EFFECTS OF TRANSFORMING GROWTH FACTOR β AND RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2 ON HUMAN PERIODONTAL LIGAMENT FIBROBLASTS

Objective To evaluate the effects of transforming growth factor β(TGF-β) and recombinant human bone morphogenetic protein 2 (rhBMP2) on human periodontal ligament fibroblasts (HPDLFs). Methods HPDLFs were done primary culture to detect the distinct concentrations of TGF-P and rhBMF2 on its proliferation, alkaline phosphatase (ALP) activity, osteocalcin (OC) synthesis and formation of the minerali-zed nodules, respectively. Results TGF-β (5～100ng/ml) significantly stimulated the proliferation of HPDLFs. The ALP activity of HPDLFs was evaluated evidently by 5ng/ml TGF-β. TGF-β( 0. 5 ～ 100ng/ml) had no effects on OC synthesis and formation of the mineralized nodules of HPDLFs. rhBMP2 (0. 25～2mg/ ml) had no remarkable effect on the proliferation of HPDLFs. The ALP activity, OC synthesis and forma-tion of the mineralized nodules of HPDLFs were significantly stimulated by 0. 5～ 2mg /ml rhBMP2. Conclusion The effects of TGF-β and rhBMP2 on HPDLFs are dose-dependent. TGF-P can stimulate HPDLFs to express the early marker of osteoblastic phenotype, and it lacks the ability to promote maturation of the osteogenic phenotype. rhBMP2 can not only stimulate the expression but also promote the maturation of osteoblas-tic phenotype of HPDLFs.

Construction of bicistronic green fluorescent protein labeled pSELECT GFPzeo human bone morphogenetic protein 2 eukaryotic expression vector

Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase

Acupotomy ameliorates subchondral bone absorption and mechanical properties in rabbits with knee osteoarthritis by regulating bone morphogenetic protein 2-Smad1 pathway

OBJECTIVE:To investigate the effects of acupotomy on the subchondral bone absorption and mechanical properties in rabbits with knee osteoarthritis(KOA).METHODS:The rabbits were divided into blank control,model,acupotomy and electroacupuncture(EA)groups,with 12 rabbits in each.Modified Videman's method was used to prepare KOA model.The acupotomy and EA group were given indicated intervention for 3 weeks.The behavior of rabbits in each group was recorded.Subsequently,cartilage–subchondral bone units were obtained and morphological changes were observed by optical microscope and micro computed tomography.Compression test was used to detect the mechanical properties of subchondral bone,Western blot and real-time polymerase chain reaction(RT-PCR)were applied to detect the expression of bone morphogenetic protein 2-Smad1(BMP2-Smad1)pathway in subchondral bone.RESULTS:Compared with the control group,rabbits in the KOA group showed lameness,knee pain,and cartilage degradation;the subchondral bone showed active resorption,the mechanical properties decreased significantly and the BMP2-Smad1 pathway downregulated significantly.Both acupotomy and EA intervention could increase the thickness of trabecular bone(Tb.Th),the bone volume fraction(BV/TV)and the thickness of subchondral bone plate,reduce the separation of trabecular bone(Tb.Sp),improve the maximum load and elastic modulus of subchondral bone,and effectively delay cartilage degeneration in KOA rabbits.This process may be achieved through upregulation the related proteins of BMP2-Smad1 pathway.The maximum load and elastic modulus of subchondral bone in the acupotomy group were slightly better than those in the EA group.CONCLUSIONS:Acupotomy could effectively protect cartilage by inhibiting abnormal bone resorption and improving mechanical properties of subchondral bone thorough the related proteins of BMP2-Smad1 pathway in KOA rabbits.

A segmental defect adaptation of the mouse closed femur fracture model for the analysis of severely impaired bone healing

Objective: To better characterize nonunion endochondral bone healing and evaluate novel therapeutic approaches for critical size defect healing in clinically challenging bone repair, a segmental defect model of bone injury was adapted from the threepoint bending closed fracture technique in the murine femur.Methods: The mouse femur was surgically stabilized with an intramedullary threaded rod with plastic spacers and the defect adjusted to different sizes. Healing of the different defects was analyzed by radiology and histology to 8 weeks postsurgery. To determine whether this model was effective for evaluating the benefits of molecular therapy, BMP-2 was applied to the defect and healing then examined.Results: Intramedullary spacers were effective in maintaining the defect. Callus bone formation was initiated but was arrested at defect sizes of 2.5 mm and above, with no more progress in callus bone development evident to 8 weeks healing. Cartilage development in a critical size defect attenuated very early in healing without bone development, in contrast to the closed femur fracture healing, where callus cartilage was replaced by bone. BMP-2 therapy promoted osteogenesis of the resident cells of the defect, but there was no further callus development to indicate that healing to pre-surgery bone structure was successful.Conclusions: This segmental defect adaptation of the closed femur fracture model of murine bone repair severely impairs callus development and bone healing, reflecting a challenging bone injury. It is adjustable and can be compared to the closed fracture model to ascertain healing deficiencies and the efficacy of therapeutic approaches.

Immunoadsorption therapy for Klinefelter syndrome with antiphospholipid syndrome in a patient:A case report

BACKGROUND Klinefelter syndrome(KS) is a genetic disease of male sex chromosome malformations that affects sperm production and reduces testosterone production. It has been reported that there is currently more than 10 cases of KS combined with antiphospholipid syndrome(APS).CASE SUMMARY Here, we describe a 31-year-old male patient with chromosome 47, XXY type, who suffered deep vein thrombosis of the lower limbs accompanied by abnormal antiphospholipid antibody, lupus anticoagulant and factor VⅢ. After treatment with immunoadsorption therapy, glucocorticoids, cyclophosphamide, intravenous immunoglobulin and anticoagulant therapy, the patient showed dramatic symptomatic improvement. During the follow-up, the patient did not develop any new thrombotic events.CONCLUSION Immunoadsorption combined with glucocorticoid and cyclophosphamide shock comprehensive treatment has achieved significant results for patients with KS combined with antiphospholipid syndrome.

CLONING AND SEQUENCING OF MATURE FRAGMENT OF HUMAN BMP4 GENE

Objective To study the cloning and sequencing of mature fragment of human bone morphogenetic protein 4 gene. Methods The template DNA was obtained from the human osteosarcoma cell line U2OS. By using RT PCR method, the cDNA coding for the mature fragment of BMP 4 was amplified, cloned into the vector pUC19, and sequenced by Sanger Dideoxy mediated Chain Termination method. Results The mature fragment of BMP4 cDNA was obtained by RT PCR and determined by sequencing. Through the computer search on Genebank, the analysis showed that the homology of nucleotides and amino acids between cDNA of rhBMP4 mature fragment of this study and the published sequence was 99%. Sequence analysis showed that there were two differences, one was at base 1154(201): G→C, which had no influence on the corresponding amino acids(Val). Another was at base1222(269):C→T, the mutation at the base 1222 had the change of Ala to Val. Conclusion The mature fragment of BMP4 gene has been cloned. The results will be of great significance in treatment of skeletal injuries and diseases.

Sox9 augments BMP2-induced chondrogenic differentiation by downregulating Smad7 in mesenchymal stem cells(MSCs)

Cartilage injuries caused by arthritis or trauma pose formidable challenges for effective clinical management due to the limited intrinsic proliferative capability of chondrocytes.Autologous stem cell-based therapies and transgene-enhanced cartilage tissue engineering may open new avenues for the treatment of cartilage injuries.Bone morphogenetic protein 2(BMP2)induces effective chondrogenesis of mesenchymal stem cells(MSCs)and can thus be explored as a potential therapeutic agent for cartilage defect repair.However,BMP2 also induces robust endochondral ossification.Although the precise mechanisms through which BMP2 governs the divergence of chondrogenesis and osteogenesis remain to be fully understood,blocking endochondral ossification during BMP2-induced cartilage formation may have practical significance for cartilage tissue engineering.Here,we investigate the role of Sox9-donwregulated Smad7 in BMP2-induced chondrogenic differentiation of MSCs.We find that overexpression of Sox9 leads to a decrease in BMP2-induced Smad7 expression in MSCs.Sox9 inhibits BMP2-induced expression of osteopontin while enhancing the expression of chondrogenic marker Col2a1 in MSCs.Forced expression of Sox9 in MSCs promotes BMP2-induced chondrogenesis and suppresses BMP2-induced endochondral ossification.Constitutive Smad7 expression inhibits BMP2-induced chondrogenesis in stem cell implantation assay.Mouse limb explant assay reveals that Sox9 expands BMP2-stimulated chondrocyte proliferating zone while Smad7 promotes BMP2-intitated hypertrophic zone of the growth plate.Cell cycle analysis indicates that Smad7 induces significant early apoptosis in BMP2-stimulated MSCs.Taken together,our results strongly suggest that Sox9 may facilitate BMP2-induced chondrogenesis by downregulating Smad7,which can be exploited for effective cartilage tissue engineering.

Can microcalcifications' characteristics predict the risk of breast cancer metastasis to bone?

Aim: To correlate the microcalcifications' characteristics, such as morphology and elemental compositions, with the occurrence of bone metastatic lesions at 5 years from diagnosis. Methods: In this retrospective study, we enrolled 70 patients from which we collected one breast biopsy each. From each biopsy, paraffin serial sections were obtained to perform histological classifications, immunohistochemical analyses and Energy Dispersive X-ray evaluation. Results: Microcalcifications analysis showed a significant association between the presence of calcium crystals made of magnesium substituted hydroxyapatite and the development of bone metastasis from 5 years from diagnosis. No significant association was observed by evaluation the morphological appearance of microcalcifications. Immunohistochemical analysis displayed a significant association between the expression of bone morphogenetic proteins 2 and pentraxin-3, two osteoblast induction factors, and the formation of bone metastatic lesions. Conclusion: Results here reported highlighted the possible use of breast microcalcifications as a negative prognostic marker of bone metastatic diseases. In particular, the association between elemental composition of breast microcalcifications and the formation of bone lesions can lay the foundation for the development of new in vivo diagnostic tools based on the analysis of microcalcifications and capable to predict the formation of bone metastasis.

Harvest of functional mesenchymal stem cells derived from in vivo osteo-organoids

Bone marrow-derived mesenchymal stem cells(BM-MSCs)play a crucial role in stem cell therapy and are extensively used in regenerative medicine research.However,current methods for harvesting BM-MSCs present challenges,including a low yield of primary cells,long time of in vitro expansion,and diminished differentiation capability after passaging.Meanwhile mesenchymal stem cells(MSCs)recovered from cell banks also face issues like toxic effects of cryopreservation media.In this study,we provide a detailed protocol for the isolation and evaluation of MSCs derived from in vivo osteo-organoids,presenting an alternative to autologous MSCs.We used recombinant human bone morphogenetic protein 2-loaded gelatin sponge scaffolds to construct in vivo osteo-organoids,which were stable sources of MSCs with large quantity,high purity,and strong stemness.Compared with protocols using bone marrow,our protocol can obtain large numbers of high-purity MSCs in a shorter time(6 days vs.12 days for obtaining passage 1 MSCs)while maintaining higher stemness.Notably,we found that the in vivo osteo-organoid-derived MSCs exhibited stronger anti-replicative senescence capacity during passage and amplification,compared to BM-MSCs.The use of osteo-organoid-derived MSCs addresses the conflict between the limitations of autologous cells and the risks associated with allogeneic sources in stem cell transplantation.Consequently,our protocol emerges as a superior alternative for both stem cell research and tissue engineering.