Objective To review the research progress of promoting the bone formation at early stage by components of the extracellular matrix (ECM). Methods Recent literature concerning the influence of these components on new bone formation and bone/implant contact was extensively reviewed and summarized. Results Coating of titanium or hydroxyapatite implants with organic components of the ECM (such as collagen type I, chondroitin sulfate, and Arg-Gly-Asp peptide) offers great potential to improve new bone formation and enhance bone/implant contact, which in turn will shorten recovery time and improve implant stability. Conclusion The increasing knowledge about the role of the ECM for recruitment, proliferation, differentiation of cells, and regeneration of tissue will eventually deal to the creating of an artificial ECM on the implant that could allow a defined adjustment of the required properties to support the healing process.
Objective To comprehend the pathological features and possible pathogenesis of avascular necrosis of the femoral head (ANFH) by morphology and immunohistochemical observation of osterix (OSX) and adiponectin through in vitro traumatic and non-traumatic ANFH specimens, so as to provide a theoretical basis for cl inical treatment. Methods Sixty-six ANFH specimens were collected from 66 cl inical cases undergoing hip replacement surgery. Twenty-four cases of traumatic ANFH (group A) included 17 males and 7 females, aged 21 to 70 years with an average of 56.5 years; 23 cases of steroid-induced ANFH (group B) included 16 males and 7 females, aged 56 to 72 years with an average of 61 years; and 19 cases of alcohol ic ANFH (group C) were males, aged 55 to 67 years with an average of 58.5 years. Bone tissue was got from weight-bearing and non-weight-bearing area of the femoral head respectively. The basic pathological changes was observed by HE staining under the optical microscope, and the percentage of empty bone lacuna and the percentage of trabecular bone area were calculated. The morphological changes of ANFH in different groups were observedby scanning electron microscope (SEM). OSX and adiponectin expression were detected by immunohistochemical technique. Results Gross of the femoral head surface in each group was rough, collapse, articular cartilage loss, osteophyte formation; cross section: dark red in group A, and yellow in groups B and C. HE staining showed that weight-bearing area of ANFH have similar morphological features in three groups. In non-weight-bearing area of groups B and C, the fat cells in bone marrow markedly increased and were hypertrophic; however there were more fibrous tissue in group A. There were statistically significant differences (P lt; 0.001) in the percentage of empty bone lacuna of the weight-bearing and non-weight-bearing area among three groups. There were no statistically significant differences (P gt; 0.05) in the percentage of trabecular bone area among three groups. The SEM observation showed that three groups had similar pathological changes. Brown granules for OSX and adiponectin positive substance were mainly located in the osteoblast of bone marrow of the femoral head. There was statistically significant difference (P lt; 0.05) in the average absorbency (A) value of OSX between group A and groups B, C, but there was no statistically significant difference (P gt; 0.05) between groups B and C. While there was no statistically significant difference (P gt; 0.05) in the A value of adiponectin among three groups. Conclusion Hormones and alcohol necrosis have more obviously fatty degeneration, but the repair capacity of traumatic femoral head necrosis is ber than that of hormones and alcohol necrosis. Alcohol and hormones have inhibitory action on the OSX-mediated osteogenic differentiation. Hormones and alcohol may not affect osteoblast expressing adiponectin and its receptors.
Objective To evaluate the effects of composite bone in strategy of tissue engineering on bone defect repair in rats. Methods Sixteen matured Wistar rats (male or female, weighing 250-300 g) were used to prepare platelet lysate (PL). PL/allogeneic decalcified bone granules (ADBG)/Col I (PAC) and ADBG/Col I (AC) were prepared by mixing Col Igel ADBG with or without PL. BMSCs of 8 Wistar rats (male or female, weighing 250-300 g) were isolated and cultured. The 5th passage of BMSCs were co-cultured with PAC at the density of 1 × 106 cells/mL to fabricate the tissue engineered composite PACB in vitro. Forty healthy Wistar rats were made bilateral bone defects in femoral condyles and divided into 4 groups (A, B, C and D, n=10). The defects were filled with equivalent PACB, PAC, AC and Col I in groups A, B, C and D respectively. At 4 weeks, the defect repair was evaluated with radiology, histology, ALP biochemical tests. Results At 4 weeks, the bone density measurement was (7.31 ± 0.54), (4.36 ± 0.67), (2.12 ± 0.47), and (1.09 ± 0.55) pixels in groups A, B, C, and D, respectively. The area of new bone formation in defect area under single view was (412.82 ± 22.31), (266.57 ± 17.22), (94.34 ± 20.22), and (26.12 ± 12.51) pixels in groups A, B, C and D respectively. The ALP contents in femoral condyles were (94.31 ± 7.54), (69.88 ± 4.12), (41.33 ± 3.46), and (21.03 ± 3.11) U/L, respectively. The above indexes of group A were significantly higher than those of groups B, C or D (P lt; 0.05). Three-color flow cytometry assay showed that the T lymphocyte subsets of CD3+CD4+CD8-, CD3+CD8+CD4-, and the ratio of CD4/CD8 displayed no significant difference among four groups (P gt; 0.05). Conclusion Tissue engineered bone PACB is capable to promote the bone defect repair.
Objective It is reported that transforming growth factor β1 (TGF-β1) has the protective effects on the articular cartilage in osteoarthritis (OA). To investigate the significance of the expressions of matrix metalloproteinase 9 (MMP-9), TGF-β1 mRNA and corresponding proteins in OA. Methods The specimens of articular cartilage and synovium were collected from voluntary donators, including 60 cases of OA (experimental group) and 20 cases of traumatic amputation,cruciate l igament rupture, discoid cartilage injury, and menisci injury (normal control group). The pathological changes were observed by HE staining. MMP-9 and TGF-β1 protein expressions were detected by immunohistochemical technique, and the mRNA expressions of MMP-9 and TGF-β1 were detected through in situ hybridization technique; and their correlation was analysed. Results HE staining showed: shrinkage, necrosis, and irregular arrange of the articular chondrocytes, extracellular matrix fracture, hypertrophy and hyperplasia synovium, infiltration of lymphoid and mononuclear cells and prol iferation of many small blood vessels in the experimental group; regular arrangement of the articular chondrocytes, the homogeneously staining matrix, and synovial tissue without chronic inflammation and significant prol iferation in the normal control group. The mRNA and protein expressions of MMP-9 and TGF-β1 were positive in 2 groups. The positive-stained cells included chondrocytes, synovial l ining cells, and vascular endothel ial cells, fibroblasts, and inflammatory infiltrated cells in subsynovial layer. The expressions of mRNA and corresponding protein of MMP-9 and TGF-β1 in the experimental group were significantly higher than those in the normal control group (P lt; 0.01). There was a positive correlation between MMP-9 mRNA and protein expression (r=0.924, P=0.000), and between TGF-β1 mRNA and protein expression (r=0.941, P=0.000) in the experimental group. There was a negative correlation between the expression of MMP-9 protein and TGF-β1 protein (r= — 0.762, P=0.000), and between the expression of MMP-9 mRNA and TGF-β1 mRNA (r= — 0.681, P=0.000) in the experimental group. Conclusion The higher expression of TGF-β1 can protect articular cartilage by down-regulating the expression of MMP-9 of chondrocytes and synoviocytes in OA, which may delay the biological behavior of OA such as occurrence and progress, etc.
Objective To investigate the expression levels and significance of vascular endothel ial growth factor (VEGF) and microvessel density (MVD) in rabbit radius defects repaired with allogeneic and autogenic bone. Methods Forty adult New Zealand rabbits were chosen, and 10 mm bone defect model was created in the bilateral radii of 28 experimental rabbits. The other 12 rabbits provided allogeneic bone under the standard of American Association of Tissue Bank. In the left side, allogeneic bone were used to repair bone defect (experimental group), equal capacity autogenous il iac bone was used in the right side (control group). Animals were sacrificed at 2, 4, 8, and 12 weeks postoperatively. Immunohistochemical method was used to determine the expression of VEGF, CD34 protein and MVD counting. Bone histomorphometric parameters, including percent trabecular area (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were measured by von Kossa staining undecalcified sl ices. The relation was analyzed between VEGF and MVD, histomorphometric parameters. Results The positive signals of VEGF protein were detected in cytoplasm of vascular endothel ial cells, chondrocytes, osteoblasts, fibroblasts and osteoclasts. At 2 weeks, there was no significant difference in VEGF protein expression between experimental group and control group (P gt; 0.05); at 4 and 8 weeks, the expression of VEGF in control group was significantly higher than that in experimental group (P lt; 0.05); and at 12 weeks, there was no significant difference between two groups (P gt; 0.05). There was a positive correlation (P lt; 0.01) between VEGF expression and MVD value in two groups at 2, 4, 8, and 12 weeks postoperatively. There was no significant difference in bone histomorphometric parameters (BV/TV, Tb.Th, Tb.N, Tb.Sp) between two groups at 12 weeks postoperatively (P gt; 0.05), but there was a positive correlation between VEGF expression and parameters of BV/TV, Tb.Th, and Tb.N (P lt; 0.01); and a negative correlation between VEGF and Tb.Sp (P lt; 0.01). Conclusion VEGF can express diversity at different time and positions, and the different expressions indicated various biology significances in the process of the bone heal ing. It can coordinate growth of cartilage and bone and profit vascular reconstruction of allogeneic bone. VEGF may participate in promoting osteogenesis in the course of allogeneic bone transplantation.
Objective To investigate the adhesiveness of osteoblasts and vascular endothel ial cells from rat BMSCs co-cultured on allogeneic freeze-dried partially bone in vitro. Methods The BMSCs were isolated from 4-week-old SD rats (weighing 100-110 g) and cultured in vitro. The third generation of BMSCs were induced into osteoblasts and vascular endothel ial cells. The osteoblasts and vascular endothel ial cells after being induced for 7 days in a ratio of 1 to 1 were directlyco-cultured (experimental group), while the second generation of uninduced BMSCs was used as a control (control group). The growth and prol iferation abil ity were analyzed by MTT examination and the growth curve was drawn at 1-8 days. The osteoblasts and vascular endothel ial cells after being induced for 14 days were implanted in the allogeneic freeze-dried partially bone coated by 20% Col I or not at different densities (0.25 × 106/mL、0.50 × 106/mL、1.00 × 106/mL、2.00 × 106/mL、4.00 × 106/mL), as modified group and unmodified group, the cell adherence rate was calculated after 24 hours. These two kinds of cells were implanted in the pre-disposal treated allogeneic freeze-dried partially bone and observed by scanning electron microscope. Results ALP staining of osteoblasts showed that there were blue grains in cytoplasm at 7 days. CD31 and CD34 immunocytochemical staining of vascular endothelial cell showed that there were positive signals in the cytoplasm at 14 days. The MTT test showed that the prol iferation level of the experimental group was lower than those of the control group. There were significant differences in absorbance value between two group from 3 days to 8 days (P lt; 0.05). The cell adherence rate increased with increasing seeding density when the seeding density was (0.25-1.00) × 106/mL. The cell adherence rate reached the peak when the seeding density was 1.00 × 106/mL. The cell adherence rate decreased when the seeding density was more than 2.00 × 106/mL. There were significant differences in cell adherence rate between modified group and unmodified group at different seeding densities (P lt; 0.05). The prol iferation of the osteoblasts and endothel ial cells presented better growth and histocompatibil ity under scanning electron microscope. Conclusion The growing behavior of two kinds of cells is good in the allogeneic freezedried partially bone coated by 20% Col I , which can be used in reconstrction of vascularized tissue engineered bone.
Objective To evaluate the effect of implantation of the complex of high viscous chitosan/glycerol phosphate with demineral ized bone matrix (HV-C/GP-DBM) in repairing cartilage defects of rabbits. Methods HV-C/ GPDBM was prepared by compounding HV-C/GP and DBM by 2:1 (W/W). Twenty-four 34-week-old New Zealand white adult rabbits, weighing 3.5-4.5 kg, were included. A bit with the diameter of 3.5 mm was used to drill 3-cm-deep holes in both sides of femoral condyle to make cartilage defects. The complex of HV-C/GP-DBM was then injected into the right holes as the experimental group and the left ones serve as the control group. The rabbits were killed at 4, 8 and 16 weeks after theoperation, respectively. The obtained specimens were observed macroscopically, microscopically and histologically. According to the International Cartilage Repair Society Histological Scoring (ICRS), the effect of cartilage repair was assessed at 16 weeks postoperatively. Results At 4-8 weeks postoperatively, in the experimental group, the defects were filled with hyal ine cartilage-l ike tissues; the majority of chitosan degradated; and the DBM particles were partly absorbed. However, in the control group, there were small quantities of discontinuous fibrous tissues and maldistributed chondrocytes at the border and the bottom of the defects. At 16 weeks postoperatively, 6 joints in the experimental group had smooth surface, and the defects were basically repaired by hyal ine cartilage-l ike tissues. The newly-formed tissues integrated well with the surrounding area. Under the cartilage, the new bone formation was still active and some DBM particles could be seen. However, the defects in the control group were repaired by fibrous tissues. The result of histological scoring of the specimens at 16 weeks showed that a total of 6 aspects including formation of chondrocytes and integration with the surrounding cartilages were superior in the experimental group to those in the control group, and there were significant differences between the two groups (P lt; 0.05). Conclusion The biodegradable and injectable complex of HV-C/GP-DBM with good histocompatibil ity and non-toxic side effects can repair cartilage defects and is a promising biomaterial for cartilage defect repair.
Objective To investigate the effect of domestic porous tantalum encapsulated with pedicled fascial flap on repairing of segmental bone defect in rabbits’ radius. Methods A total of 60 New Zealand white rabbits (aged 6- 8 months and weighing 2.5-3.0 kg) were randomly divided into the experimental group and control group (30 rabbits each group). A 1.5 cm segmental bone defect in right radius was established as the animal model. The porous tantalums encapsulated with pedicled fascial flaps (30 mm×20 mm) were implanted in the created bone defect in the experimental group, and the porous tantalums were only implanted in the control group. X-ray films were observed at the day after operation and at 4, 8, and 16 weeks after operation. Specimens were taken out at 4, 8, and 16 weeks after operation for HE staining and toluidine blue staining observation. The maximum load force and bending strength were detected by three point bending biomechanical test, and the Micro-CT analysis and quantitative analysis of the new bone volume fraction (BV/TV) were performed at 16 weeks after operation to compare the bone defect repair abilityin vivo in 2 groups. Results All incisions healed by first intention without wound infection. At 4, 8, and 16 weeks after operation, the X-ray films showed that the implants were well maintained without apparent displacement. As followed with time, the combination between the implants and host bone became more and more closely, and the fracture line gradually disappeared. HE staining and toluidine blue staining showed that new bone mass and maturity gradually increased at the interface and inside materials in 2 groups, and the new bone gradually growed from the interface to internal pore. At 16 weeks after operation, the three point bending biomechanical test showed that the maximum load force and bending strength in the experimental were (96.54±7.21) N and (91.26±1.76) MPa respectively, showing significant differences when compared with the control group [(82.65±5.65) N and (78.53±1.16) MPa respectively] (t=3.715, P=0.004; t=14.801, P=0.000). And Micro-CT analysis exhibited that there were a large amount of new bone at the interface and the surface of implant materials and inside the materials. The new bone BV/TV in the experimental group (32.63%±3.56%) was significantly higher than that in control group (25.07%±4.34%) (t=3.299, P=0.008). Conclusion Domestic porous tantalum encapsulated with pedicled fascial flap can increase local blood supply, strengthen material bone conduction ability, and promote the segmental bone defect repair.