Preliminary Study On The Application Of An Injectable Tissue Engineered Bone Based On Chitosan And PB-MSCs To Dental Implants In Osteoporotic Rabbits

Osteoporosis is a systematic bone metabolic disease characterized by decrease of bone mass and degeneration of bone microstructure.Jaw bones,as a part of systematic skeletal tissues,are also affected by osteoporosis.Quantity and quality of bone tissues in jaw bones are both decreased under osteoporotic status which result in a prolonged healing period and lower BIC,BD,BA aider dental implants are implanted.These unfavorable changes threaten the long and stable function of dental implants.Moreover osteoporosis enhances residual ridge resorption and confronts augmentation of alveolar bone with a higher risk of failure.Studies are in progress on how to obtain faster,better osseointegration of dental implants and successful augmentation of alveolar bone.In this paper,several efforts were managed to solve these problems. Mesenchymal stromal cells were obtained from peripheral blood and acted as seed cells.A well-cytoactivity-preserving hydrogel based on chitosan was prepared and acted as scaffold.An injectable tissue engineered bone constructed by these two components was applied to dental implants in an osteoporotic rabbit model.10w later,new bone formed around the extraosseous part of dental implants and contacted tightly with them.The radiographs showed more X-ray radiopaque shadow at the proximal endosseous part of dental implants,which hinted more new bone formed.All of these results indicated the potential osteogenic ability of this injectable tissue engineered bone.It could be applied to augmentation of alveolar bone and improve the lowered BD,BA under osteoporotic status. However,detailed and further studies were necessary to evaluate the overall effects of this kind of bone graft.Experimentâ… Culture of rabbit bone marrow and peripheral blood derived mesenchymal stromal cells.Objective To investigate whether peripheral blood could act as a source of seed cells for bone tissue engineering like bone marrow.Methods Peripheral blood of lm old New Zealand rabbits was aspirated from abdominal aorta.Mononuclear cells were separated out using density centrifugalization and cultured in vitro.At the same time bone marrow in long bones was washed out and cultured.P₄ cells were used to perform identification of cell markers and differentiation experiments.RT-PCR was used to identify the cell markers.Oil Red o stain was performed to the observe the formation of lipid droplets when adipo-induced.Histological staining was performed to detect the expression of ALP,typeâ… collagen and deposition of mineralized nodes.Result Cell colonies formed early in primary culture of bone marrow.Cells in the colonies were uniform fibroblast-like cells and positive of CD29,negtive of CD34.When adipo-induced,most of the cells differentiated to lipocytes.When osteo-induced,these cells became positive of ALP at lw,positive of typeâ… collagen at 3w,and formed numerous bulk calcium nodes at 4w.However,Cell colonies formed relatively late in primary culture of peripheral blood.There were several different morphological cells and thus,forming different morphological cell colonies.These cells were positive of CD29 and weakly positive of CD34. When adipo-induced,part of the cells differentiated to lipocytes.When osteo-induced, they became positive of ALP at 2w,positive of typeâ… collagen at 4w, and formed calcium nodes at 5w.Although no vascular inducer were added into the culture medium,tubule-like structures were seen.Conclusions Peripheral blood contains multipotent stem cells.When osteo-induced,they can differentiated to osteoblasts and form calcium nodes. Peripheral blood can act as a source of seed cells for bone tissue engineering.Experimentâ…¡Preparation of a well eyto-eompatible hydrogel based on chitosan.Objective To prepare a novel injectable hydrogel based on chitosan with physiological osmolality and well-cytoactivity- preserving ability.Materials and methods Chitosan solutions were prepared with hydrochloric acid of different concentrations:group A,0.1M;group B,0.09M, group C,0.08M;group D,0.07M;group E,0.07M.Group A to D were prepared at room temperature and group E at 60℃.All the groups were stirred continuously for 6h and then autoclave sterilized.Aqueousβ-disodium glycerol phosphate was prepared in three concentrations(w/v):groupâ… ,11%;groupâ…¡,25%;groupâ…¢,50%.Chitosan based hydrogel were prepared by 4ml chitosan solution and 1 ml aqueousβ-disodium glycerol phosphate.PH of every hydrogel was detected and its gelatification time at 37 was recorded.Three groups of chitosan hydrogel (E+â… ,D+â…¡,D+â…¢)were mixed with PB-MSCs.They were cultured in vitro for 3d.Live cells were detected by Formazan crystals formed by deoxidized MTT。Result Lower hydrochloric acid concentration resulted in less autoclaving-induced viscosity loss of chitosan solution and higher PH of chitosan based hydrogel which led the hydrogel to be gelable or to gel in a shorter time at 37℃.Most of the cells in E+â… hydrogel were live while part of the cells were live in D+â…¡hydrogel and few cells were live in D+â…¢hydrogel.Conclusions E andâ… constructed hydrogel contained 2.2%β-disodium glycerol phosphate which induced a physiological osmolality.It possessed a PH of 7.13,gelatification time of 20min and well-preserved viscosity.All of these properties it to be a suitable injectable hydrogel for cell-encapsulating. ovariectomy and the changes of mandibular alveolar bone.Objective To establish an osteoporotic rabbit model for the next study just by ovariectomy and observe the changes of mandibular alveolar bone.Methods 20 New Zealand rabbits were equally divided into 2 groups randomly:operation group whose bilateral ovaries were ectomized and shame operation group as a control.OC and TRAP in serum,BMD of femur and lumbar vertebra were detected before,3m and 6m after operation.All the animals were sacrificed 6m after operation.Their demineralized mandibular alveolar bones were performed histological staining in 5μm slices to observe the change of bone near mental foramen.Thickness of medial osteoplaque beside mandibular incisor was measured.Bone percentage of medial side inferior mandibular incisor were measured on computer by photoshop7.01 after slices had been photographed.Result Operation group showed higher OC level than sham operation group 3m(7.39±0.63μg/L versus6.46±0.68μg/L,P＜0.05),6m(7.22±0.58μg/L versus6.57±0.45μg/L,P＜0.05)after operation.OD value of TRAP was also higher in operation group 3m(0.389±0.061 versus 0.332±0.039,P＜0.01), 6m(0.371±0.051 versus0.308±0.029,P＜0.01)after operation.BMD of femur and lumbar vertebra showed no statistical difference in these 2 groups 3m after operation.Femur BMD was lower in operation group(0.349±0.056g/cm³)than sham operation group(0.481±0.086g/cm³,P＜0.01)6m after operation and lumbar vertebra had the same case(0.257±0.042g/cm³ versus 0.360±0.042g/cm³, P＜0.01).Bone trabeculas of cancellous bone around mandibular incisor were thinner,less even vanished in operation group 6m after operation.Thickness of medial osteoplaque beside mandibular incisor in operation group(0.52±0.16mm) was thinner than that in sham operation group(0.85±0.14mm,P＜0.01).Bone percentage of medial side inferior mandibular incisor in operation group(51.7±11.6%)was lower than that in sham operation group(82.6±9.2%,P＜0.01).Conclusions Rabbit model of osteoporosis was successfully established. Mandibular alveolar bone showed typical osteoporotic changes.This indicated that special treatments must be considered to ensure the success of dental implants procedures under osteoporotic status.Experimentâ…£Preliminary application of the injectable tissue engineered bone to osteoporotic dental implants.Objective To apply an injectable tissue engineered bone safely,efficiently to dental implants procedures and observe its effect.Methods(1)9 healthy New Zealand rabbits were equally divided into 3 groups,lml chitosan hydrogel was applied to the implant site in different ways. Methodâ… :chitosan hydrogel was injected into the implant site before preparing the implant hole.Methodâ…¡:chitosan hydrogel was injected into the newly prepared implant hole,and the dental implant was placed immediately.Methodâ…¢:coagulated chitosan hydrogel was placed into the prepared implant hole.The reaction of rabbits was observed in 2h.(2)Injectable tissue engineered bone was constructed by autologous peripheral blood mesenchymal stromal cells and well-cytoactivity -preserving chitosan hydrogel described in experimentâ…¡.Dental implants used in this paper were BAM HA-coated,round,non-threaded dental implants.They were implanted into the lateral metaphysis of femurs.12 osteoporotic rabbits were equally divided into 3 groups.Blank control group: dental implants were implanted only.Control group:coagulated chitosan hydrogel was placed around the endosseous and extraosseous part of dental implants. Experimental group:coagulated injectable tissue engineered bone was placed around the endosseous and extraosseous part of dental implants.10w later,all the osteoporotic rabbits were sacrificed and the femurs were harvested The specimens were observed macroscopicly and radiographicly.Result(1)All the rabbits applied by methodâ… died in 10～30min after the chitosan hydrogel was injected into the femurs.The rabbits applied by methodâ…¡or methodâ…¢seemed to be unaffected and stayed alive.(2)No new bone formed around the extraosseous part of dental implants in blank control and control groups while new bone formed around the extraosseous part of dental implants and contacted tightly with them in experimental group.The radiographs showed black radiolucent shadow at the proximal endosseous part of dental implants in blank control group,while nebulous X-ray radiopaque shadow in control group.In the radiographs of experimental group,more striated or alveolate X-ray radiopaque shadow located proximally to the dental implants,which hinted more new bone formed.Conclusions It was rather hazardous to inject an injectable hydrogel,which could coagulate spontaneously under physical status,into the closed bone circumstance.The injectable tissue engineered bone possessed potential osteogenic ability.It could be applied to augmentation of alveolar bone and improve the lowered BD,BA under osteoporotic status.However,detailed and further studies were to be performed in the future.