OBJECTIVE: To sum up the clinical results of bio-derived bone transplantation in orthopedics with tissue engineering technique. METHODS: From January 2000 to May 2002, 52 cases with various types of bone defect were treated with tissue engineered bone, which was constructed in vitro by allogeneous osteoblasts from periosteum (1 x 10(6)/ml) with bio-derived bone scaffold following 3 to 7 days co-culture. Among them, there were 7 cases of bone cyst, 22 cases of non-union or malunion of old fracture, 15 cases of fresh comminuted fracture of bone defect, 4 cases of spinal fracture and posterior route spinal fusion, 3 cases of bone implant of alveolar bone, 1 case of fusion of tarsotarsal joint. The total weight of tissue engineered bone was 349 g in all the cases, averaged 6.7 g in each case. RESULTS: All the cases were followed up after operation, averaged in 18.5 months. The wound in all the case healed by first intention, but 1 case with second intention. Bone union was completed within 3 to 4.5 months in 50 cases, but 2 cases of delayed union. Six cases were performed analysis of CD3, CD4, CD8, ICAM-1 and VCAM-1 before and after operation, and no obvious abnormities were observed. CONCLUSION: Bio-derived tissue engineered bone has good osteogenesis. No obvious rejection and other complications are observed in the clinical application.
Objective To investigate the effect of homograft of marrow mesenchymal stem cells (MSCs) seeded onto poly-L-lactic acid (PLLA)/gelatin on repair of articular cartilage defects. Methods The MSCs derived from36 Qingzilan rabbits, aging 4 to 6 months and weighed 2.5-3.5 kg were cultured in vitroand seeded onto PLLA/gelatin. The MSCs/ PLLA/gelatin composite was cultured and transplanted into full thickness defects on intercondylar fossa. Thirty-six healthy Qingzilan rabbits were made models of cartilage defects in the intercondylar fossa. These rabbits were divided into 3 groups according to the repair materials with 12 in each group: group A, MSCs and PLLA/gelatin complex(MSCs/ PLLA/gelatin); group B, only PLLA/gelatin; and group C, nothing. At 4,8 and 12 weeks after operation, the gross, histological and immunohistochemical observations were made, and grading scales were evaluated. Results At 12 weeks after transplantation, defect was repaired and the structures of the cartilage surface and normal cartilage was in integrity. The defects in group A were repaired by the hylinelike tissue and defects in groups B and C were repaired by the fibrous tissues. Immunohistochemical staining showed that cells in the zones of repaired tissues were larger in size, arranged columnedly, riched in collagen Ⅱ matrix and integrated satisfactorily with native adjacent cartilages and subchondral bones in group A at 12 weeks postoperatively. In gross score, group A(2.75±0.89) was significantly better than group B (4.88±1.25) and group C (7.38±1.18) 12 weeks afteroperation, showing significant differences (P＜0.05); in histological score, group A (3.88±1.36) was better than group B (8.38±1.06) and group C (13.13±1.96), and group B was better than group C, showing significant differences (P＜0.05). Conclusion Transplantation of mesenchymal stem cells seeded onto PLLA/gelatin is a promising way for the treatment of cartilage defects.
Objective To investigate the morphology and biomechanics of in vivo osteogensis after repairing rabbit skull defects with plastic engineered bone which was prefabricated with alginate gel, osteoblasts and bone granules. Methods Twenty-eight rabbits were divided into group A (n=16), group B(n=8) and group C(n=4).The bilateral skull defects of 1 cm in diameter were made. Left skull defects filled with alginate gel-osteoblasts-bone granules(group A1) and right skull defects filled withalginate gel-bone granules(group A2).The defects of group B was left, as blank control and group C had no defect as normal control. The morphological change and bone formation were observed by methods of gross, histology and biomechanics. Results In group A1, the skull defects were almost entirely repaired by hard tissue 12 weeks after operation. The alginate gel-osteoblasts-bone granule material had changed into bone tissue with fewbone granules and some residuary alginate gel. The percentage of bone formation area was 40.92%±19.36%. The maximum compression loading on repairing tissue ofdefects was 37.33±2.95 N/mm; the maximum strain was 1.05±0.20 mm; andloading/strain ratio was 35.82±6.48 N/mm. In group A2, the alginate and bone granules material partially changed into bone tissue 12 weeks after operation. The percentage of bone formation area was 18.51%±6.01%. The maximum compression loading was 30.59±4.65 N; the maximum strain was 1.35±0.44 mm; and the loading/strainratio was 24.95±12.40 N/mm. In group B, the skull defects were mainly repaired bymembrane-like soft tissue with only few bone in marginal area；the percentage of bone formation area was 12.72%±9.46%. The maximum compression loading was 29.5±2.05 N; the maximum strain was 1.57±0.31mm；and the loading/strainratio was 19.90±5.47 N/mm.In group C, the maximum compression loading was 41.55±2.52 N; the maximum strain was 095±017 mm; and the l oading/strain ratio was 47.57±11.22 N/mm. 〖 WTHZ〗Conclusion〓〖WTBZ〗The plastic engineered bone prefabricated with algina te gelosteoblastsbone granule may shape according to the bone defects and ha s good ability to form bone tissue, whose maximum compression loading can reach 89 % of normal skull and the hardness at 12 weeks after operation is similar to that of normal skull.
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 vascularization of the compositeof bio-derived bone and marrow stromal stem cells(MSCs) in repairing goat tibial shaft defect.Methods Bio-derived bone was processed as scaffold material. MSCs were harvested and cultured in vitro. The multiplied and induced cells were seeded onto the scaffold to construct tissue engineered bone. A 20 mm segmental bone defect inlength was made in the middle of the tibia shaft in 20 mature goats and fixed with plate. The right tibia defect was repaired by tissue engineered bone (experimental side), and the left one was repaired by scaffold material (control side).The vascularization and osteogenesis of the implants were evaluated by transparent thick slide, image analysis of the vessels, and histology with Chinese ink perfusion 2, 4, 6, and 8 weeks after operation.Results More new vessels were found in control side than in experimental side 2 and 4 weeks after implantation (Plt;0.05). After 8 weeks, there was no significant difference in number of vessels between two sides(Pgt;0.05), and the implants were vascularized completely. New bone tissue was formed gradually as the time and the scaffold material degraded quickly after 6 and 8 weeks in the experimental side. However, no new bone tissue was formed andthe scaffold degraded slowly in control side 8 weeks after operation.Conclusion Bio-derived bone has good quality of vascularization. The ability of tissue-engineered bone to repair bone defect is better than that of bio-derived bone alone.
ObjectiveTo investigate the feasibility of adipose-derived mesenchymal stem cells (ADMSCs) differentiating into corneal epithelium-like cells after transfection with Pax6 gene. MethodsThe adipose tissue from bilateral inguinal of healthy C57BL/6 mice (5-6 weeks old) was used to isolate and culture ADMSCs.The 3rd passage ADMSCs were subjected to treatments of non-transfection (group A),pcDNA3.1 empty vector transfection (group B),and recombinant plasmid of pcDNA3.1-Pax6 transfection (group C),respectively.At 48 hours after transfection,the cells in groups B and C were selected with G418.The cell morphology changes were observed under the inverted microscope.Pax6 protein and level of corneal epithelial cells specific molecular-cytokeratin 12 (CK-12) were measured by Western blot.Real-time fluorescence quantitative PCR was applied to measure the mRNA expression of CK-12. ResultsNo morphology change was observed in groups A and B.Two different cell clones were found in group C.No.1 selected clone showed a flagstone-like appearance that was similar to that of corneal epithelial cells;No.2 selected clone showed a net-like appearance,with 3-7 cell processes.The Western blot results showed the Pax6 protein expression in 2 clones of group C,but no expression in groups A and B; and CK-12 protein expression was only observed in No.1 selected clone of group C,and no expression in the others.The real-time fluorescence quantitative PCR results showed that the CK-12 mRNA expression level of No.1 selected clone of group C was 8.64±0.73,which was significantly higher than that of No.2 selected clone of group C (0.55±0.42),group B (1.36±0.40),and group A (1.00±0.00) (P<0.05),and there was no significant difference among groups A,B and No.2 selected clone of group C (P>0.05). ConclusionPax6 gene transfection could induce differentiation of ADMSCs into corneal epithelium-like cells which express CK-12 at both the mRNA and protein levels.This result provides a promising strategy of generating corneal epithelilcm-like cells for construction of tissue engineered cornea.
ObjectiveTo explore the clinical application and effectiveness of a personalized tissue engineered cartilage with seed cells derived from ear or nasal septal cartilage and poly-glycolic acid (PGA)/poly-lactic acid (PLA) as scaffold in patients with nasal reconstruction. MethodsBetween March 2014 and October 2015, 4 cases of acquired nasal defects and 1 case of congenital nasal deformity were admitted. The patient with congenital nasal deformity was a 4-year-old boy, and the source of seed cells was nasal septal cartilage. The other 4 patients were 3 males and 1 female, aged 24-33 years, with an average of 28.5 years. They all had multiple nasal subunit defects caused by trauma and the source of seed cells was auricular cartilage. The tissue engineered cartilage framework was constructed in the shape of normal human nasal alar cartilage and L-shaped silicone prosthesis with seed cells from cartilage and PGA-PLA compound biodegradable scaffold. The boy underwent nasal deformity correction and silicone prosthesis implantation in the first stage, and the prosthesis was removed and implanted with tissue engineered cartilage in the second stage; the remaining 4 adult patients all used expanded forehead flaps for nasal reconstruction. All 5 patients underwent 1-4 nasal revisions. The implanted tissue engineered cartilage was observed during the operation and taken from 2 patients for histological examination.ResultsAll the incisions healed by first intention after the tissue engineered cartilage implantation, and the expanded forehead flaps survived. Postoperative low fever occurred in 3 patients. No complications such as infection, obvious immune rejection response, and tissue engineered cartilage protrusion were found in all patients. All patients were followed up 9-74 months (mean, 54.8 months). During follow-up, the patients had no obvious discomfort in the nose and the ventilation function were good. All patients were satisfied with the nasal contour. Early-stage histological examination showed the typical cartilage characteristics in 1 patient after the implantation of tissue engineered cartilage. Late-stage histological examination in 1 patient of tissue engineered cartilage showed the characteristics of fibrous connective tissue; and the other showed there was remaining cartilage.ConclusionThe safety of tissue engineered cartilage constructed in vitro for reconstruction is preliminarily confirmed, but the effectiveness still needs further verification.
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 study the allograft effect of two kinds of tissue engineered oral mucosa lamina proprias on skin fullthickness wounds. Methods The cultured Wistar rat oral mucosa fibroblasts (OMF) were incorporated into collag en or chitosancollagen to construct the tissue engineered oral mucosa laminaproprias, and then the OMFs were labeled with BrdU. The fullthickness round skin defects were made with a round knife (diameter, 0.8 cm) on the backs of 36 Wistar rats (2125 weeks old), which were divided into 2 experimental groups: the fibroblastpopulated collagen lattices (FPCL) group (grafted by FPCLs) and the fibroblastpopulated chitosan collagen lattices (FPCCL) group (grafted by FPCCLs), and the control group (only covered with gauges). All the wounds were observed by the naked eyes or the light microscope, and were measured 4, 7, 14, and 21 days postoperatively. Results There were no infection during the wound healing period. At 7 days after the grafting, the wounds in the 3 groups were covered by scab and/or gauze; at 14 days, the gauze and scab on the wounds in the three groups were all replaced by the new epidermis naturally except one scab each in the FPCCL group and the control groups,which was replaced at 17 days.All the centers of the new epidermis were measurable as the pink red points. At 21 days, all the new skins were smooth without hairs, and their color was similar to the normal one. At 4, 7, and 14 days,there was an indication that the wound diameters became significantly smaller in the three groups; but after the 14th day, there was no significant indication of this kind. At 7 days, the wound diameter in the FPCL group was significantly smaller than that in the FPCCL group and the control group (Plt;0.01). Under the lightmicroscope, at 4 days postoperatively, the decayed tissue on the surfaces of the recipient wounds in the FPCL group and the FPCCL group was separated from the lower granular tissue in which there were many inflammatory cells, fibroblasts, and new vessels. There was a similar-phenomenon in the control group. Each skin wound in the three groups was only partly keratinocyted at 7 days postoperativel y. The recipient wounds were wholly keratinocyted with when rete ridges observed at 14 and 21 days, but in the control group the wounds were keratinocyted with no rete ridges. Fibers in the new dermis were thin. The OMFs with Brdu appeared in the granular tissue and new dermis at 4, 7, 14, and 21 days postoperatively, which could be illustr ated by the immunohistochemical staining. The positive OMFs and the granular tissue joined in the repair of the skin defe cts without any allergic reaction during the period of the wound healing. Conclusion The oral mucosa fibroblasts as the new seed cells can join i n the repair of the skin defects effectively and feasibly. The fibroblastpopul ated collagen lattices and the fibroblastpopulated chitosan collagen lat tices can repair skin defects effectively and feasibly, too. And the quality of the new skins was better in the two experimental groups than in the control group.
Objective To evaluate the tensile mechanical characteristics of decalcified cortical bone matrix with different thicknesses so as to provide an experimental basis for the scaffold of tissue engineering. Methods Decalcified cortical bone matrix was prepared from fresh bovine tibia with rapid decalcification techniques. Its physical characteristics including colour, texture, and so on, were observed. Then the decalcified rate was calculated. Decalcified cortical bone matrices were radially cut into sl ices with different thicknesses along longitudinal axis and divided into 4 groups: group A (100- 300 μm), group B (300-500 μm), group C (500-700 μm), and group D (700-1 000 μm). Then the sl ice specimens of each group were characterized with tensile test and histological examination. Results General observation showed that decalcified cortical bone matrix with hydrogen peroxide treatment was ivory white with good elasticity and flexibil ity. The decalcified rate was 97.6%. The tensile strength and elastic modulus of groups B, C, and D were significantly higher than those of roup A (P lt; 0.05); there was no significant difference among groups B, C, and D (P gt; 0.05). The stiffness in 4 groups increased gradually with the increasing thickness, it was significantly lower in group A than those in groups B, C, and D (P lt; 0.05), and in groups B and C than that in group D (P lt; 0.05). While there was no significant difference in ultimate strain within 4 groups (P gt; 0.05). Histologically, intact osteon was observed in every group, with an average maximum diameter of 182 μm (range, 102- 325 μm). Conclusion The mechanical properties of decalcified cortical bone matrix might depend on the integrity of the osteons. Sl ices with thickness of 300 μm or more could maintain similar mechanical properties when decalcified cortical bone matrix is used as a scaffold for tissue engineering.