ObjectiveTo evaluate the effect of leucocyte- and platelet-rich plasma (L-PRP) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in treating avascular necrosis of the femoral head (ANFH) in rabbits. MethodsTwenty-four New Zealand white rabbits (4-6 months old, both genders, weighing 2.0-3.0 kg) were used for the establishment of bilateral ANFH models and divided into 4 groups (n=6). BMSCs were isolated from the bone marrow of iliac crest, cultured and identified. L-PRP was prepared by Landesberg method. Core decompression only (group A), core decompression and L-PRP implantation (group B), core decompression and BMSCs implantation (group C), and core decompression and implantation of BMSCs and L-PRP were performed in 4 groups. To evaluate bone formation and remodeling of the defects, X-ray photography was taken at 2, 4, and 8 weeks postoperatively. The modified Lane-Sandhu scoring system was used to evaluate the bone formation. Two rabbits were sacrificed at 2, 4, 8 weeks after operation to harvest the specimens for histological observation, new blood vessel count and new bone area ratio. ResultsThe observations of radiology and histology displayed different degrees of bone regeneration at bone defect sites in each group. At 2, 4, and 8 weeks postoperatively, the results of Lane-Sandhu X-ray photography scoring, new blood vessel count, and new bone area ratio showed that groups C and D were significantly better than groups A and B, group D was significantly better than group C. and group B was significantly better than group A (P<0.05). ConclusionThese findings demonstrate that L-PRP can promote osteogenic differentiation of BMSCs in treating ANFH in rabbits, and core decompression associated with BMSCs and L-PRP is an effective and feasible method to treat ANFH.
ObjectiveTo explore the effect of fetal bovine serum (FBS) of different concentrations in the culture medium on osteogenic growth peptide (OGP) promoting bone marrow mesenchymal stem cells (BMSCs) proliferation and differentiation. MethodsBMSCs were separated from limb bones of 8 Sprague Dawley rats (5 weeks old) and purified by adherence method, and BMSCs at passage 3 were divided into 4 groups according to OGP concentration: OGP 1×10-10 mol/L group, OGP 1×10-9mol/L group, OGP 1×10-8 mol/L group, and control group without OGP; and 0, 2%, 5%, 8%, and 10%FBS concentration gradient was used in each group. The cell proliferation rate was detected by MTT method at 1, 3, 5, 7, 9, and 12 days after culture, and the activity of intracellular alkaline phosphatase (ALP) was determined by the method of p-nitrophenyl phosphate disodium at 9 days after culture. ResultsBMSCs showed adherent growth, rapid proliferation, long fiber vortex, and typical morphology. MTT analysis showed that cells could not sustain proliferation when FBS concentration was less than 5% in each group; when FBS concentration was above 8%, cells proliferated continually. Proliferation promoting effect of OGP 1×10-8 mol/L and 1×10-9 mol/L groups was significantly higher than that of the control group in all serum concentrations (P<0.05); when FBS concentration was lower than 10%, the proliferation promoting effect of OGP 1×10-8 mol/L group was significantly higher than that of the other 2 OGP groups (P<0.05), but when FBS concentration was 10%, OGP 1×10-8 mol/L group had no advantage of promoting proliferation. ALP test results showed that as the FBS concentration increased, ALP activity of all groups also significantly increased (P<0.05). Under the condition of 5%FBS and 8%FBS, the ALP activity of each OGP group was significantly greater than that of the control group, and it was the highest in OGP 1×10-8 mol/L group (P<0.05). Under the condition of 10%FBS, the ALP activity of each OGP group was still greater than that of the control group (P<0.05), but no significant difference was found between the OGP 1×10-8 mol/L group and OGP 1×10-9 mol/L group (P>0.05). ConclusionThe concentration of 8%FBS is the best concentration of serum for OGP promoting the proliferation and differentiation of BMSCs, and the most suitable concentration of promoting the proliferation and differentiation of BMSCs is OGP 1×10-8 mol/L.
Abstract：Pulmonary metastasectomy is an important curative option for patients with osteogenic and softtissue sarcoma spread to the lungs. Complete surgical removal of pulmonary metastases can improve survival and is recommended under certain criteria. Specific issues that require consideration when planning pulmonary metastasectomy include: preoperative assessment of the operation index and contraindications, choice of surgical strategies, pulmonary parenchymal preservation, and the role of lymphadenectomy. With the development of iconography and chemotherapy, the emergence of targeted drugs, and the innovation of radiotherapy, the concept of the diagnosis and treatment for pulmonary metastases from osteogenic and softtissue sarcoma is also undergoing great changes.
Objective To review the osteogenic mechanism and osteogenic effects of bone morphogenetic protein 6 (BMP-6) so as to provide the basis for further research of BMP-6. Methods The related articles about the osteogenic mechanism and the osteogenic effects of BMP-6 in experimental animals were extensively summarized. Results BMP-6 from bone matrix can transduct the osteogenic signal to bone marrow mesenchymal stem cells (BMSCs) by means of Smad protein signal transduction pathway. And the BMSCs which received the signals will differentiate into osteoblasts and chondroblasts. Therefore, BMP-6 plays an important role in the development and maturation of bone and cartilage. In addition, BMP-6 has a close relation with bone diseases, such as fracture, osteoporosis, and bone tumor. Conclusion The deep research of BMP-6 is expected to provide a new therapeutic approach for treating bone diseases of nonunion, osteoarthritis, and osteoporosis.
Objective To review the research progress of the osteogenic effect of strontium (Sr) and its application in the orthopaedics. Methods The recent literature concerning the osteogenic effect of Sr and its application in orthopaedics at home and abroad was extensively reviewed, and the research and development were summarized. Results Both in vivo and in vitro studies showed that Sr could enhance bone formation and inhibit bone resorption. Clinically, Sr was applied for treatment of osteoporosis, composite biomaterials in tissue engineering, and treatment of bone tumors and bone metastases. Conclusion Sr is one important combined element of alternative materials in bone tissue engineering, and can strengthen the mechanical and biological properties of the bone replacement material, so it has some development potential in bone tissue engineering.
Objective To review the progress and clinical application of malleable bone paste/putty. MethodsRecent literature about malleable bone paste/putty was reviewed and analyzed. ResultsThe preparation and clinical application of malleable bone paste/putty have become increasingly mature. Many kinds of malleable bone paste/putty have been applied extensively and the good clinical results have been achieved in the treatment of the irregular bone defects. The materials and methods for preparing malleable bone paste/putty are different. Then they have different bone repair abilities. ConclusionMalleable bone paste/putty provides effective method to treat irregular bone defects. But the malleable bone paste/putty still has some shortage, so further researches should be carried out.
Objective To clarify the trends of expression levels of several up-regulated micro RNA (miRNA) in tissues of atrophic bone nonunion and mRNAs and proteins of their related target genes in osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and to explore their biological functions. Methods The hBMSCs were isolated from bone marrow of il iac bone by gradient centrifugation, and cultured. Osteogenic culture medium was used for osteogenic differentiation of the 4th generation of hBMSCs. The changes of corresponding miRNAs, mRNA and protein expression levels of related target genes were observed at 0 hour, 12 hours, 1 day, 2 days, 4 days, 7 days, and 14 days, by quantitative real-time PCR and Western blot. Results In the process of hBMSCs osteogenic differentiation, the mRNA and protein expression levels of osteoblastic target genes [alkal ine phosphatase l iver/bone/kidney (ALPL), bone morphogeneticprotein 2 (BMP-2), and platelet-derived factor alpha polypeptide (PDGF-A)] at most time points increased significantly whencompared with the values at 0 hour except that of BMP-2 decreased at 12 hours and 1 day, with maximum changes at 1 to 7 days. The miRNA expression levels, mRNA and protein expression levels changed significantly at different time points, while the trends of hsa-miRNA-149 and hsa-miRNA-654-5p changes were negatively correlated with the trends of ALPL and BMP-2 mRNA and protein expression changes respectively (P lt; 0.05). There was no obviously negative correlation between the trends of hsa-miRNA-221 change and PDGF-A change (P gt; 0.05). Conclusion In the osteogenic differentiation process of hBMSCs, hsa-miRNA-149 and hsa-miRNA-654-5p are closely related with the mRNA and protein regulation of ALPL and BMP-2, respectively.
Objective Dexamethasone is one of the basic agents which could induce osteogenic differentiation of mesenchymal stem cells. To investigate the optimal concentration of dexamethasone in osteogenic differentiation of adiposederivedstem cells (ADSCs) so as to provide the theoretical basis for further bone tissue engineering researches. Methods FiveNew Zealand rabbits (2-3 kg) of clean grade, aged 3 months and male or female, were obtained. ADSCs were isolated from the subcutaneous adipose tissue of inguinal region, and cultured with collagenase digestion, then were detected and identified by CD44, CD106 immunofluorescence staining and adi pogenic differentiation. ADSCs at passage 3 were used and the cell density was adjusted to 1 × 105 cells/mL, then the cells were treated with common cultural medium (group A) and osteogenic induced medium containing 0 (group B), 1 × 10-9 (group C), 1 × 10-8 (group D), 1 × 10-7 (group E), 1 × 10-6 (group F), and 1 × 10-5 mol/ L (group G) dexamethasone, respectively. The cell prol iferation and the mRNA expressions of osteocalcin (OC) and core binding factor α1 (Cbfα1) were detected by MTT and RT-PCR, respectively. The activity of alkal ine phosphatase (ALP) was measured, and the percentage of mineral area was calculated. The mineral nodules were also detected by al izarin red staining. Results ADSCs mostly presented fusiform and polygon shape with positive expression of CD44 and negative expression of CD106. The result of oil red O staining was positive after ADSCs treated with adipogenic induced medium. The result of MTT revealed that the absorbance (A) value decl ined with the ascending of the concentration of dexamethasone, and there was significant difference in A value between groups D and E at 5 and 7 days after osteogenic induction (P lt; 0.05). The mRNA expressions of OC and Cbfα1 reached the peak in groups E and D at 7 days after osteogenic induction, respectively. The activity of ALP and the percentage of mineral area had the maximum value in group D at 14 days, then decl ined gradually. There was no significant difference in the mRNA expressions of OC and Cbfα1, the activity of ALP, and the percentage ofmineral area between groups D and E (P gt; 0.05), but significant differences were found between groups D and E and other groups (P lt; 0.05). After 14 days, the cells of group G died, and the result of al izarin red staining was positive in groups B, C, D, E, and F. Conclusion When the concentration of dexamethasone in osteogenic medium is 1 × 10-8 mol/L, it could not only reduce the inhibitive effect on cells prol iferation, but also induce osteogenic differentiation of ADSCs more efficiently.
Objective Simvastatin has been reported to be effective on stimulation of bone formation. To investigate the effects of simvastatin on bone formation relative factors of proximal tibia trabecular bone and on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods Fourty 1-week-old male Sprague Dawley rats were divided randomly into 2 groups, 20 rats per group. Rats in experimental group received subcutaneous injection of simvastatin [(5 mg/ (kg• d)], and the rats in control group received injection of normal sal ine at the same dose. The expressions of bone morphogenetic protein 2 (BMP-2), matrix metalloproteinase 13 (MMP-13), and vascular endothel ial growth factor (VEGF) of trabecular bone were analyzed in the tibia by immunohistochemical staining at 1 and 3 weeks after injection. BMSCs from the rat femur at 1 and 3 weeks after injection were cultured under condition of osteogenic induction. ALP staining wasperformed on the 14th day after culture; real-time fluorescent quantitative PCR was used to detect the mRNA expressions of BMP-2, Runx2, Osterix, Msx2, Dlx3, and Dlx5 on the 21st day after culture; and von Kossa staining was performed on the 28th day after culture. Results There was no significant difference in the expressions of BMP-2, MMP-13, and VEGF betweenthe experimental group and control group at 1 and 3 weeks after injection (P gt; 0.05). There was no significant difference in the percentages of ALP positively-stained cells between the experimental group and the control group on the 14th day after culture (P gt; 0.05). The mRNA expressions of BMP-2, Runx2, Osterix, Msx2, Dlx3, and Dlx5 in osteogenic differentiation-inducedBMSCs had also no significant difference between the experimental group and the control group at 1 and 3 weeks after culture (P gt; 0.05). No significant difference in biomineral ization was found between the experimental group and control group at 1 and 3 weeks after culture (P gt; 0.05). Conclusion Subcutaneous injection of simvastatin [(5 mg/(kg•d)] for 1 or 3 weekscan affect neither the expressions of bone formation relative factors of proximal tibia trabecular bone nor the osteogenic differentiation of the BMSCs.
Objective Calcium phosphate bioceramics has a broad appl ication prospect because of good biocompatibil ity, but porous scaffolds with complex shape can not be prepared by the traditional methods. To fabricate porous calcium phosphate ceramics by rapid prototyping and to investigate the in vitro osteogenic activities. Methods The porous calcium phosphate ceramics was fabricated by rapid prototyping. The bone marrow mesenchymal stem cells (BMSCs)were isolated from bone marrow of Beagle canine, and the 3rd passage BMSCs were seeded onto the porous ceramics. The cell/ceramics composite cultured in osteogenic medium were taken as the experimental group (group A) and the cell/ceramics composite cultured in growth medium were taken as the control group (group B). Meanwhile, the cells seeded on the culture plate were cultured in osteogenic medium or growth medium respectively as positive control (group C) or negative control (group D). After 1, 3, and 7 days of culture, the cell prol iferation and osteogenic differentiation on the porous ceramics were evaluated by DNA quantitative analysis, histochemical staining and alkal ine phosphatase (ALP) activity. After DiO fluorescent dye, the cell adhesion, growth, and prol iferation on the porous ceramics were also observed by confocal laser scanning microscope (CLSM). Results DNA quantitative analysis results showed that the number of BMSCs in all groups increased continuously with time. Plateau phase was not obvious in groups A and B, but it was clearly observed in groups C and D. The CLSM observation indicated that the activity of BMSCs was good and the cells spread extensively, showing good adhesion and prol iferation on the porous calcium phosphate ceramics prepared by rapid prototyping. ALP quantitative analysis results showed that the stain of cells on the ceramics became deeper and deeper with time in groups A and B, the staining degree in group A were ber than that in group B. There was no significant difference in the change of the ALP activity among 4 groups at the first 3 days (P gt; 0.05); the ALP activity increased obviously in 4 groups at 7 days, group A was significantly higher than other groups (P lt; 0.05) and groups C, D were significantly higher than group D (P lt; 0.05). Conclusion The porous calcium phosphate ceramics has good cytocompatibil ity and the designed pores are favorable for cell ingrowth. The porous ceramicsfabricated by rapid prototyping has prominent osteogenic differentiation activity and can be used as a choice of scaffolds for bone tissue engineering.