)
Immunohistochemical results showed no significant expression of bmp-2 in normal group and control group (Figure 9a,b,e&f). In the materials group and the MSCs group, bmp-2 was mainly concentrated on osteocytes and osteoclasts at the edge of trabecular bone (Figure 9c,d,g&h). The expression level of bmp-2 in the material group and the MSCs group at 3 months after surgery was significantly higher than that at 6 months after surgery. The expression level of bmp-2 was the highest in MSCs group. The results showed that the ability of active collagen particles combined with hUC-MSCs to induce the generation of BMP2 was better than that of bone collagen particles alone.

Proliferation and apoptosis analysis
Ki67 ( Figure 10B) and TUNEL ( Figure 10A) methods were used to detect cell proliferation and apoptosis in each group at 3 and 6 months after surgery. TUNEL assay ( Figure 10C1) showed that the apoptosis rate of the control group was similar to that of the normal group of 3 months after surgery. The apoptosis rate of the material group was lower than that of the normal group. The apoptosis rate of MSCs group was significantly higher than that of normal group. The immunohistochemical test results of Ki67 ( Figure 10C2) showed that the proliferation rate of the control group and the material group was significantly lower than that of the normal group at 3 months after the surgery. The proliferation rate of the MSCs group was similar to that of the normal group. At 6 months after the surgery, there was no significant change in the proliferation rate of the control group, which was still lower than that of the normal group. The proliferation rate of the material group increased obviously but did not exceed that of the normal group. The proliferation rate of the MSCs group was still close to that of the normal group.
Comprehensive analysis showed that the apoptosis rate and proliferation rates of the normal group remained unchanged. The control group showed high apoptosis rate and low proliferation rate in both periods. In the material group, the apoptosis rate and proliferation rate were significantly increased. The MSCs group showed that the proliferation rates remained unchanged while the apoptosis rates decreased significantly.
Therefore, it can be speculated that the combination of bone collagen particles of MSCs can further to promote cell proliferation and apoptosis, thus promoting bone regeneration.

Discussion
HUC-MSCs were taken from neonatal umbilical cords, which are medical waste. Therefore, it has the advantages of abundant source, low cost, no harm to donors and no ethical issues involved [18]. This makes it an ideal candidate for the potential for stem cells in medical applications [22]. Although hUC-MSCs have good bone induction, they are easy to be absorbed and degraded in vivo. Therefore, an ideal scaffold material is needed to enable hUC-MSCs to better act on bone defect sites [20]. Therefore, future studies can start from how to improve the bone conversion to hUC-MSCs to different degrees of bone defect.

Conclusions
First, we established the animal model of alveolar clefts based on the existing experience.
The bone collagen particles combined with hUC-MSCs were then implanted into the rabbits. Bone regeneration and repair to each group were observed at 3 and 6 months after implantation. The effect of bone collagen particles combined with hUC-MSCs in inducing bone repairs and regeneration is far better than that of bone collagen particles alone. This study provides a new method of the repair of alveolar cleft.

Isolation & culture of hUC-MSCs
HUC-MSCs were isolated from Wharton's jelly. In this study, tissue blocks adherent culture was used to isolate hUC-MSCs. The blood vessels in the umbilical cord were first removed and the tissue cut into about 1 cubic millimeter pieces. Finally, the tissue blocks were evenly spread on the bottom of the petri dish for primary culture. HUC-MSCs within five generations were collected to incubate bone collagen particles.

Preparation of implant materials
Bone collagen particles are made of bovine cancellous bone by special decellularization and degreasing process, which can reduce its immunogenicity. It also preserves the natural structure of the bon, which is good for the growth of cells and blood vessels. The bone collagen particles were provided by zhenghai biotechnology co. LTD Yantai, China .  Figure 1f-h). The normal group did not receive any treatment. In the control group, collagen membrane was directly covered and skin was sutured after incisors was removed.
In the material group, after the incisions were removed, the holes were filled with bone collagen granules, and then the collagen membrane was covered and the skin was sutured. In the MSCs group, after the incisions were removed, the holes were filled with bone collagen particles incubated by hUC-MSCs, and then the collagen membrane was covered and the skin was sutured. The rabbits were treated with antibiotics for 1 week to prevent infection. Blood was collected from each group at 3 and 6 months after surgery.
All rabbits were euthanized and the upper jaw was examined and obtained for further evaluation.

Blood analysis
At 3 months after the surgery, 3 rabbits were randomly selected from each group. Blood routine, liver function, kidney function and BGP of rabbits were detected by collecting 3.5ml of venous blood from the ears. At 6 months after the surgery, the indexes of the remaining 3 rabbits were detected.

Micro CT analysis
3 months after the surgery, one rabbit was randomly selected from each group to make a skull model. The procedure was repeated 6 months after surgery. The skull was photographed to record its appearance. Bone regeneration in skull was evaluated by micro CT. Bone regeneration in the alveolar cleft was evaluated using a micro CT system (SIEMENS Inveon Research Workplace 4.2, Beijing). The three-dimensional repair of the injuries to each group was observed, and the trabecular bone and bone density values were recorded.

Histology staining
Rabbits were euthanized and histological evaluated at 3 or 6 months postoperatively. The specimens were immersed in 4% paraformaldehyde for 24h and then decalcified in 10% EDTA. After decalcification, the tissue was first embedded with paraffin, and then paraffin sections with a thickness of 4um were prepared by the microtome. Primary anti-Ki67 (ab15580,1:1000 dilution; Abcam Ltd) was used to detect cell proliferation by immunofluorescence. TUNEL can be used to locate apoptotic cells. Three regions were randomly selected and the percentage of Ki67 and TUNEL positive cells was quantified using Image J software.

Statistical analysis
All values are expressed as the mean ± SD. P < 0.05 indicates statistical significance.

Ethics approval and consent to participate
The study was approved by the ethics committee of the National Research Institute for Family Planning.

Availability of data and materials
All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.

Funding
This work was funded by grants from the National Key Research and Development Program of China (2016YFC1000803).

Authors' contributions
XCS, XM and HFX designed the study. XCS, HW and JHL were responsible for the vivo surgery and performing the procedure. XCS was responsible for in vitro experiments. XCS, HW were responsible for hUC-MSCs culture. XCS, HW and HFX prepared the manuscript.
XCS, HW, DZ and HFX were responsible for revising the manuscript critically for important intellectual content. All authors read and approved the final manuscript.    Mean SD values were calculated for each group. * represents the statistical difference between each group and the normal group. # represents the statistical difference between each group and the control group.*, # P0.05; **, ## P0.01. BUN: blood urea nitrogen CR: creatinine UA: uric acid.     The general appearance of the skull and the red box is the surgical area. A sampling group 3 months after surgery; B sampling group 6 months after surgery.
The red box and red plus sign assist in displaying the recovery of the transplanted area. All groups were compared with the control group, and the difference was expressed as *.The red box represents the surgical area.      cells that are proliferating. * represents the statistical difference between each group and the normal group at 3 months. # represents the statistical difference between each group and the normal group at 3 months.