| Restoration of lost alveolar bone support remains as one of the main objectives of periodontal surgery. Amongst the various types of bone grafts available for grafting procedures, autogenous bone grafts are considered to be the gold standard in alveolar defect reconstruction. However, autologous bone collection brings a series of complications and amounts of patients treated with transplantation materials neither artificial bone graft nor autogenous bone were observed to be suffering from resorption, which often results in additional bone grafts. Therefore, there is an urgent need to develop an ideal scaffold material to replace autogenous bone. Because of high biocompatibility and osteocompatibility, tricalcium phosphate is widely used in orthopedic and maxillofacial surgery. a-TCP particles have outstanding bone regeneration ability, but the clinical findings a-tricalcium phosphate material tends to remain in the defect, which can lead to inflammation or delaying bone healing. On the other hand, as an important component of bone tissue, collagen is also an ideal bone substitute material. However, the mechanical strength of collagen is not enough and the degradation speed is rapid. Tricalcium phosphate bone material because of its excellent bone conductivity, biocompatible, and less possibility to cause inflammation, is widely used in bone defects. Tricalcium phosphate materials are divided into low temperature α-phase and high temperature β-phase. Although having the same chemical composition, it shares different molecular structure. α-TCP has higher hardness and better bone repair effect, but residues could be remained in bone tissue and because of difficult shaping, clinical application is influenced. This study mixed α-tricalcium phosphate with collagen to form an a-tricalcium phosphate/collagen composite material, which has good elasticity and is easy for osteoblast adhesion. Our previous study compared 20mg/ml,50mg/ml, 100mg/ml,200mg/ml concentration of tricalcium phosphate/collagen and found the hardness of material and bone regeneration effect is positive correlation with the content of tricalcium phosphate but have negative correlation of elasticity. Therefore, in this study, we used 150mg/ml tricalcium phosphate/collagen ratio, which concluding high tricalcium phosphate and also have moderate hardness. In our previous study we found that α-tricalcium phosphate/collagen material can completely repair non-critical bone defect in six weeks with no residual in bone tissue. However, whether α-tricalcium phosphate/collagen can repair large bone defects is still unknown. In this study,9mm × 9mm critical bone defect was created on rat skull and due to anatomical factors, skull defects are difficult to have infection and negative osteogenic factors is relatively small, which is easy to analyze. Simultaneously, we use the Micro-CT to observe bone tissue growth, and by acquiring TRAP, ALP non-decalcified tissue sections to observe osteoclast and osteoblast activity. The CT results showed six weeks after α-TCP/CS implantation, the bone defect had amounts of new bone formation. However,8 weeks after surgery, some fibrous tissue space still insisted, which showed the defect didn’t heal entirely. Autologous bone showed good osteogenic capacity in 4-6 weeks, but a large number of osteoclasts caused bone resorption at eight weeks, which greatly affect the bone repair effect. From the experimental results, the α-tricalcium phosphate/collagen composite can quickly achieve bone formation and increase the rate of material degradation. After the completion of the new structure and function of bone reconstruction, the material can be replaced by bone tissue.8 weeks in tissue sections of bone defect, material had almost completely absorbed and new bone repaired bone defects. This shows that the degradation rate of α-tricalcium phosphate/collagen composite materials matched new bone formation rate. Meanwhile, the material have a porous structure which is similar to natural bone and this structure is conducive to the ingrowth of cells, blood vessels and deposition of bone matrix. In this study, α-tricalcium phosphate/collagen composite materials to repair critical bone defects in rat models showed calcium phosphate material mixed with collagen composite material is suitable for bone repair and the degradation rate is moderate, which can quickly form a new bone tissue to repair large bone defects. Meanwhile it suggests that in biomaterials research, different materials can be mixed to improve single material properties. However, the hardness and elastic modulus of the experimental material need for further testing, material composite may further reduce the ability of the loading, affecting the application of the material. Meanwhile, in the actual alveolar bone defects environment, the decomposition rate of the material may further accelerated so that the clinical effect will be our next experiment problem to solve. Few studies have reported the bone regeneration effect of α-tricalcium phosphate mixed with collagen and in the present study, we fabricated a porous α-tricalcium phosphate/collagen soponge (α-TCP/CS) for the regeneration of bone defects. Then, we evaluated the effects of α-TCP/CS on bone regeneration in critical rat calvarial defects to provide experimental evidence for the clinical application.In periodontal surgery, autogenous bone and artificial bone substitutes are frequently used for bone transplantation. However, the application of autogenous bone technique is limited by the lack of sufficient donor sites and high possibility of significant resorption. In this study, we attempted to use the α-tricalcium phosphate/atelocollagen sponge composite to repair calvarial defects in rat models compared with that using autogenous bone.Materials and MethodsExperiment Ⅰ:Porous α-TCP granules, either 10 mm or 200 mm in diameter and possessing a continuous pore structure, were prepared by pulverization of an α-TCP block bearing 80% total pores. Collagen was extracted from porcine skin by enzymatic treatment with pepsin (Nippon Meat Packers; Osaka, Japan). α-TCP particles were mixed with the homogenized collagen solution at a ratio of 150 mg α-TCP particles/mL collagen solution. This mixture was poured into plastic molds and immediately frozen to -80℃ and freeze-dried for 24 h. The freeze-dried α-TCP/collagen composites resembled sponge-like structures and were subsequently cross-linked in vacuo at 140℃ for 24 h.Experiment Ⅱ:Cells form on the composite material pseudopodia, cell growth state, which proves the adhesion and growth of cells in collagen α-tricalcium phosphate/collagen composite contained in the local microenvironment and play osteogenic properties.Experiment Ⅲ:A total of 28 male Sprague-Dawley rats (8 weeks old,250-270 g, SHIMIZU Laboratory Supplies Co.; Kyoto, Japan), which provided for 28 defect sites, were used for transplantation. Calvarial skin flaps were ploughed up after administration of anesthesia. A critical-size bone defect (diameter:9 mm, depth:1.0 mm) was created at the center of the skull using a bone trephine bur under saline irrigation. Three groups were prepared as follows:8-week control without implantation; 4-,6-, and 8-week α-TCP/CS group and autogenous bone group. The calvarial osteocomma was extracted from the defect in the autogenous bone groups and the osteocomma was shattered into 0.1-1-mm3 pieces by using a bone crusher (YDK Co.; Tokyo, Japan). The shattered osteocomma was replaced into the bone defect. In all groups, the bone grafts were covered using GORE-TEX1 GTR membranes (Japan Gore-Tex Co.; Tokyo, Japan). Calvarial flaps were repositioned and sutured following the operational procedure.Calvarial bones were scanned immediately after euthanization by micro-CT (65 kV,90 mA; SMX-130CT, Shimadzu; Kyoto, Japan). Linearity of the micro-CT scanner was established by scanning a phantom containing several densities of a standard calibration material. The calvarial bones were measured in three dimensions, and their structural indices were calculated using a morphometric program (TRI/3D-BON; Ratoc System Engineering; Tokyo, Japan).Experiment Ⅳ:Samples were fixed with 4% paraformaldehyde containing phosphate buffer solution for 16 h. Four-micrometer-thick undecalcified frozen sections were obtained by using the Kawamoto method. Hematoxylin and eosin stain (HE), alkaline phosphatase (ALP), and tartrate-resistant acid phosphatase (TRAP) staining were applied for histological studies. HE staining (Hematoxylin and eosin; Muto Pure Chemicals Co., Tokyo, Japan) was applied for defect tissue observations. TRAP and ALP staining (TRAP/ALP KIT, Wako Pure Chemical Industries Co.; Osaka, Japan) were used for osteoclast identification and for monitoring the activity of osteoblasts following transplantation. Sections were observed after staining by digital microscopy (BZ-9000; Keyence Co.; Osaka, Japan).4,6,8 weeks after the first surgery, injection of calcein (5 mg/kg), tetracycline (25 mg/kg) and 25 mg/kg of alizarin red marked bone growth between the circumferential speed. Get a non-decalcified bone specimens were stained with Sichuan Act, and sections under the microscope laser fluorescence labeling conditions.Experiment Ⅴ:4,6,8 In the first week after surgery specimen sections were RANKL (receptor activator of nuclear factor κ B ligand factor) by immunostaining; images were taken under the microscope.4,6,8 In the first week after surgery specimen sections were TNF-α (Tumor Necrosis Factor-α) by immunostaining; images were taken under the microscope.Results1. α-tricalcium phosphate/collagen composite as white porous sponge-like material. Scanning electron microscopy showed that the experimental group of materials for coralline porous structure, pore size of about 100 to 300 microns, and seen a lot of filamentous structure of collagen as a scaffold support material.2. Cells form on the composite material pseudopodia, cell growth state, which proves the adhesion and growth of cells in collagen α-tricalcium phosphate/ collagen composite contained in the local microenvironment and play osteogenic properties.3. Micro-CT images showed that both a-TCP/CS group and autogenous bone group had obvious bony union in the defect region at 6 weeks. Obvious bone volume reduction could be seen at 8-weeks in autogenous bone group and in 8-weeks α-TCP/CS group, no significant resorption could be seen.After statistical analysis,8-weeks bone volume fraction (BV/TV) of a-TCP/CS group and autologous bone group has statistically significant difference (p<0.01). α-TCP/CS group was higher (mean 69.7%) than autogenous bone group (mean of 42.475%).6 weeks after surgery, CT imaging vertical surface image visible autologous bone graft group showed the bone tissue formation defects, reduced bone defect area, close to the surrounding bone mineral density newborn; not clearly distinguish the edge of the bone defect and implantation of autologous bone material. Cross-sectional images visible defect portion implanted autologous bone interconnected pieces of bone formation and reduce bone defect density image area, α-tricalcium phosphate/collagen composite group images visible vertical surfaces to reduce bone defect area, the material has a significant bone formation images. Autologous bone and α-tricalcium phosphate/collagen composite material implants no infection and rejection after animals. Animals were sacrificed at different time points after the naked eye graft site without purulent exudate.6 weeks seen a lot of bone tissue formation in both groups,8 weeks visible autologous bone resorption group obvious phenomenon.8 weeks, CT image shows the vertical face image group autologous bone defect area increases, significant bone resorption phenomenon, the central part see a small amount of residual bone. High-density bone cross-sectional images visible reduction in low-density soft tissue image area increases, α-tricalcium phosphate/collagen composite material group no significant vertical surface image resorption phenomenon, most of new bone defect repair department. I saw the edge of the control group a small amount of bone defects in bone formation.After eight weeks of bone volume fraction material group (BV/TV) and autologous bone group difference was statistically significant (p<0.01), material group (mean 69.7%) than autologous bone group (mean of 42.475%). At 4 and 6 weeks, group and autologous bone material group BV/TV value, BMD values, BMC/TV value difference was not statistically significant. At 8 weeks, significant BV/TV values between the two groups, BMD, BMC/TV have significant difference.4. HE staining:4 weeks, HE staining showed that autologous bone and α- tricalcium phosphate/collagen composite was surrounded by fibrous connective tissue, α-tricalcium phosphate/collagen composite center visible new bone nodules (Figure 5).6 weeks, HE staining a-tricalcium phosphate/collagen composite and autologous bone the groups were more new bone formation between the trabecular bone marrow cavity.8 weeks, HE staining showed that autologous bone graft group, autologous bone and surrounding new bone is absorbed by the majority of the surrounding fibrous connective tissue ingrowth of bone absorption lacunae in; α-tricalcium phosphate/collagen composite group seen a lot of bone formation, and no a-tricalcium phosphate/collagen composite material remains, new bone and the surrounding bone defect is difficult to distinguish the boundaries; the control group shows a small amount of bone defects marginal bone formation, Most of the defect was filled with fibrous connective tissue.ALP staining:4 weeks ALP staining pictures visible autologous bone group and material group were seen surrounding the bone defect portion material ALP expression, implant material around a large display of active osteoblasts secrete large amounts of bone matrix involved in bone defect.6 weeks ALP staining pictures show and the material group compared with autologous bone ALP expression group is strong, show more active this time of osteoblasts in α-tricalcium phosphate/ collagen composite group.8 weeks in both groups show less ALP expression, showing eight weeks osteogenesis slowed down bone defect growth arrest.TRAP staining:4 weeks See autologous bone material group are visible and a small amount of bone marrow cavity osteoclast resorption occurred participation; material visible cells tend to group attached to the bone tissue, while the material is attached osteoclasts less, implanted α-tricalcium phosphate/collagen composite material degradation and self-absorption of bone part, both groups saw a handful of TRAP staining of osteoclasts.6 weeks when TRAP stained sections showed a large number of osteoclasts appear at this time of autologous bone marrow cavity group of new bone and the formation of resorption pits, while α-tricalcium phosphate/ collagen composite material group saw a few new bone in osteoclasts.8 weeks TRAP staining show autologous bone incision group still shows a large number of osteoclasts, bone resorption activity; α-tricalcium phosphate/collagen composite materials group saw few osteoclasts, no significant bone resorption phenomenon.Autologous bone defect portion group and material group similar bone growth rate of 4-6 weeks between the fast pace of rat bone mineralization, osteoblast activity, mineralized bone surface longitudinal growth of new bone formation and rapid; 6-8 weeks in rats bone mineralization rate decreased osteoblast activity, less mineralized surface of new bone formation, bone growth is slow.5. RANKL immunostaining:From RANKL staining can be seen each week are visible after autologous bone loss group fibrous tissue surrounding the Ministry of RANKL expression, and the material group was stronger and weaker expression of eight weeks at 4 and 6 weeks. RANKL plays an important role in promoting osteoclast differentiation and promote bone resorption. Autologous bone seen in RANKL expression can lead to the accumulation of a large number of osteoclast formation and absorption implantation of autologous bone and new bone. In α-tricalcium phosphate/collagen composite group RANKL expression is weak, causing material RANKL expression can explain less, causing less of osteoclast formation, bone resorption and less.TNF-α immunostaining:Seen in the figure, six weeks after autologous bone of TNF-α expression is also causing a lot of osteoclast formation and bone resorption caused by one of the major reasons for the newborn. Further, in the group of materials, from six weeks showed no expression of TNF-α, was not evident in osteogenesis by TNF-α.Conclusions1.α-tricalcium phosphate/collagen composite has good biocompatibility, biodegradability, and biological activity of bone conduction.2. Porous spongy α-tricalcium phosphate/collagen composite has a similar three-dimensional porous structure with bone tissue, and the material is easy to absorb, in repairing bone defects as well as autogenous bone graft substitutes.3. a-tricalcium phosphate/collagen composite less likely to cause inflammation in the newborn showed less bone osteoclast formation and the osteogenesis was stable. |
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