Objective Tissue engineered bone (TEB) lacks of an effective and feasible method of storage and transportation. To evaluate the activity of osteogenesis and capabil ity of ectopic osteogenesis for TEB after freeze-dried treatment in vitro and in vivo and to explore a new method of preserving and transporting TEB. Methods Human bone marrow mesenchymal stem cells (hBMSCs) and decalcified bone matrix (DBM) were harvested from bone marrow and bone tissue of the healthy donators. TEB was fabricated with the 3rd passage hBMSCs and DBM, and they were frozen and dried at extremely low temperatures after 3, 5, 7, 9, 12, and 15 days of culture in vitro to obtain freeze-dried tissue engineered bone (FTEB). TEB and FTEB were observed by gross view and scanning electron microscope (SEM). Western blot was used to detect the changes of relative osteogenic cytokines, including bone morphogenetic protein 2 (BMP-2), transforming growth factor β1 (TGF-β1), and insul in-l ike growth factor 1 (IGF-1) between TEB and FTEB. The ectopic osteogenesis was evaluated by the methods of X-ray, CT score, and HE staining after TEB and FTEB were transplanted into hypodermatic space in athymic mouse. Results SEM showed that the cells had normal shape in TEB, and secretion of extracellular matrix increased with culture time; in FTEB, seeding cells were killed by the freeze-dried process, and considerable extracellular matrix were formed in the pore of DBM scaffold. The osteogenic cytokines (BMP-2, TGF-β1, and IGF-1) in TEB were not decreased after freeze-dried procedure, showing no significant difference between TEB and FTEB (P gt; 0.05) except TGF-β1 15 days after culture (P lt; 0.05). The ectopic osteogenesis was observed in TEB and FTEB groups 8 and 12 weeks after transplantation, there was no significant difference in the calcified level of grafts between TEB and FTEB groups by the analysis of X-ray and CT score. On the contrary, there was no ectopic osteogenesis in group DBM 12 weeks after operation. HE staining showed that DBM scaffold degraded and disappeared 12 weeks after operation. Conclusion The osteogenic activity of TEB and FTEB is similar, which provides a new strategy to preserve and transport TEB.
Objective To examine the mRNA expression of activin A(ACT A) and follistatin(FS) during mandibular lengthening and to elucidate the regulating pattern of during mandibular distractionosteogenesis.Methods Skeletally mature-white New Zealand rabbits were established right mandibular distraction osteogenesis models and the mandibles were lengthened 7 days after osteomy. Atthe end of latency period and the end of distraction period, 10,20, 30, 40 and60 days after fixation, the regenerating tissue of animals’ lengthened mandibles and that of the other side normal mandibles were harvested to extract RNA andto analyse ACT A, FS mRNA by RT-PCR.Results The expression of ACT A mRNA was not detectable in normal bone tissue and ACT A mRNA began to express at the end of latency period. The expression of ACT AmRNA increased gradually along with the beginning of distraction and reached the peak on the 10th and 20th days of distraction which was 5.04 and 4.98 times as much as that of the end of latency period, respectively. The trend of expression of FS mRNA during mandibular distraction osteogenesis was the same as expression of ACT A mRNA. Conclusion ACT A/FS play an important role during rabbit mandibular distraction osteogenesis.
Objective To study the mechanism of ectopic osteogenesis of nacre/Polylactic acid (N/P) artificial bone combined with allogenic osteoblasts, and to explore the possibility as a scaffold material of bone tissue engineering. Methods The allogenic- osteoblasts seeded onto N/P artificial bone were co-cultured in vivo 1 week.The N/P artificial bone with allogenic osteoblasts were implanted subcutaneously into the left back sites of the New Zealand white rabbits in the experimental group and the simple N/P artificial bone into the right ones in the control group. The complexes were harvested and examined by gross observation, histologic analysis and immunohistochemical investigation 2, 4 and 8 weeks after implantation respectively.Results In experimental group, the osteoid formed after 4 weeks, and the mature bone tissue withbone medullary cavities formed after 8 weeks; but in control group there was nonew bone formation instead of abundant fibrous tissue after 4 weeks, and more fibrous tissue after 8 weeks.Conclusion N/P artificial bone can be used as an optical scaffold material of bone tissue engineering.
ObjectiveTo review antibacterial/osteogenesis dual-functional surface modification strategy of titanium-based implants, so as to provide reference for subsequent research. MethodsThe related research literature on antibacterial/osteogenesis dual-functional surface modification strategy of titanium-based implants in recent years was reviewed, and the research progress was summarized based on different kinds of antibacterial substances and osteogenic active substances. ResultsAt present, the antibacterial/osteogenesis dual-functional surface modification strategy of titanium-based implants includes: ① Combined coating strategy of antibiotics and osteogenic active substances. It is characterized in that antibiotics can be directly released around titanium-based implants, which can improve the bioavailability of drugs and reduce systemic toxicity. ② Combined coating strategy of antimicrobial peptides and osteogenic active substances. The antibacterial peptides have a wide antibacterial spectrum, and bacteria are not easy to produce drug resistance to them. ③ Combined coating strategy of inorganic antibacterial agent and osteogenic active substances. Metal ions or metal nanoparticles antibacterial agents have broad-spectrum antibacterial properties and various antibacterial mechanisms, but their high-dose application usually has cytotoxicity, so they are often combined with substances that osteogenic activity to reduce or eliminate cytotoxicity. In addition, inorganic coatings such as silicon nitride, calcium silicate, and graphene also have good antibacterial and osteogenic properties. ④ Combined coating strategy of metal organic frameworks/osteogenic active substances. The high specific surface area and porosity of metal organic frameworks can effectively package and transport antibacterial substances and bioactive molecules. ⑤ Combined coating strategy of organic substances/osteogenic active substancecs. Quaternary ammonium compounds, polyethylene glycol, N-haloamine, and other organic compounds have good antibacterial properties, and are often combined with hydroxyapatite and other substances that osteogenic activity. ConclusionThe factors that affect the antibacterial and osteogenesis properties of titanium-based implants mainly include the structure and types of antibacterial substances, the structure and types of osteogenesis substances, and the coating process. At present, there is a lack of clinical verification of various strategies for antibacterial/osteogenesis dual-functional surface modification of titanium-based implants. The optimal combination, ratio, dose-effect mechanism, and corresponding coating preparation process of antibacterial substances and bone-active substances are needed to be constantly studied and improved.
Objective To investigate the effectiveness of tunnel osteogenesis technique combined with locking plate in the treatment of aseptic non-hypertrophic nonunion of femoral shaft. MethodsThe clinical data of 23 cases of aseptic non-hypertrophic nonunion of femoral shaft treated with tunnel osteogenesis technique combined with locking plate between January 2017 and December 2020 were retrospectively analysed. There were 17 males and 6 females with an average age of 41.4 years (range, 22-72 years). There were 22 cases of closed fracture and 1 case of open fracture. The types of internal fixation at admission included intramedullary nail in 14 cases and steel plate in 9 cases. The number of nonunion operations received in the past was 0 to 1; the duration of nonunion was 6-60 months, with an average of 20.1 months. Among them, there were 17 cases of aseptic atrophic nonunion of the femoral shaft and 6 cases of dystrophic nonunion. Twenty-two cases were fixed with 90° double plates and 1 case with lateral single plate. The operation time, theoretical blood loss, hospitalization stay, nonunion healing, and postoperative complications were recorded. Harris hip function score, Lysholm knee function score, lower extremity function scale (LEFS), and short-form 36 health survey scale (SF-36) were used at last follow-up to evaluate hip and knee functions. Visual analogue scale (VAS) score was used to evaluate the relief of pain at 1 day after operation and at last follow-up. ResultsThe average operation time was 190.4 minutes, the average theoretical blood loss was 1 458.4 mL, and the average hospitalization stay was 8.2 days. All the 23 patients were followed up 9-26 months, with an average of 18.2 months. The healing time of nonunion in 22 patients was 3-12 months, with an average of 5.6 months. There were 8 cases of limb pain, 8 cases of claudication, 6 cases of limitation of knee joint movement, and 2 cases of limitation of hip joint movement. At last follow-up, the imaging of 1 patient showed that the nonunion did not heal, accompanied by pain of the affected limb, lameness, and limitation of knee joint movement. At 1 day after operation, the VAS score of 23 patients was 6.5±1.8, the pain degree was good in 7 cases, moderate in 12 cases, and poor in 4 cases; at last follow-up, the VAS score was 0.9±1.3, the pain degree was excellent in 21 cases and good in 2 cases, which were significantly better than those at 1 day after operation (t=12.234, P<0.001; Z=–5.802, P<0.001). At last follow-up, the average Harris hip function score of 22 patients with nonunion healing was 94.8, and the good rate was 100%; Lysholm knee function score averaged 94.0, and the excellent and good rate was 90.9%; LEFS score averaged 74.6; SF-36 physical functioning score averaged 85.0 and the mental health score averaged 83.6. ConclusionTunnel osteogenesis technique combined with locking plate in the treatment of aseptic non-hypertrophic nonunion of femoral shaft has a high healing rate and fewer complications, which can effectively relieve pain and improve lower limb function and quality of patients’ life.
Objective To discuss the role of heparan sulfate (HS) in bone formation and bone remodeling and summarize the research progress in the osteogenic mechanism of HS. Methods The domestic and abroad related literature about HS acting on osteoblast cell line in vitro, HS and HS composite scaffold materials acting on the ani-mal bone defect models, and the effect of HS proteoglycans on bone development were summarized and analyzed. Results Many growth factors involved in fracture healing especially heparin-binding growth factors, such as fibroblast growth factors, bone morphogenetic protein, and transforming growth factor β, are connected noncovalently with long HS chains. HS proteoglycans protect these proteins from protease degradation and are directly involved in the regulation of growth factors signaling and bone cell function. HS can promote the differentiation of stem cells into osteoblasts and enhance the differentiation of osteoblasts. In bone matrix, HS plays a significant role in promoting the formation, maintaining the stability, and accelerating the mineralization. Conclusion The osteogenesis of HS is pronounced. HS is likely to become the clinical treatment measures of fracture nonunion or delayed union, and is expected to provide more choices for bone tissue engineering with identification of its long-term safety.
ObjectiveTo detect the difference in the osteogenesis ability of biphasic calcium phosphate (BCP) ceramic granular materials with different mesoporous diameters prepared at different sintering temperatures through in vivo and in vitro experiments, so as to provide evidence for screening BCP materials with better clinical application parameters.MethodsThree kinds of BCP (materials 1, 2, 3) were prepared by mixing hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) at a ratio of 8∶2 and sintered at 1 050, 1 150, and 1 250℃ for 3 hours, respectively. The internal porosity and the diameter, volume, and area of the mesopore were measured by Brunauer-Emmett-Teller test (BET); the composition of the material was evaluated by X-ray diffraction (XRD); the microscopic surface morphology of the material was observed by scanning electron microscopy (SEM). The 3rd generation bone marrow mesenchymal stem cells (BMSCs) from Sprague-Dawley rats were co-cultured with the materials 1, 2, and 3 for 7 days in vitro respectively (groups A, B, and C), and the cells adhesion on the materials was observed by SEM and phalloidine staining, respectively. Cell proliferation activity was measured by cell counting kit 8 method. In vivo, 9 muscle bags were made in dorsal muscles of 9 beagles, respectively. The muscle bags were randomly divided into 3 groups (3 per beagle in each group) and materials 1, 2, and 3 were placed into the muscle bags of groups A, B, and C, respectively. After 1, 2, and 3 months of operation, 3 beagles were anesthetized and the samples were stained with HE, Masson, and Safranin, and the bone formation area ratio in the BCP gap was calculated. Real-time fluorescence quantitative PCR (qRT-PCR) was performed to detect the expressions of bone-related genes [including alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OC)].ResultsThe BET test showed that with the increase of sintering temperature, the internal porosity of the particles did not change significantly, but the diameter, volume, and area of the mesopores gradually decreased. The XRD detection showed that the XRD waves of HA and β-TCP could be seen in all 3 kinds of materials; SEM showed that there were widely distributed macropores on the surface of 3 kinds of BCPs, and the interpores connected with the others. In vitro, BMSCs adhered and proliferated on the surfaces of 3 kinds of BCPs, and the cell biocompatibility of the materials in groups B and C was better than that in group A. In vivo, obvious osteoid tissue deposition could be observed in the intergranular space of 3 kinds of BCPs from 2 months after implantation. The bone formation area ratio of each group increased with time. The bone formation area ratio in group A was significantly higher than that in groups B and C at 2 and 3 months after implantation, and in group A than in group B at 1 month (P<0.05). qRT-PCR showed that the expressions of osteogenic related genes peaked at 2 months in group A, and gradually increased with time in groups B and C. The relative expressions of ALP and OPN mRNAs in group A were significantly higher than those in groups B and C at 1 month after implantation, the relative expression of OC mRNA in group A was significantly higher than that in groups B and C at 2 months after operation, the relative expression of ALP mRNA in groups B and C and the relative expression of OPN mRNA in group B were significantly higher than those in group A, all showing significant differences (P<0.05); there was no significant difference in the relative expression of each gene among the other groups at each time point (P>0.05).ConclusionThe mesoporous diameter of BCP decreases with the increase of sintering temperature. Different mesoporous diameters lead to different ectopic osteogenesis of BCP materials. BCP material with mesoporous diameter of 12.57 nm has better osteogenic ability which can activate the osteogenic gene earlier. The mesoporous diameter is expected to be an adjustable index for optimizing the osteogenic capacity of BCP materials.
ObjectiveAfter using hyaluronic acid (HA) to modify curcumin (CUR), the effects of calcium phosphate cement (CPC) combined with HA/CUR on the proliferation and osteogenesis of osteoblasts were investigated.MethodsFirst, HA and CUR were esterified and covalently combined to prepare HA/CUR, and the characteristics were observed and the infrared spectrum was tested. Then, HA, CUR, and HA/CUR were mixed with CPC according to 5% (W/W) to prepare HA-CPC, CUR-CPC, and HA/CUR-CPC, respectively. Setting time detection, scanning electron microscope observation, injectable performance test, and compression strength test were conducted; and the CPC was used as a control. Osteoblasts were isolated and cultured from the skull of newborn Sprague Dawley rats, and the 2nd generation cells were cultured with the 4 types of bone cement, respectively. The effects of HA/CUR-CPC on the proliferation and osteogenesis of osteoblasts were estimated by the scanning electron microscopy observation, live/dead cell fluorescence staining, cell counting, osteopontin (OPN) immunofluorescence staining, alkaline phosphatase (ALP) staining,and alizarin red staining.ResultsInfrared spectroscopy test showed that HA and CUR successfully covalently combined. The HA/CUR-CPC group had no significant difference in initial setting time, final setting time, injectable rate, and compressive strength when compared with the other 3 groups (P>0.05); scanning electron microscope observation showed that HA/CUR was scattered on CPC surface. After co-culture of bone cement and osteoblasts, scanning electron microscopy observation showed that the osteoblasts, which had normal morphology and the growth characteristics of osteoblasts, clustered and adhered to HA/CUR-CPC. There was no significant difference in cell survival rate between HA/CUR-CPC group and other groups (P>0.05), and the number of cells significantly increased (P<0.05); the degrees of OPN immunofluorescence staining, ALP staining, and alizarin red staining were stronger than other groups.ConclusionHA/CUR-CPC has good biocompatibility and mechanical properties, which can promote the proliferation and osteogenesis of osteoblasts.
OBJECTIVE: To establish the animal models of mandibular distraction osteogenesis in rabbits and study its osteogenetic mechanism. METHODS: The right mandibles just anterior to the first molars of 12 rabbits were performed osteotomies, and the mandibles were positioned with distractors. The left mandibles were control group without operation. After 1 week, the distractors were stretched 0.9 mm every day for 10 days progressively. One day, 2, 4, 8 weeks after distraction, the mandibles were studied with gross measurement, X-ray, and histological examination. RESULTS: The right mandible were lengthened 8.3 mm on average without bone nonunion and deformity healing. It was observed that the gaps between the distracted bone edges were first occupied by fibrous tissue. Two weeks after distraction, it was found that the gaps were bridged by callus in X-ray, the new bone and the normal bone could not be differentiated clearly after 8 weeks. In histological sections, there were collagen bundles in early distraction, then those collagen bundles were calcificated and become trabeculaes. No Cartilage was found during distraction. CONCLUSION: It suggests that the rabbit mandible can be lengthened by distraction osteogenesis, and the new bone is formed by intramembranous ossification.
Objective To summarize the regulatory effect of non-coding RNA (ncRNA) on type H vessels angiogenesis of bone. Methods Recent domestic and foreign related literature about the regulation of ncRNA in type H vessels angiogenesis was widely reviewed and summarized. ResultsType H vessels is a special subtype of bone vessels with the ability to couple bone formation. At present, the research on ncRNA regulating type H vessels angiogenesis in bone diseases mainly focuses on microRNA, long ncRNA, and small interfering RNA, which can affect the expressions of hypoxia inducible factor 1α, platelet derived growth factor BB, slit guidance ligand 3, and other factors through their own unique ways of action, thus regulating type H vessels angiogenesis and participating in the occurrence and development of bone diseases. ConclusionAt present, the mechanism of ncRNA regulating bone type H vessels angiogenesis has been preliminarily explored. With the deepening of research, ncRNA is expected to be a new target for the diagnosis and treatment of vascular related bone diseases.