Application of Bone Collagen Matrix combined with HUC-MSCs for Alveolar Process Cleft in a Rabbit Model

Background: Most materials used clinically for lling severe bone defects lack the ability to induce bone regeneration or low bone conversion rate, therefore, their therapeutic effects are limited. Human umbilical cord mesenchymal stem cells (HUC-MSCs) have good osteoinduction. The mechanism of heterogeneous bone collagen matrix combined with HUC-MSCs in repairing bone defects of alveolar process cleft is still unclear. Methods: Here, a rabbit alveolar process cleft model was established to evaluate the repair effect and molecular mechanism of bone collagen matrix and its combination with HUC-MSCs on critical-sized bone defects in the alveolar process. We randomly divided 48 young Japanese white rabbits (JWRs) into normal, control, material and MSCs groups. The alveolar process cleft model was established by removing the bone of equal volume at the left maxillary. Bone collagen matrix combined with HUC-MSCs that were then implanted in the defect area. X-ray analysis and blood analysis were analyzed 3 months after surgery. Skull tissues were collected for micro-focus computerized tomography (micro-CT) analysis and histochemical staining. The above experiment was repeated 6 months after surgery.Results: the main ndings Results: It was found that bone collagen matrix and HUC-MSCs showed good biocompatibility. Heterogeneous bone collagen matrix combined with HUC-MSCs signicantly enhanced osteoinduction and osteoconduction. Conclusions: This experiment provides a new method for repairing alveolar process cleft. Trial registration: Not applicable.


Introduction
Alveolar process cleft is common in clinic. Alveolar bone defects caused by birth defect trauma and in ammation seriously affect the patient's facial and oral functions. It also brings great di culties to the patient's subsequent denture repair. The goal of alveolar bone reconstruction is to obtain bone with physiological function and eventually restore facial morphology and occlusal function. At present, autogenous bone transplantation is still the clinical gold standard, the common autogenous bone has skull [1], cancellous bone [2], ilium [3] and so on. In addition, there are dozens of bone substitutes, such as allogeneic bone [4], allogeneic bone [5] and tissue-engineered bone [6], that have been applied in clinical practice.
The ideal bone repair material needs to consider a number of factors. On the one hand, it should have a wide range of sources, high biocompatibility and safety. On the other hand, it must have certain plasticity and degradability. In addition, it also need to have good bone conductivity and bone inductance. The collagen matrix used in this study was made from bovine cancellous bone through a series of decellularization and degreasing processes. Its main ingredients are hydroxyapatite and collagen. The material not only greatly reduced its immunogenicity, but also maintained the natural structure of the bone. The material has a suitable pore size to facilitate the growth of cells and blood vessels. The environment of bone regeneration is complex, so it is di cult to meet the need of bone regeneration simply using Bone collagen matrix. Studies have shown that HUC-MSCs play an important role in inducing bone regeneration [7][8][9]. HUC-MSCs have the advantages of wide availability, rapid proliferation and low immunogenicity [9]. Fetal umbilical cord belongs to medical waste, so the HUC-MSCs have low ethical controversy. Therefore, in this study, HUC-MSCs were inoculated into bone collagen matrix to induce bone regeneration and repair.
How to make a suitable animal model of alveolar bone defect according to the clinical characteristics of oral bone grafting surgery is the basis and focus of studying alveolar bone repair and evaluating the osteogenic ability of bone grafting materials. Many researchers have used monkey [10], beagle [11][12], miniature pig [13][14], rabbit [15], rat [16] and other experimental animals to make bone defect models in different parts of their jaws. After the implantation of bone material, the ability to reconstruct the jaw bone was assessed by pathological methods. Compared with other animals, rabbits have the advantages of short growth cycle, easy feeding and low cost. And the rabbit body size is moderate, gentle temperament, easier to operate. So many researchers have also used rabbits to make models of alveolar process cleft [17][18][19].
In this study, JWRs were selected as experimental animals based on our previous research [20], and part of their left maxilla was removed by surgery to prepare the alveolar process cleft model. Considering that rabbit has more blood supply sources and stronger self-healing ability than human, the bone defect should meet the CSD (critical size defect) [21]. This provides an important model basis for evaluating the osteogenic capacity of Bone collagen matrix after inoculation with HUC-MSCs.

Isolation and culture of HUC-MSCs
HUC-MSCs were isolated from Wharton's jelly using tissue block adherent culture. First, the blood vessels of the human umbilical cord were removed, and then the cord was cut into about one cubic millimeter of tissue. The tissue block was attached to the bottom of the cell culture dish and cultured in a carbon dioxide incubator for 20 days. The primary cells were collected and then passed for culture. The HUC-MSCs used in this study were previously identi ed by our lab [22].

Preparation of implant materials
The bone collagen matrix is the heterogeneous bone matrix made from bovine cancellous bone after a series of processes, retaining its natural three-dimensional porous structure. The main ingredients are hydroxyapatite and collagen. The collagen membrane that covers the site of the injury after adding the Bone collagen matrix were derived from bovine skin. The thickness of the collagen membrane is about 0.8 mm. Before use, cut the collagen membrane into small pieces equal to the defect area. The Bone collagen matrix and the collagen membrane were provided by zhenghai biotechnology co. LTD Yantai, China . HUC-MSCs (concentration : 10 7 cells/mL) within 5 generations were collected to be inoculated with bone collagen matrix and cultured in carbon dioxide incubator for 0.5 h.

Groups and treatment
In this study, we used 48 female JWRs (bodyweight: 2000±300 g). These JWRs were purchased from huafukang biotechnology co. LTD Beijing, China . All these animals were kept in the animal room of National Research Institute for Family Planning. Provide them with clean water and food. Indoor conditions are as follows: temperature (24±1℃), air humidity (55%±5%), noise (less than 60dB), lighting time (12 h). Keep the room clean, dry and ventilated at all times. The experiment was approved by the local research and ethics committee.
The study was designed to be divided into four groups, each of which was randomly assigned to twelve JWRs. They were normal, control, material and MSCs groups. Rabbits were anesthetized by intravenous injection of serazine hydrochloride into the ear margin (concentration: 1-2 mg/kg). Look for the left maxilla after the rabbit is anesthetized. The alveolar process cleft model was established by removing the equal volume of the jaw bone with the rongeur (Fig. 1).
The normal group was fed normally. In the control group, after removing part of jaw bone, collagen membrane was directly covered and the muscles and skin at the injured site were sutured. In the material group, after removing part of jaw bone, the bone collagen matrix of the same volume was lled, and then the collagen membrane was directly covered and the muscles and skin of the injured site were sutured. In the MSCs group, after removing part of jaw bone, the bone collagen matrix of the same volume composite HUC-MSCs was lled, and then the collagen membrane was directly covered and the muscles and skin of the injured site were sutured. Conventional anti-in ammatory therapy was given for 1 week to prevent postoperative infection. Three rabbits were taken from each group at 3 and 6 months postoperatively. All rabbits were euthanized, and the transplant site was examined. Fresh skull tissue was rst xed in 4% paraformaldehyde for 24h, then decalci ed with 10% EDTA for 1 month, and nally embedded in para n. Para n sections (6 mm) were prepared by rotary microtome (Leica RM2245, Leica, Gmbh, Germany). For histology staining, para n sectionswere depara nized in xylene and rehydrated through graded alcohol solutions to water.

X-ray analysis
X-ray analysis was performed on the surgical sites of each group 3 and 6 months after surgery. We used a Softex M-60 x-ray machine (Kanagawa, Japan) at the Beijing ornamental animal hospital. The examination parameters were set as follows: 80 kV, 125 mA and an exposure time of 40 ms.

Blood analysis
Three rabbits were randomly selected from each group 3 months and 6 months after surgery. About 3.5ml of blood was collected by ear vein sampling, some of which were used for direct detection and the other for serum separation. Blood routine, liver function, renal function and serum bone gla protein (BGP) of rabbits were detected. Routine blood tests were performed using an LH 750 automated haematology analyser (Beckman Coulter, USA). The blood biochemistry test was performed using a DXC 800 automated biochemical analyser (Beckman Coulter, USA).

Micro CT analysis
Three rabbits were randomly selected from each group at 3 months and 6 months after surgery. It was euthanized and a skull model was made. The general appearance of the skull is recorded from lateral and vertical perspectives. The maxillary region of the skull was scanned by micro CT. Bone regeneration in each group was evaluated by micro CT. Bone regeneration in the material transfer area was evaluated by a micro CT system (SIEMENS Inveon Research Workplace 4.2, Beijing). The repair of maxillary region in each group was observed from a stereoscopic perspective. The bone trabeculae and bone mineral density of bone regeneration area in each group were analyzed.

Hematoxylin and eosin (HE) staining
HE staining was used to observe tissue morphology. The nucleus is stained blue by hematoxylin and the other areas are colored red by eosin. TE2000-U inverted phase contrast microscope (Nikon, Tokyo, Japan) was used to observe bone histomorphological changes.

Periodic Acid-Schif (PAS) staining
PAS staining was used to assess the glycogen content. PAS staining was performed using a commercial kit (SenBeiJia Biological Technology Co. Ltd, NanJing, Jiangsu, China) according to the manufacturer's instructions. Brie y, the sections were incubated with periodic acid solution in dark for 5 min, then incubated with the Schiff reagent in dark for 20 min at room temperature. The sections were counterstained with Lillie-Mayer's hematoxylin. The samples were visualized by using differential interference contrast (DIC) optics microscopy (DM IL LED, Leica Gmbh, Germany).The structure of cartilage can be dyed deep purple or deep red.

Sirius redstaining
Sirius red staining was used to detected different collagen bers. Sirius red staining was performed using a commercial kit (SenBeiJia Biological Technology Co. Ltd, NanJing, Jiangsu, China) according to the manufacturer's instructions. Brie y, the sections were incubated with Sirius red for 1 h at room temperature, then counterstained with Mayer's hematoxylin. Sirius red can dye type 1 collagen bright orange. Image J software was used to calculate the relative percentage of type 1 collagen staining area under different elds in each group.
2.10 Bone-speci c Alkaline Phosphatase (ALP) assay Bone-speci c ALP is one of the phenotypic markers of osteoblasts, which can directly re ect the activity or function of osteoblasts. Calcium-cobalt staining of alkaline phosphatase was used to detected the bone-speci c ALP content by a commercial kit (KeyGEN BioTECH Co.Ltd, NanJing, Jiangsu, China) according to the manufacturer's instructions. Brie y, the sections were incubated with ALP solution for 5 min, then incubated with Cobalt nitrate solution for 2 min at room temperature. The sections were counterstained with eosin. Osteoblasts can be dyed black.
2.12 Detection of apoptosis assay by TdT-mediated dUTP nick-end Labelling (TUNEL) TUNEL was performed by using an in-situ Cell Death Detection Kit, Fluorescein (Roche, Mannheim, Germany) according to the manufacturer's instructions. For the correlation of TUNEL with nuclear morphology, sections were counterstained with Hoechst 33258. Apoptotic cells were counted in 3 different optical elds (magni cation ×400) selected in a random manner. Samples were viewed at excitation 488 nm/emission 512 nm by uorescence microscopy (ZEISS LSM 510 META, Germany). At least 500 cells for each sample were evaluated for apoptosis in different optical elds (magni cation ×400) randomly selected. The results were expressed as the ratio of TUNEL-positive cells to total cells. Each treatment was repeated at least three times.

Statistical analysis
The results are presented as mean ± SD and P < 0.05 was considered statistically signi cant. Data were analyzed statistically by One-way ANOVA and the Student's t-test with GraphPad Prism software (GraphPad Prism 6).

Morphometric analysis of alveolar process tissues
General visual observation showed that the injury side was compared with the normal side in each group. At 3 months, no obvious repair was found in the bone defect area of the material group and the control group, while partial repair was found in the bone defect area of the MSCs group ( Fig. 2A). At 6 months, there was no obvious repair in the bone defect area of the material group, partial repair in the bone defect area of the control group, and basic repair in the bone defect area of the MSCs group (Fig. 2B).
In order to evaluate bone repair of alveolar process cleft, X-ray was used for preliminary analysis (Fig. 3), in which bone density was positively correlated with brightness. Compared with the normal group, we found that the bone mineral density of the control group was the lowest, that of the MSCs group was the highest, and that of the material group was between the two groups at 3 months. At 6 months, the bone density of the MSCs group was still higher than that of the other groups. Therefore, we can infer that the osteogenic rate of bone collagen matrix combined with HUC-MSCs is signi cantly higher than the effect of bone collagen matrix alone.
In order to further analyze the absorption of bone materials and bone formation of alveolar process cleft, micro-CT scan was used to detect skull tissues from different perspectives at 3 and 6 months after surgery. The red box is the material transplantation area. The injury side was compared with the normal side in each group. The results of micro-CT scan were consistent with those of the General observation. A signi cant amount of new bone tissue was found on the injury side of the MSCs group 6 months after surgery (Fig. 4B). We calculated the percentage of bone density and the percentage of trabecular bone in each group (Fig. 4C). At 3 months after surgery (Fig. 4Ca) (Fig. 4Cb). The percentage of bone density in MSCs group (82.936 ± 2.112 %) was signi cantly higher than that of the other two groups (material group: 43.858 ± 0.522 %, control group: 1.29 ± 0.522 %) at 6 months after surgery (Fig. 4Cd). Therefore, the osteogenic ability of bone collagen matrix combined with HUC-MSCs was signi cantly better than that of bone collagen matrix alone.
3.2 Microscopic structure of bone defect site detected by HE staining HE staining showed that no signi cant bone repair was observed in the control group in both periods ( Fig.  5 Aa1 & Ae1). 3 months after the surgery, a few bone bers, bone marrow, bone trabeculae and a large number of cavitation structures were observed in the damaged area of the material group (Fig. 5 Ac1). At 6 months after the surgery, trabecular bone, bone marrow and cavitation structures were observed in a small number of bone repair areas in the material group (Fig. 5 Ag1). In the MSCs group, no cavitation was observed in the damaged area at 3 months after surgery, and a large number of bone trabeculae and brous tissues were observed (Fig. 5 Ad1). At 6 months after the surgery, bone trabeculae in the damaged area of the MSCs group had been connected to form bone tissue (Fig. 5 Ah1). The results of HE staining indicated that MSCs group had a better repair effect.

Measurement of serum Bone gla protein (BGP)
BGP is a bone-speci c protein. It is synthesized by the osteoblast and incorporated into the bone matrix. Serum BGP results showed that the BGP content of the control group and the material group was close to that of the normal group, while that of the MSCs group was signi cantly higher than that of the normal group (Fig. 5 B1 & B2). The results showed that the osteogenic ability of bone collagen matrix combined with HUC-MSCs was signi cantly better than that of bone collagen matrix alone.
3.4The effect of bone collagen matrix combined with hUC-MSCs on osteoblasts, collagen and saccharides associated with bone formation ALP staining was used to detected the osteoblasts that were stained black color. The results of ALP staining showed that the normal maxillary was a uniform bone matrix without osteoblasts (Fig. 6 a & e).
By comparing the results of PAS staining in the two periods, we found that there were no obvious black areas in the control group (Fig. 6 b & f). In the material group, black appeared on the edge of the bone trabecular at 3 months after surgery (Fig. 6 c), and no black area was observed at 6 months after surgery (Fig. 6 g). In the MSCs group, black appeared at the edge of the bone trabecular at 3 months after surgery (Fig. 6 d), and remained at 6 months after surgery (Fig. 6 h). The results showed that bone collagen matrix combined with HUC-MSCs could promote osteoblasts better than bone collagen matrix alone.
Sirius red staining was used to analyze the collagen distribution. Sirius red can dye type 1 collagen into bright orange. The Sirius red stain showed the uniform distribution of type 1 collagen in the normal maxillary ( Fig. 7 Aa & Ae). In the control group, only a very small amount of type 1 collagen was present (Fig. 7 Ab & Af). In the material group, a small amount of collagen type 1 was observed in the bone defect area after the implantation of bone collagen matrix alone (Fig. 7 Ac & Ag). However, after the implantation of bone collagen matrix combined with HUC-MSCs, a large amount of collagen type 1 was observed in the bone defect area (Fig. 7 Ad & Ah). The statistical results showed that the content of collagen type 1 in the MSCs group was similar to that in the normal group (Fig. 7 B & C). The content of type 1 collagen in the control group and the material group was signi cantly lower than that in the normal group. The results showed that bone collagen matrix combined with HUC-MSCs could induce type 1 collagen more effectively than bone collagen matrix alone.
PAS staining was used to assay the saccharide content. The saccharide is a constituent part of matrix of cartilage. The results of PAS staining showed that the normal maxillary was a uniform bone matrix without chondrocytes (Fig. 8 a & e). By comparing the results of PAS staining in the two periods, we found that there were no obvious red or fuchsia areas in the control group (Fig. 8 b & f). At 3 months after the surgery, the bone trabecular border of the material group appeared red or fuchsia (Fig. 8 c), which was signi cantly reduced at 6 months after the surgery (Fig. 8 g). At 3 months after the surgery, the bone trabecular of the MSCs group showed large areas of fuchsia (Fig. 8 d), while at 6 months only the edges of the new bone tissue showed fuchsia (Fig. 8 h). The results showed that bone collagen matrix combined with HUC-MSCs could promote chondrocytes better than bone collagen matrix alone.

3.5The effect of bone collagen matrix combined with hUC-MSCs on BMP-2
The expression of BMP-2 in tissues is detected by immunochemistry. Since there was no obvious bone repair in the control group, BMP-2 expression was not observed in the two periods ( Fig. 9 b & f). In the material group, BMP-2 was mainly concentrated in osteocytes and osteoclasts at the edge of the bone trabecular ( Fig. 9 c & g). In the MSCs group, BMP-2 was mainly expressed at the edge of bone trabecular at 3 months after surgery (Fig. 9 d), and at 6 months after surgery, BMP-2 was mainly expressed in the growth area of new bone tissue (Fig. 9 h). The results showed that the ability of bone collagen matrix combined with HUC-MSCs to induce BMP-2 generation was better than that of bone collagen matrix alone.
3.6 Proliferation and apoptosis analysis of bone defect site detected TUNEL assay was used to detect apoptosis in the bone defect center of each group, and the apoptotic nuclei were stained brown (Fig. 10 A). Ki67 immunohistochemistry was used to detect the proliferation of cells in the bone defect center of each group, and the proliferation cells were labeled with green uorescence (Fig. 10 B). Since no bone tissue was formed in the bone defect center of the control group and the material group, the cell proliferation rate and apoptosis rate were statistically 0. TUNEL results showed that the apoptosis rate of the MSCs group was signi cantly higher than that of the normal group at 3 months after surgery. The apoptosis rate at 6 months after surgery was signi cantly lower than that of the normal group (Figure 10 C1). The immunohistochemical results of Ki67 showed that the proliferation rate of the MSCs group was close to that of the normal group in both periods (Figure 10 C2).
The proliferating cells of MSCs group were mainly concentrated in the bone growth area (Figure 10Bh1). Therefore, bone collagen matrix combined with HUC-MSCs can effectively promote bone regeneration and repair by regulating cell proliferation and apoptosis.

Assessment of postoperative health status of rabbits
Blood routine (Table 1& Table 4), liver function ( Table 2 & Table 5) and renal function ( Table 3 & Table 6) were used to estimate health status of rabbits at 3 and 6 months after the surgery. Then the values of each group were compared with those of the normal group and the control group. Blood routine results showed that lymphocyte (LYM) content decreased and neutrophil granulocyte (NEUT) content increased in the MSCs group at 3 months, and returned to normal at 6 months. C-reactive protein (CRP) content in the material group was higher than that in the normal group at 3 and 6 months, but the value decreased. CRP content in the control group was higher than that in the normal group at 3 months and close to that in the normal group at 6 months. It was suggested that the control group and MSCs group had mild in ammatory response at 3 months and tended to be normal at 6 months. The material group remained in a state of mild in ammation. Liver function results showed mildly abnormal aspartate aminotransferase (AST) and alanine aminotransferase (ALT) values in the material group at 3 months and normal values at 6 months. This suggests that the control group had a mild in ammatory response at 3 months and tended to be normal at 6 months. ALP content in the control, material and the MSCs groups was higher than that in the normal group at 3 months and lower than that in the normal group at 6 months. This suggests that osteoblasts increase substantially at 3 months and become normal at 6 months. Renal function results showed that CR was higher in the control group and the material group, and blood urea nitrogen (BUN) and creatinine (CR) were higher in the MSCs group at 3 months. All indicators were normal at 6 months. To sum up, compared with the normal group, the control group, the material group and the MSCs group all had mild in ammatory responses at 3 months. At 6 months, all the indicators in each group were basically normal. CRP in the material group was relatively high at 6 months, compared with that in the MSCs group, adding HUC-MSCs reduced the in ammatory response.

Discussion
Bone induction refers to the induction of connective tissue adjacent to the bone graft area by bone growth factors or seed cells in the bone material. By affecting undifferentiated bone progenitor cells, promoting their differentiation and proliferation and eventually becoming osteoblasts, the formation of new bone is facilitated.
In recent years, MSCs have been widely used in the treatment of various organ and tissue injuries [23][24][25].
In vivo experiments have con rmed that HUC-MSCs can repair vascular and tissue epithelial injury [26]. Studies have also found that HUC-MSCs play an important role in inducing bone regeneration [7][8][9]. HUC-MSCs are characterized by easy extraction, multidirectional differentiation, short proliferation time, low immunogenicity, and long survival time after transplantation, have become the preferred seed cells for transplantation [27][28].HUC-MSCs were obtained from neonatal umbilical cords. Neonatal umbilical cords are clinical waste, so there are few ethical implications [29]. The obtained stem cells showed rapid selfrenewal [30]. HUC-MSCs also express low levels of major histocompatibility complex II and costimulatory molecules, thereby reducing rejection [31]. HUC-MSCs are easy to be absorbed and degraded in vivo, so appropriate scaffold materials are needed to play a better role. Collagen scaffolds is an ideal scaffold material to enhance the action of HUC-MSCs on bone defect sites [8].
The bone collagen matrix used in this study is bone matrix made from bovine cancellous bone after degreasing and decellularization process. After decalci cation and deproteinization, the heterogeneous bone matrix is mainly composed of type 1 collagen, which is insoluble and highly hinged. The heterogeneous bone matrix has good bone-guiding activity, toughness and strength. The material preserves the natural structure of the bone and has the right pore size for cells and blood vessels to grow in. Due to the removal of antigens, the antigenicity of the material is very weak, and the rejection after implantation is not obvious. The heterogeneous bone matrix is more widely derived than the allogeneic bone, and can be degraded and absorbed in a shorter time, all of which meet the requirements of ideal carrier.
Studies have shown that the function of collagen biomaterials alone no obvious effective in bone repair [32]. The combination of bone collagen matrix can slowdown the degradation rate of HUC-MSCs, thus prolonging the bone repair time.
Therefore, we believe that HUC-MSCs combined with bone collagen matrix has obvious osteogenic induction ability. The joint transplantation of both contributed to the repair of maxillary injury and restoration of occlusal function, likely through promote the expression of osteoblasts, chondrocytes, type 1 collagen and BMP-2. The results of blood analysis suggested that HUC-MSCs could reduce in ammatory response. Studies have suggested that HUC-MSCs may play a repair role by inhibiting in ammation [22]. Therefore, its application to the human body caused by the in ammatory response needs further research.

Conclusions
With the development of tissue engineering methods, the HUC-MSCs combined with collagen materials is a promising strategy for bone repair. The method of HUC-MSCs combined with bone collagen matrix to ll l a bone defect site is simple, rapid and suitable for the defective maxilla. This study shows that the combination of HUC-MSCs with bone collagen matrix is a promising strategy in the eld of regenerative medicine and bone repair.

Consent for publication
Not applicable.
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.*, # P 0.05; **, ## P 0.01. BUN: blood urea nitrogen; CR: creatinine; UA: uric acid. Table 4 Blood routine test results at 6 months postoperatively.
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.*, # P 0.05; **, ## P 0.01. RBC red blood cell; HCT: hematocrit; RDW-CV: red blood cell volume distribution width; RDW-SD: red blood cell distribution width; MCV: mean corpuscular volume; HBG: hemoglobin; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; platelet; PDW: platelet distribution width; MPV: mean platelet volume; PLCR: platelet-large cell ratio; PCT: platelet volume; CRP: C reactive protein. Table 5 Liver function test results in blood at 6 months postoperatively.
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.*, # P 0.05; **, ## P 0.01. ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase; TP: total protein; ALB: albumin; GLB: globulin; TBIL: total bilirubin; DBIL: bilirubin direct; IBIL: indirect bilirubin. Table 6 Renal function results test in blood at 6 months postoperatively.
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.*, # P 0.05; **, ## P 0.01. BUN: blood urea nitrogen; CR: creatinine; UA: uric acid.    Micro CT results. A CT images from different angles 3 months after surgery; B CT images from different angles 6 months after surgery; C-a The percentage of bone trabeculae 3 months after surgery; C-b The percentage of bone density 3 months after surgery; C-c The percentage of bone trabeculae 6 months after; C-d The percentage of bone density 6 months after surgery. The injury side of each group was compared with that of the control group and the difference was denoted by *. The difference between the injury side and the normal side was expressed as #. #### , **** P 0.0001. The red box is the surgical area.   Sirius red staining results (40×). A a-d Sirius red staining results 3 months after surgery; e-h Sirius red staining results 6 months after surgery. a, e Normal group; b, f Control group; c, g Material group; d, h MSCs group. Mark the area of the positive signal with a red arrow. B The percentage of type 1 collagen 3 months after surgery. C The percentage of type 1 collagen 6 months after surgery. All groups were compared with the Normal group, and the statistical difference was denoted by *. *** P 0.001 ,**** P 0.0001.   The percentage of proliferative cells in each group at 3 or 6 months. Cells labeled in green represent cells that are proliferating. * Represents the statistical difference between each group and the normal group. # represents the statistical difference between each group and the normal group. * P < 0.05; # # P < 0.01.