To summarize the medium-term cl inical result of bio-derived bone transplantation in orthopedics with tissue engineering technique. Methods From December 2000 to June 2001, 10 cases of various types of bone defect were treated with tissue engineered bone, which was constructed in vitro by allogenous osteoblasts from periosteum (1 × 106/ mL) with bio-derived bone scaffold following 3 to 7 days co-culture. Six men and 4 women were involved in this study, aged from 14 to 70 years with a median of 42 years. Among them, there were 2 cases of bone cyst, 1 case of non-union of old fracture, 6 cases of fresh comminuted fracture with bone defect, and 1 case of chronic suppurative ostemyel itis. The total weight of tissue engineered bone was 3-15 g in all the cases, averaged 7.3 g in each case. Results The wound in all the case healed by first intention. For 7 year follow up, bone union was completed within 3.0 to 4.5 months in 9 cases, but loosening occurred and the graft was taken out 1 year after operation in 1 case. The X-ray films showed that 9 cases achieved union except one who received resection of the head of humerus. No obvious abnormities were observed, and the function of affected l imbs met daily l ife and work. Conclusion Bio-derived tissue engineered bone has good osteogenesis. No obvious rejection and other compl ications are observed in the cl inical appl ication.
Objective To explore the histological changes of bio-derived bone prepared by different methods after implantation, and to provide the scaffold material from xenogeneic animal for tissue engineering. Methods Theextremities of porcine femur were cut into 0.5 cm×0.5 cm×0.5 cm. Then they were divided into 5 groups according to different preparation methods: group A was fresh bone just repeatedly rinsed by saline; group B was degreased; group C was degreased and decalcificated; group D was degreased, acellular and decalcificated; group E wasdegreased and acellular. All the materials were implantated into femoral muscle pouch of rabbit after 25 kGy irradiation sterilization. The cell counting ofinflammatory cells and osteoclasts, HE and Masson staining, material degradation, collagen and new bone formation were observed at 2, 6, and 12 weeks postoperatively. Results The residue level of trace element in biomaterials prepared by different methods is in line with the standards. All the animals survived well. There were no tissue necrosis, fluid accumulation or inflammation at all implantation sites at each time point. The inflammatory cells counting was most in group A, and there was significant difference compared with other groups(P＜0.05). There was no significant difference in osteoclasts counting among all groups. For the index of HE and Masson staining, collagen and new bone formation, groups C and D were best, group E was better, and groups A and B were worse. Conclusion The degreased, acellular and decalcificated porcine bone is better in degradation,bone formation, and lower inflammatory reaction, it can be used better scaffold material for tissue engineered bone.
OBJECTIVE: To compare the clinical results of repairing bone defect of limbs with tissue engineering technique and with autogeneic iliac bone graft. METHODS: From July 1999 to September 2001, 52 cases of bone fracture were randomly divided into two groups (group A and B). Open reduction and internal fixation were performed in all cases as routine operation technique. Autogeneic iliac bone was implanted in group A, while tissue engineered bone was implanted in group B. Routine postoperative treatment in orthopedic surgery was taken. The operation time, bleeding volume, wound healing and drainage volume were compared. The bone union was observed by the X-ray 1, 2, 3, and 5 months after operation. RESULTS: The sex, age and disease type had no obvious difference between groups A and B. all the wounds healed with first intention. The swelling degree of wound and drainage volume had no obvious difference. The operation time in group A was longer than that in group B (25 minutes on average) and bleeding volume in group A was larger than that in group B (150 ml on average). Bone union completed within 3 to 7 months in both groups. But there were 2 cases of delayed union in group A and 1 case in group B. CONCLUSION: Repair of bone defect with tissue engineered bone has as good clinical results as that with autogeneic iliac bone graft. In aspect of operation time and bleeding volume, tissue engineered bone graft is superior to autogeneic iliac bone.
Objective To investigate the feasibility of repairing goat tibia defect with marrow stromal cells (MSCs).Methods MSCs were cocultured with the bio-derived bone in vitro, and the 20 mm tibia defectswere made and fixed with plate in 35 goats, and they were divided into the experimental group, control group and blank group. The defects on the right side were filled with tissue engineering bone as the experimental group, the defects onthe left side with bio-derived bone as the control group in 33 goats, and the defect on the both sides were not filled with any materials as the blank group in 2 goats. Threpair capability was assessed physically, histopathologically and biomechanically at 2, 4, 6, 8, 12,16 and 24 weeks after operation in 3 groups.Results By physical, histopathological and biomechanical examinations, the bio-derived bone was partially absorbed in the experimental group and was rarely absorbed in the control group in the 4th week; the defects were partially repaired in the experimental group, and in the control group, few new bones were observed in the two ends of the implants, in which there was fibrous tissue. The effects of biomechanics had no statistically significant difference between the experimental group and the control group(P＞0.05) in the 8th week; the defects were perfectly repaired in the experimental group and the effects of biomechanics had statistically significant difference between two groups (P＜0.05) in the 12th weeks. The defects were not repaired in the 24th week in the blank group.Conclusion The tissue engineering bone can efficiently repair bone defect, and itsrepair capability is better than that of bio-derived bone alone both in quantity and in quality of bone formation.
Objective To study the osteogenic effects of a new type of peptides anchored aminated-poly-D, L-lactide acid (PA/PDLLA) scaffold in repairing femoral defect in rats. Methods The PDLLA scaffolds were treated by ammonia plasma and subsequent anchor of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides via amide linkage formation. Thus PA/PDLLA scaffolds were prepared. The bone marrow was harvested from the femur and tibia of 4 4-week-old Sprague Dawley (SD) rats, and bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured by whole bone marrow adherence method. BMSCs-scaffold composites were prepared by seeding osteogenic-induced BMSCs at passages 3-6 on the PA/PDLLA and PDLLA scaffolds. The right femoral defects of 8 mm in length were prepared in 45 adult male SD rats (weighing, 350-500 g) and the rats were divided into 3 groups (n=15) randomly. BMSCs-PA/PDLLA (PA/PDLLA group) or BMSCs-PDLLA (PDLLA group) composites were used to repair defects respectively, while defects were not treated as blank control (blank control group). General state of the rats after operation was observed. At 4, 8, and 12 weeks after operation, general, radiological, histological, micro-CT observations and real-time fluorescent quantitative PCR were performed. Results Two rats died after operation, which was added; the other rats survived to the end of the experiment. At each time point after operation, general and radiological observations showed more quick and obvious restoration in PA/PDLLA group than in PDLLA group; no bone repair was observed in blank control group. The X-ray scores were the highest in PA/PDLLA group, higher in PDLLA group, and the lowest in blank control group; showing significant difference in multiple comparison at the other time (P lt; 0.05) except between blank control group and PDLLA group at 4 weeks (P gt; 0.05). The X-ray scores showed an increasing trend in PDLLA group and PA/PDLLA group with time (P lt; 0.05). Histological and micro-CT observations showed the best osteogenesis in PA/PDLLA group, better in PDLLA group, and worst in blank control group. Comparison between groups had significant differences (P lt; 0.05) in bone mineral density, bone volume/total volume of range of interest, trabecular number, and structure model index. Significant differences (P lt; 0.05) were found in the expression levels of osteogenesis-related genes, such as osteocalcin, alkaline phosphatase, collagen type I, bone morphogenetic protein 2, and osteopontin when compared PA/PDLLA group with the other groups by real-time fluorescent quantitative PCR analysis. Conclusion The PA/PDLLA scaffolds can accelerate the repair of femoral defects in rats.
Objective To study the ectopic osteogenesis and vascularization ofthe tissue engineered bone promoted by an artificial bone composite that consists of coral hydroxyapatite (CHA), 1,25-(OH)2 D3, human marrow stromal osteoblast (hMSO), and human umbilical vein endothelial cell (hUVEC).Methods After the isolation and the culture in vitro, hMSO and hUVEC were obtained. Then, hMSO (5×105/ml) and hUVEC (2.5×105/ml) were seeded at a ratio of 2∶1 onto the CHA scaffolds coated with 1,25-(OH)2 D3 (the experimental group) or onto the CHA scaffolds without 1,25-(OH)2 D3 (the control group). The scaffolds were culturedin vitro for 3 days, and then the scaffolds were implanted into the pockets that had beenmade on the backs of 18 nude mice. Then, 6 of the mice were implanted with one experimental engineered bone bilaterally; another 6 mice were implanted with onecontrol engineered bone bilaterally; the remaining 6 mice were implanted with one experimental engineered bone and one control engineered bone on each side. At4, 8 and 12 weeks after operation, the retrieved scaffolds and cells were examined by the nake eye and histology as well as by the scanning electron microscopy. The quantitative assessment of the newly-formed bone and the quantitative analysis of the newly-formed blood vessels were performed. Results The evaluationsby the histology revealed that at 4 weeks the original bone tissues grew into the scaffolds in all the groups, but significantly more newly-formed bone tissuesand newly-formed blood vessels were found in the experimental group. At 12 weeks the newly-formed bone tissues were found in all the groups, but there was a typical bone unit found in the experimental group. There was a significantly smaller amount of capillary vessels in the control group than in the experimental group at all the time points. The evaluations by the scanning electron microscopy revealed that at 4 weeks in the experimental group there were great amounts of extracelluar matrix that embedded the cells, and plenty of capillary vessels were found on the surface of the implanted bone materials and some of them grew into the materials; however, in the control group there was a smaller amount of capillary vessels although much extracelluar matrix was still found there. At 8 weeks sarciniform osteoids were found on some of the implanted materials, with much extracelluar matrix and many newly-formed capillary vessels in the experimental group; however, in the control group there were fewer capillary vessels and lower degrees of the bone maturity. The quantitative assessment of the newly-formed bone showed that the newformed bones were 3.1±0.52 in the experimental group but2.30±0.59 in the control group at 8 weeks (Plt;0.05), and 4.63±0.55 vs. 3.53±0.62 at 12 weeks. There was a significant difference at these two time points between the two groups (Plt;0.05). The quantitative analysis of the newly-formed blood vessels showed that the vascular areas were 28.74％±7.81％i n the experimental group but 19.52％±4.57% in the control group at 4 weeks (Plt;0.05), and 24.66％±7.38％ vs. 1784%±5.22% at 12 weeks. There was a significant difference at these two time points between the two groups (Plt;0.05). Conclusion 1,25-(OH)2 D3 as an active factor can increase the interaction between hMSO and hUVEC, and thus promote the ectopic osteogenesis and vascularization in the tissue engineered bone.
Objective To evaluate the adhesion, prol iferation and osteogenic differentiation of rabbit BMSCs after cultured on freeze-dried demineral ized bone matrix (FDBM) modified with type II cadherin ectodomain (Cad- II). Methods BMSCs isolated from 10 Japanese white rabbits (male and female, 4-week-old, 0.61-0.88 kg) were cultured. The second generation of BMSCs (cell density 1 × 106 /mL) were seeded onto the Cad-II modified allogenic FDBM (experimental group) and only FDBM (control group) respectively, and then cocultured in vitro. The densities of seeded cells, the adhesion rate and their ALP activity were measured. The complex was observed through inverted phase contrast microscope and scanning electron microscope to evaluate the interaction between cells and FDBM. Another group of second generation of BMSCs (cell density 5 × 105 /mL) were seeded onto the Cad-II modified FDBM (experimental group) and only FDBM (control group) respectively, and then cocultured in vitro too. The ALP activity and osteocalcin immunohistochemical was measured. Results There was no significant difference in cell prol iferation between experimental group and control group. The adhesion rate of cells in the experimental group was 87.41% ± 5.19%, higher than that in the the control group 35.56% ± 1.75% (P lt; 0.01); the densities of seeded cells reached 5.0 × 105, showing significant difference compared with the control group (2.6 × 104, P lt; 0.05). Inverted phase contrast microscope showed that in the experimental group, more cultured BMSCs pasted in the hole and edge of the scaffold than that in the control group. HE staining showed the densities of seeded cells in the experimental group was higher than that in the control group. Scanning electron microscope showed that in the experimental group, a lot of cultured BMSCs adhered, spreaded in the scaffold, in the control group only a few BMSCs unevenly distributed in the scaffold. After 7 days of culture, the cultured BMSCs on modified FDBM expressed higher ALP activity; after 14 days of culture, the ALP activity (29.33 ± 1.53) was higher than that cultured on unmodified FDBM (18.31 ± 1.32), the positive rates of osteocucl in were 83% ± 7% in the experimental group and 56% ± 7% in the control group, showing significant difference (P lt; 0.01). Conclusion Cad-II enhanced cell adhesion to FDBM and promoted BMSCs differentiate to osteoblast, but no obvious effects were observed in cell prol iferation.
Objective To investigate the effect of astragalus polysaccharides(AP) on chitosan／polylactic acid(AP／C／PLA)scaffolds and marrow stromal cells(MSCs)tissue engineering on periodontal regeneration of horizontal alveolar bone defects in dogs． Methods MSCs were isolatedfrom the bone marrow and then cultured in conditioned medium to be induced to become osteogenic．The MSCs were harvested and implanted into AP／C／PLA and C／PLA scaffolds．A horizontal alveolar bone defect(5 mm depth, 2 mm width)were produced surgically in the buccal side of the mandibular premolar 3 and 4 of 10 dogs．The defects were randomly divided into 4 groups(n=10)：Group A, root planning only(blank contro1); group B, AP／C／PLA with conditioned medium(medium contro1);group C, C／PLA with MSCs(scaffolds contro1); and group D, AP／C／PLA with MSCs(experimental group)．Eight weeks after surgery， block sections of the defects were collected for gross, histological and X-ray analysis. Results MSCs induced in vitro exhibited an osteogenic phenotype with expressingcollagen I and alkaline phosphatase． X-ray film observation showed that the bone density and height had no changes in group A; in group B, the bone density was increased to a certain extent and furcation area reached a few height, but no height was increased in interdental septum; in group C，the bone density was increased and furcation area nearly reached the native height,but interdental septum reached a few height;in group D,the bone density was increased significantly and furcation area and interdental septum reached the native height. Histological evaluation showed that there was greater tissue formation in group D than that in groups A, B and C, in which new alveolar bone, new cementum, periodontal ligament with Sharpey’s fibers, and new bone tissue was similar to native periodontal tissues. Ingroup A，B, C and D respectively, the amount of new alveolar bone regeneration was 0.83±0.30, 1.46±0.55, 2.67±0.26 and 2.90±0.41 mm; new cementum regeneration was 0.78±0.45，1.30±0.60，2.29±0.18 and 2.57±0.22 mm; the amount of connective tissue adhesion was 0.80±0.22，1.33±0.34，2.23±0.42 and 2.64±0.27 mm; all showing significant differenecs between group D and groups A, Band C (Plt;0.05)．Conclusion The technology of tissue engineering with AP／C／PLAscaffolds and induced MSCs may contribute to periodontal regeneration．
Objective To provide the chosen scaffold materials for experiment and application of tissue engineering and to detect the properties of the collagenbio-derived bone scaffold material loading WO-1. Methods The purebio-derived bone scaffold material, bio-derived bone scaffold material loading collagen, collagen bio-derived bone scaffold material loading WO-1 were made by use of allograftbone, and typeI collagen, and WO-1. The morphological features, constitute components and mechanical properties were examined by scanning electron microscopy,X- rays diffraction and mechanical assay. Results The bio-derived bone scaffold material maintained natural network pore system; the bio-derived bone scaffold material loading collagen maintained natural network pore system, the surface of network pore system was coated by collagen membrane; the collagen bio-derived bone scaffold material loading WO-1 maintained natural network pore system, thesurface of network pore system was coated by collagen membrane. The pore sizes of the 3materials were 90-700 μm, 75-600 μm and 80-600 μm, respectively, and the porosities were 87.96%, 80.47%, 84.2%. There was no significant difference between them(P＞0.05).The collagen bio-derived bone scaffold material loading WO-1 consisted of ［HA,Ca10(OH)2(PO4)6］. There was no significant difference in the mechanical strength of the three scaffold materials. Conclusion The bio-derived bone scaffold material loading WO-1 is as good as bio-derived bone scaffold material and collagen bio-derived bone scaffold material, and it is an effective scaffold material for tissue engineering bone.
Objective To evaluate the osteogenesis of bi phasic ceramic-l ike biologic bone (BCBB) with tissue engineering in repairing segmental bone defects. Methods BMSCs isolated from the femoral and tibial marrow of 2-weekold Japanese white rabbit were cultured to passage 3. Then 20 μL of the cell suspension at a concentration of 1 × 107 cells/mLwere seeded into 15 mm × 15 mm × 5 mm BCBB block; the construction of tissue engineered BCBB was completed after 8 days of compound culture. Forty-eight adult Japanese white rabbits were randomly divided into groups A, B, C and D, then BCBBs cultured with BMSCs in vitro for 8 days (group A) and only BCBBs without BMSCs (group B) were respectively implanted into the radius segmental bone defects of rabbits, autogenous il iac bone graft (group C) and empty defect (group D) were used as controls. The specimens were examined after 4, 8, 12 and 24 weeks, the osteogenesis was evaluated through X-ray radiograph and histology examination. Results X-ray examination: the border between the material and host’s bone was clear after 4 weeks, and blurred after 8 weeks in group A and group B; the density of some part of the edge of the material was similar to that of radius and there was high density imaging in the materials of group A after 12 weeks; there was much high density imaging in the materials of group B after 12 weeks. The medullary cavity of bone was formed and l ittle high density imaging in the materials of group A after 24 weeks. Some high density imaging still existed in the materials of group B after 24 weeks. The X-ray evaluated scores showed that the scores of group A was higher than that of group B, and there was significant difference between group A and group B after 12 and 24 weeks (P lt; 0.05). Histological examination: there was new bone formation in the materials and also new bone grew adhesively on the surface of BCBB in group A. While in group B only new bone grew and attached to the surface of BCBB. BCBB degraded more with the time and more new bone formed. The histological evaluation showed that the bone forming area in group A was more than that in group B, and there was significant difference between group A and group B (P lt; 0.05). Conclusion The osteogenesis of BCBB with tissue engineering was superior to only BCBB, BCBB could be used as a scaffold of bone tissue engineering.