Relationships between blood leukocyte mitochondrial DNA copy number and inflammatory cytokines in knee osteoarthritis / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Osteoarthritis (OA) is a degenerative articular disorder manifested by cartilage destruction, subchondral sclerosis, osteophytes, and synovitis, resulting in chronic joint pain and physical disability in the elderly. The purpose of this study was to investigate mitochondrial DNA copy number (mtDNACN) and inflammatory cytokines in primary knee OA patients and healthy volunteers. A total of 204 knee OA patients and 169 age-matched healthy volunteers were recruited. Their relative blood leukocyte mtDNACN was assessed by quantitative real-time polymerase chain reaction (qRT-PCR), and ten inflammatory cytokines in their plasma were detected by multiplex immunoassay. Blood leukocyte mtDNACN in the OA group was significantly lower than that in the control group. Leukocyte mtDNACN in the control group was negatively correlated with their age (r=-0.380, P<0.0001), whereas mtDNACN in the OA group was positively correlated with their age (r=0.198, P<0.001). Plasma interleukin-4 (IL-4) and IL-6 were significantly higher in the knee OA group than in the control group. The plasma IL-6 level was positively correlated with blood leukocyte mtDNACN in the OA group (r=0.547, P=0.0014). IL-5 showed as a major factor (coefficient 0.69) in the second dimension of principle components analysis (PCA)-transformed data and was significantly higher in the OA group (P<0.001) as well as negatively correlated with mtDNACN (r=-0.577, P<0.001). These findings suggest that elevation of plasma IL-4 and IL-6 and a relative reduction in mtDNACN might be effective biomarkers for knee OA. IL-5 is a plausible factor responsible for decreasing blood leukocyte mtDNACN in knee OA patients.

RhBMP-2 and -7 combined with absorbable collagen sponge carrier enhance ectopic bone formation: An in vivo bioassay

Background: Recombinant human bone morphogenetic proteins (rhBMPs) have been characterized especially chondrogenic and osteogenic activity both in vitro and in vivo studies. However, delivery of more than onegrowth factor by sustained release carrier to orthopedic site has yet been questionable in term of efficacy and synergism.Objective: Evaluate osteoinductivity and synergistic effect of rhBMP-2 and -7 using absorbable collagen sponge (ACS) carrier system in vivo.Methods: cDNA of BMP-2 and -7 active domains were cloned and expressed in Escherichia coli BL21 StarTM (DE3) using pRSETc expression system. Then, the purified rhBMPs were loaded onto ACS and evaluated by in vivo rat subcutaneous bioassay. Two and eight weeks postoperatively, all treated groups were histologically verified for evidence of new bone formation and neovascularization by hematoxylin-eosin staining and light microscopy.Results: The Wistar rat treated with rhBMP-2 or -7/ACS exhibited new bone formation, compared to ACS control. The group treated with ACS supplemented with both rhBMP-2 and -7 significantly showed the osteoid matrix very well-organized into trabeculae-like structure with significant blood vessel invasion.Conclusion: The osteogenic induction of rhBMPs was combined with ACS carrier in the in vivo bioassay. In addition, the combination of both two potent recombinant osteoinductive cytokines, rhBMP-2 and -7, with ACS carrier demonstrated synergistic effect and might be a more promising and effective choice for therapeutic applications.Keywords: Absorbable collagen sponge, bone morphogenetic protein, ectopic bone formation, ratsubcutaneous model

Osteoinductive potential of small intestinal submucosa/demineralized bone matrix as composite scaffolds for bone tissue engineering

Background: Demineralized bone matrix (DBM) is extensively used in orthopedic, periodontal, and maxillofacial application and investigated as a material to induce new bone formation. Small intestinal submucosa (SIS) derived from the submucosa layer of porcine intestine has widely utilized as biomaterial with minimum immune response.Objectives: Determine the osteoinductive potential of SIS, DBM, SIS/DBM composites in the in vitro cell culture and in vivo animal bioassays for bone tissue engineering.Materials and methods: Human periosteal (HPO) cells were treated in the absence or presence SIS, DBM, and SIS/DBM. Cell proliferation was examined by direct cell counting. Osteoblast differentiation of the HPO cells was analyzed with alkaline phosphatase activity assay. The Wistar rat muscle implant model was used to evaluate the osteoinductive potential of SIS, DBM, and SIS/DBM composites.Results: HPO cells could differentiate along osteogenic lineage when treated with either DBM or SIS/DBM. SIS/DBM had a tendency to promote more cellular proliferation and osteoblast differentiation than the other treatments. In Wistar rat bioassay, SIS showed no new bone formation and the implants were surrounded by fibrous tissues. DBM demonstrated new bone formation along the edge of old DBM particles. SIS/DBM composite exhibited high osteoinductivity, and the residual SIS/DBM was surrounded by osteoid-like matrix and newlyformed bone.Conclusion: DBM and SIS/DBM composites could retain their osteoinductive capability. SIS/DBM scaffolds may provide an alternative approach for bone tissue engineering.Keywords: Demineralized bone matrix, human periosteal cells, small intestinal submucosa, osteoblast differentiation 

Gene expression characteristics of osteoblast differentiation in human umbilical cord mesenchymal stem cells induced by demineralized bone matrix

Background: Mesenchymal stem cells are multipotential cells capable of differentiating into osteoblasts, chondrocytes, adipocytes, tenocytes, and myoblasts. Wharton’s jelly contains stem cells that are a rich source of primitive multipotent mesenchymal cells. Demineralized bone matrix (DBM) has been extensively utilized as a biomaterial to promote new bone formation. Objective: To isolate and characterize umbilical cord mesenchymal stem (UCMS) cells derived from Wharton’s jelly and examine the biological activity of DBM in this cell line. Methods: Osteoblast differentiation of the UCMS cells was determined using alkaline phosphatase activity assay. To examine differential gene expression during osteoblast differentiation, total RNA was isolated from UCMS cells in the absence or presence of DBM on day 7 and analyzed using osteogenesis cDNA gene array. The selected genes were verified using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Results: Wharton’s jelly derived cells could differentiate along an osteogenic lineage after treatment of DBM. Alkaline phosphatase activity assay showed that human UCMS cells could differentiate into osteogenic lineage. Gene expression of human UCMS cells treated with DBM for 7 days was analyzed by using cDNA array and RT- PCR analyses. We found that expression of runx2 and smad2 was upregulated whereas smad7 expression was downregulated as confirmed by RT-PCR. Conclusion: UCMS cells from a Wharton’s jelly of human umbilical cord could express osteogenesis genes for treatment with DBM. Wharton’s jelly from umbilical cord is a new source of mesenchymal stem cells that are readily available for application to bone tissue engineering.