Cell-recruited microspheres for OA treatment by dual-modulating inflammatory and chondrocyte metabolism.

Osteoarthritis (OA) is a degenerative disease potentially exacerbated due to inflammation, cartilage degeneration, and increased friction. Both mesenchymal stem cells (MSCs) and pro-inflammatory macrophages play important roles in OA. A promising approach to treating OA is to modify multi-functional hydrogel microspheres to target the OA microenvironment and structure. Arginyl-glycyl-aspartic acid (RGD) is a peptide widely used in bioengineering owing to its cell adhesion properties, which can recruit BMSCs and macrophages. We developed TLC-R, a microsphere loaded with TGF-ß1-containing liposomes. The recruitment effect of TLC-R on macrophages and BMSCs was verified by in vitro experiments, along with its function of promoting chondrogenic differentiation of BMSCs. And we evaluated the effect of TLC-R in balancing OA metabolism in vitro and in vivo. When TLC-R was co-cultured with BMSCs and lipopolysaccharide (LPS)-treated macrophages, it showed the ability to recruit both cells in substantial numbers. As the microspheres degraded, TGF-ß1 and chondroitin sulfate (ChS) were released to promote chondrogenic differentiation of the recruited BMSCs, modulate chondrocyte metabolism and inhibit inflammation induced by the macrophages. Furthermore, in vivo analysis showed that TLC-R restored the narrowed space, reduced osteophyte volume, and improved cartilage metabolic homeostasis in OA rats. Altogether, TLC-R provides a comprehensive and novel solution for OA treatment by dual-modulating inflammatory and chondrocyte metabolism.

Microenvironment-responsive bilayer hydrogel microspheres with gelatin-shell for osteoarthritis treatment.

Osteoarthritis is a long-term degenerative condition of the joints that is characterized by the breakdown of cartilage and inflammation of the synovial membrane. The presence of an inflammatory microenvironment and the degradation of the extracellular matrix produced by chondrocytes leads to the aggravation of cartilage injury, hindering the treatment of osteoarthritis. A promising approach to address this issue is to apply a combined strategy that is sensitive to the specific conditions in osteoarthritic joints and possesses properties that can reduce inflammation and promote cartilage healing. Here, inspired by the structure of chocolate-covered peanuts, we developed an injectable, environment-responsive bilayer hydrogel microsphere using microfluidics technology. The microsphere applied chondroitin sulfate methacryloyl (ChsMA) as its core and was coated with a methacryloyl gelatin (GelMA) shell that was loaded with celecoxib (CLX) liposomes (ChsMA+CLX@Lipo@GelMA). CLX was released from the liposomes when the GelMA shell rapidly degraded in response to the osteoarthritic microenvironment and suppressed the generation of inflammatory agents, demonstrating a beneficial impact of the outer shell in reducing inflammation. While the inner methacryloyl microsphere core degraded, chondroitin sulfate was released to promote chondrocyte anabolism and facilitate cartilage repair. Thus, the synthesized bilayer hydrogel microspheres hold great potential for treating osteoarthritis.

BMP-2 functional polypeptides relieve osteolysis via bi-regulating bone formation and resorption coupled with macrophage polarization.

Osteolysis caused by wear debris around the prosthesis is the main reason for aseptic loosening. Extending prosthetic service life is still challenging. In this study, we first synthesized a bone morphogenetic protein-2 (BMP-2) functional polypeptide (BMP2pp), and evaluated the effects of BMP2pp on macrophage polarization and impaired osteogenesis caused by titanium (Ti) particles in vitro. Then, we delineated the impact of BMP2pp on bone formation and resorption in a mouse calvarial bone osteolysis model induced by Ti particles. The results showed that BMP2pp not only alleviated the Ti-induced inhibition of osteoblastic differentiation in human placenta-derived mesenchymal stem cells (hPMSCs) but also prevented Ti-induced M1 macrophage polarization and promoted M2 macrophage differentiation in mice. Conditioned medium from BMP2pp-activated macrophages increased the osteogenesis of hPMSCs. The western blot results indicated a significant decrease in the expression of NF-κB inducing kinase (NIK) and phospho-NF-κB p65 in bone marrow-derived macrophages treated with BMP2pp. Furthermore, we clarified the protective effect of BMP2pp on bone formation and the reduction in bone resorption coupled with the immunomodulatory properties of calvarial osteolysis in mice. In summary, BMP2pp ameliorated the Ti-mediated impairment in osteogenic potential of hPMSCs, suppressed the M1 polarization of macrophages by inhibiting the activation of the NF-κB signaling pathway, and ameliorated Ti-induced bone osteolysis. Our research suggests that BMP2pp may be a potential option for treating prosthetic loosening induced by wear debris from prostheses.

Bone-Targeted Bortezomib Inhibits Bortezomib-Resistant Multiple Myeloma in Mice by Providing Higher Levels of Bortezomib in Bone.

Limited treatment options exist for cancer within the bone, as demonstrated by the inevitable, pernicious course of metastatic and blood cancers. The difficulty of eliminating bone-residing cancer, especially drug-resistant cancer, necessitates novel, alternative treatments to manipulate tumor cells and their microenvironment, with minimal off-target effects. To this end, bone-targeted conjugate (BP-Btz) was generated by linking bortezomib (Btz, an anticancer, bone-stimulatory drug) to a bisphosphonate (BP, a targeting ligand) through a cleavable linker that enables spatiotemporally controlled delivery of Btz to bone under acidic conditions for treating multiple myeloma (MM). Three conjugates with different linkers were developed and screened for best efficacy in mouse model of MM. Results demonstrated that the lead candidate BP-Btz with optimal linker could overcome Btz resistance, reduced tumor burden, bone destruction, or tumor metastasis more effectively than BP or free Btz without thrombocytopenia and neurotoxicity in mice bearing myeloma. Furthermore, pharmacokinetic and pharmacodynamic studies showed that BP-Btz bound to bone matrix, released Btz in acidic conditions, and had a higher local concentration and longer half-life than Btz in bone. Our findings suggest the potential of bone-targeted Btz conjugate as an efficacious Btz-resistant MM treatment mechanism. © 2021 American Society for Bone and Mineral Research (ASBMR).

Bioclickable Mussel-Derived Peptides With Immunoregulation for Osseointegration of PEEK.

Polyether ether ketone (PEEK)-based biomaterials have been widely used in the field of spine and joint surgery. However, lack of biological activity limits their further clinical application. In this study, we synthesized a bioclickable mussel-derived peptide Azide-DOPA4 as a PEEK surface coating modifier and further combined bone morphogenetic protein 2 functional peptides (BMP2p) with a dibenzylcyclooctyne (DBCO) motif through bio-orthogonal reactions to obtain DOPA4@BMP2p-PEEK. As expected, more BMP2p can be conjugated on PEEK after Azide-DOPA4 coating. The surface roughness and hydrophilicity of DOPA4@BMP2p-PEEK were obviously increased. Then, we optimized the osteogenic capacity of PEEK substrates. In vitro, compared with the BMP2p-coating PEEK material, DOPA4@BMP2p-PEEK showed significantly higher osteogenic induction capability of rat bone marrow mesenchymal stem cells. In vivo, we constructed a rat calvarial bone defect model and implanted PEEK materials with a differently modified surface. Micro-computed tomography scanning displayed that the DOPA4@BMP2p-PEEK implant group had significantly higher new bone volume and bone mineral density than the BMP2p-PEEK group. Histological staining of hard tissue further confirmed that the DOPA4@BMP2p-PEEK group revealed a better osseointegrative effect than the BMP2p-PEEK group. More importantly, we also found that DOPA4@BMP2p coating has a synergistic effect with induced Foxp3+ regulatory T (iTreg) cells to promote osteogenesis. In summary, with an easy-to-perform, two-step surface bioengineering approach, the DOPA4@BMP2p-PEEK material reported here displayed excellent biocompatibility and osteogenic functions. It will, moreover, offer insights to engineering surfaces of orthopedic implants.

Immunopolarization-regulated 3D printed-electrospun fibrous scaffolds for bone regeneration.

Three-dimension (3D)-printed bioscaffolds are precise and personalized for bone regeneration. However, customized 3D scaffolds may activate the immune response in vivo and consequently impede bone formation. In this study, with layer-by-layer deposition and electrospinning technology to control the physical structure, 3D-printed PCL scaffolds with PLLA electrospun microfibrous (3D-M-EF) and nanofibrous (3D-N-EF) composites were constructed, and their immunomodulatory effect and the subsequent osteogenic effects were explored. Compared to 3D-N-EF scaffolds, 3D-M-EF scaffolds polarized more RAW264.7 cells toward alternatively activated macrophages (M2), as demonstrated by increased M2 and deceased classically activated macrophage (M1) phenotypic marker expression in the cells. In addition, the 3D-M-EF scaffolds shifted RAW264.7 cells to the M2 phenotype through PI3K/AKT signaling and enhanced VEGF and BMP-2 expression. Conditional medium from the RAW264.7 cells seeded in 3D-M-EF scaffolds promoted osteogenesis of MC3T3-E1 cells. Furthermore, in vivo study of repairing rat calvarial defects, the 3D-M-EF scaffolds increased the polarization of M2 macrophages, enhanced angiogenesis, and accelerated new bone formation. Collectively, our data suggested that well-designed 3D-M-EF scaffolds are favorable for osteogenesis through regulation of M2 polarization. Therefore, it is potential to utilize the physical structure of 3D-printed scaffolds to manipulate the osteoimmune environment to promote bone regeneration.

Surface bioengineering of diverse orthopaedic implants with optional functions via bioinspired molecular adhesion and bioorthogonal conjugations.

In this work, we reported an upgraded mussel-inspired strategy for surface bioengineering of osteoimplants by combination of mussel adhesion and bioorthogonal click chemistry. The main idea of this strategy is a mussel-inspired synthetic peptide containing multiple 3,4-dihydroxy-L-phenylalanine (DOPA) units and a dibenzocyclooctyne (DBCO) terminal (DOPA-DBCO). According to the mussel adhesion mechanism, the DOPA-DBCO peptide could stably adhere onto a variety of material surface, leaving the residual DBCO groups on the surface. Then, the DBCO residues could be employed for a second-step bioorthogonal conjugation with azide-capping biomolecules through bioorthogonal click chemistry, finally leading to the biomodified surfaces. To demonstrate the generality of our strategy for surface biomodification of diversified orthopaedic materials including metallic and polymeric substrates, we here conceptually conjugated some typical azide-capping biomolecules on both metal and polymeric surfaces. The results definitely verified the feasibility for engineering of functional surfaces with some essential requirements of osteoimplants, for example, the ability to facilitate cell adhesion, suppress bacterial infection, and promote osteogenesis. In a word, this study indicated that our novel surface strategy would show broad applicability for diverse osteoimplants and in different biological scenarios. We can also image that the molecular specificity of bioorthogonal conjugation and the universality of mussel adhesion mechanism may jointly provide a versatile surface bioengineering method for a wider range of biomedical implants.

Substrate stiffness modulates bone marrow-derived macrophage polarization through NF-κB signaling pathway.

The stiffness of the extracellular matrix (ECM) plays an important role in regulating the cellular programming. However, the mechanical characteristics of ECM affecting cell differentiation are still under investigated. Herein, we aimed to study the effect of ECM substrate stiffness on macrophage polarization. We prepared polyacrylamide hydrogels with different substrate stiffness, respectively. After the hydrogels were confirmed to have a good biocompatibility, the bone marrow-derived macrophages (BMMs) from mice were incubated on the hydrogels. With simulated by the low substrate stiffness, BMMs displayed an enhanced expression of CD86 on the cell surface and production of reactive oxygen species (ROS) in cells, and secreted more IL-1ß and TNF-α in the supernatant. On the contrary, stressed by the medium stiffness, BMMs expressed more CD206, produced less ROS, and secreted more IL-4 and TGF-ß. In vivo study by delivered the hydrogels subcutaneously in mice, more CD68+CD86+ cells around the hydrogels with the low substrate stiffness were observed while more CD68+CD206+ cells near by the middle stiffness hydrogels. In addition, the expressions of NIK, phosphorylated p65 (pi-p65) and phosphorylated IκB (pi-IκB) were significantly increased after stimulation with low stiffness in BMMs. Taken together, these findings demonstrated that substrate stiffness could affect macrophages polarization. Low substrate stiffness promoted BMMs to shift to classically activated macrophages (M1) and the middle one to alternatively activated macrophages (M2), through modulating ROS-initiated NF-κB pathway. Therefore, we anticipated ECM-based substrate stiffness with immune modulation would be under consideration in the clinical applications if necessary.

Gelatin Templated Polypeptide Co-Cross-Linked Hydrogel for Bone Regeneration.

Polypeptides with short chains of amino acid monomers have been widely applied in the clinic because of their various biological functions. However, the easily-inactivated characteristics and burst releasing of the peptides limit their application in vivo. Here, a novel osteogenic polypeptide hydrogel (GelMA-c-OGP) is created by co-cross-linking template photo-cross-linked gelatin (GelMA) with photo-cross-linkable osteogenic growth peptides (OGP) using ultraviolet radiation. GelMA enables the formation of hydrogel with photo-cross-linkable OGP with good mechanical properties and also promotes bone regeneration. GelMA-c-OGP hydrogel accelerates the bone formation procedure of osteogenic precursor cells by significantly enhancing the expression of osteogenic-related genes BMP-2, OCN, and OPN, and increasing the precipitation of calcium salts in osteoblasts. Similarly, GelMA-c-OGP hydrogel promotes bone regeneration in vivo. Furthermore, it is observed that more collagen fibers connect cortical bones in the GelMA-c-OGP implanted group than the control group by hematoxylin-eosin and immunohistochemical staining of Collagen I and TGF-ß. The co-cross-linked OGP polypeptide converts from liquid to solid hydrogel with transient UV light in situ, which also can strengthen the mechanical property of the defect bone and avoid burst osteogenic peptide, releasing during the bone defect healing period. Overall, this hydrogel delivering system has a significant impact on bone defect healing compared with traditional methods.

Increased lactic acid content associated with extracellular matrix depletion in a porcine disc degeneration induced by superficial annular lesion.

BACKGROUND: Degenerative intervertebral disc (IVD) disease can cause lower back pain. However, the change of lactic acid content during disc degeneration process still unclear. The objective of this study was to investigate whether the change of the content of lactic acid is associated with depletion of degenerative intervertebral disc extracellular matrix. METHODS: A total of 18 miniature pigs were equally divided into annular lesion surgery (AL) and sham group. The lateral superficial annulus fibrosus (AF) of T12-L4 discs in AL group were penetrated by 3.5 mm trepan with the depth of 3 mm, the same IVD were only exposed without any injury in the sham group. At 4, 8 and 12 weeks after surgery, the degree of intervertebral disc degeneration was evaluated by magnetic resonance, histological and biochemical analysis. RESULTS: No obvious degeneration was found in sham group. However, disc degeneration was found and gradually worsened in AL group after surgery. Histological analysis showed that the AF was rupture and disorder, the number of cells in nucleus pulposus (NP) was decreased in AL group. Compared with the sham group, the extent of type II collagen (Col-II) and aggrecan in NP tissue was dramatically decreased in AL group, consistent with the results of Col -II immunohistochemistry staining and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Besides, the gene expression of matrix metallopeptidase 3 and 13 also continuous increased in AL group. The amount of lactic acid and nerve growth factor in NP tissue was gradually increased after operation in AL group. CONCLUSIONS: The content of lactic acid gradually increased after annular lesion, associated with the damage of AF structural and the decrease of Col -II and aggrecan in NP tissue, which leading to the disc degeneration. Depletion of extracellular matrix is consistent with lactic acid accumulation inside of IVD.

B cells inhibit bone formation in rheumatoid arthritis by suppressing osteoblast differentiation.

The function of B cells in osteoblast (OB) dysfunction in rheumatoid arthritis (RA) has not been well-studied. Here we show that B cells are enriched in the subchondral and endosteal bone marrow (BM) areas adjacent to osteocalcin+ OBs in two murine RA models: collagen-induced arthritis and the TNF-transgenic mice. Subchondral BM B cells in RA mice express high levels of OB inhibitors, CCL3 and TNF, and inhibit OB differentiation by activating ERK and NF-κB signaling pathways. The inhibitory effect of RA B cells on OB differentiation is blocked by CCL3 and TNF neutralization, and deletion of CCL3 and TNF in RA B cells completely rescues OB function in vivo, while B cell depletion attenuates bone erosion and OB inhibition in RA mice. Lastly, B cells from RA patients express CCL3 and TNF and inhibit OB differentiation, with these effects ameliorated by CCL3 and TNF neutralization. Thus, B cells inhibit bone formation in RA by producing multiple OB inhibitors.

The Notch Ligand Jagged1 Regulates the Osteoblastic Lineage by Maintaining the Osteoprogenitor Pool.

Notch signaling is critical for osteoblastic differentiation; however, the specific contribution of individual Notch ligands is unknown. Parathyroid hormone (PTH) regulates the Notch ligand Jagged1 in osteoblastic cells. To determine if osteolineage Jagged1 contributes to bone homeostasis, selective deletion of Jagged1 in osteolineage cells was achieved through the presence of Prx1 promoter-driven Cre recombinase expression, targeting mesenchymal stem cells (MSCs) and their progeny (PJag1 mice). PJag1 mice were viable and fertile and did not exhibit any skeletal abnormalities at 2 weeks of age. At 2 months of age, however, PJag1 mice had increased trabecular bone mass compared to wild-type (WT) littermates. Dynamic histomorphometric analysis showed increased osteoblastic activity and increased mineral apposition rate. Immunohistochemical analysis showed increased numbers of osteocalcin-positive mature osteoblasts in PJag1 mice. Also increased phenotypically defined Lin- /CD45- /CD31- /Sca1- /CD51+ osteoblastic cells were measured by flow cytometric analysis. Surprisingly, phenotypically defined Lin- /CD45- /CD31- /Sca1+ /CD51+ MSCs were unchanged in PJag1 mice as measured by flow cytometric analysis. However, functional osteoprogenitor (OP) cell frequency, measured by Von Kossa+ colony formation, was decreased, suggesting that osteolineage Jagged1 contributes to maintenance of the OP pool. The trabecular bone increases were not due to osteoclastic defects, because PJag1 mice had increased bone resorption. Because PTH increases osteoblastic Jagged1, we sought to understand if osteolineage Jagged1 modulates PTH-mediated bone anabolism. Intermittent PTH treatment resulted in a significantly greater increase in BV/TV in PJag1 hind limbs compared to WT. These findings demonstrate a critical role of osteolineage Jagged1 in bone homeostasis, where Jagged1 maintains the transition of OP to maturing osteoblasts. This novel role of Jagged1 not only identifies a regulatory loop maintaining appropriate populations of osteolineage cells, but also provides a novel approach to increase trabecular bone mass, particularly in combination with PTH, through modulation of Jagged1. © 2017 American Society for Bone and Mineral Research.

Silencing miR-106b accelerates osteogenesis of mesenchymal stem cells and rescues against glucocorticoid-induced osteoporosis by targeting BMP2.

Osteoporosis is a serious health problem worldwide. MicroRNA is a post-transcriptional regulator of gene expression by either promoting mRNA degradation or interfering with mRNA translation of specific target genes. It plays a significant role in the pathogenesis of osteoporosis. Here, we first demonstrated that miR-106b (miR-106b-5p) negatively regulated osteogenic differentiation of mesenchymal stem cells in vitro. Then, we found that miR-106b expression increased in C57BL/6 mice with glucocorticoid-induced osteoporosis (GIOP), and that silencing of miR-106b signaling protected mice against GIOP through promoting bone formation and inhibiting bone resorption. At last, we showed that miR-106b inhibited osteoblastic differentiation and bone formation partly through directly targeting bone morphogenetic protein 2 (BMP2) both in vitro and in the GIOP model. Together, our findings have identified the role and mechanism of miR-106b in negatively regulating osteogenesis. Inhibition of miR-106b might be a potential new strategy for treating osteoporosis and bone defects.

Lymphatic Endothelial Cells Produce M-CSF, Causing Massive Bone Loss in Mice.

Gorham-Stout disease (GSD) is a rare bone disorder characterized by aggressive osteolysis associated with lymphatic vessel invasion within bone marrow cavities. The etiology of GSD is not known, and there is no effective therapy or animal model for the disease. Here, we investigated if lymphatic endothelial cells (LECs) affect osteoclasts (OCs) to cause a GSD osteolytic phenotype in mice. We examined the effect of a mouse LEC line on osteoclastogenesis in co-cultures. LECs significantly increased receptor activator of NF-κB ligand (RANKL)-mediated OC formation and bone resorption. LECs expressed high levels of macrophage colony-stimulating factor (M-CSF), but not RANKL, interleukin-6 (IL-6), and tumor necrosis factor (TNF). LEC-mediated OC formation and bone resorption were blocked by an M-CSF neutralizing antibody or Ki20227, an inhibitor of the M-CSF receptor, c-Fms. We injected LECs into the tibias of wild-type (WT) mice and observed massive osteolysis on X-ray and micro-CT scans. Histology showed that LEC-injected tibias had significant trabecular and cortical bone loss and increased OC numbers. M-CSF protein levels were significantly higher in serum and bone marrow plasma of mice given intra-tibial LEC injections. Immunofluorescence staining showed extensive replacement of bone and marrow by podoplanin+ LECs. Treatment of LEC-injected mice with Ki20227 significantly decreased tibial bone destruction. In addition, lymphatic vessels in a GSD bone sample were stained positively for M-CSF. Thus, LECs cause bone destruction in vivo in mice by secreting M-CSF, which promotes OC formation and activation. Blocking M-CSF signaling may represent a new therapeutic approach for treatment of patients with GSD. Furthermore, tibial injection of LECs is a useful mouse model to study GSD. © 2017 American Society for Bone and Mineral Research.

BMI-1 Mediates Estrogen-Deficiency-Induced Bone Loss by Inhibiting Reactive Oxygen Species Accumulation and T Cell Activation.

Previous studies have shown that estrogen regulates bone homeostasis through regulatory effects on oxidative stress. However, it is unclear how estrogen deficiency triggers reactive oxygen species (ROS) accumulation. Recent studies provide evidence that the B lymphoma Mo-MLV insertion region 1 (BMI-1) plays a critical role in protection against oxidative stress and that this gene is directly regulated by estrogen via estrogen receptor (ER) at the transcriptional level. In this study, ovariectomized mice were given drinking water with/without antioxidant N-acetyl-cysteine (NAC, 1 mg/mL) supplementation, and compared with each other and with sham mice. Results showed that ovariectomy resulted in bone loss with increased osteoclast surface, increased ROS levels, T cell activation, and increased TNF and RANKL levels in serum and in CD4 T cells; NAC supplementation largely prevented these alterations. BMI-1 expression levels were dramatically downregulated in CD4 T cells from ovariectomized mice. We supplemented drinking water to BMI-1-deficient mice with/without NAC and compared them with each other and with wild-type (WT) mice. We found that BMI-1 deficiency mimicked alterations observed in ovariectomy whereas NAC supplementation reversed all alterations induced by BMI-1 deficiency. Because T cells are critical in mediating ovariectomy-induced bone loss, we further assessed whether BMI-1 overexpression in lymphocytes can protect against estrogen deficiency-induced osteoclastogenesis and bone loss by inhibiting oxidative stress, T cell activation, and RANKL production. When WT and Eµ-BMI-1 transgenic mice with BMI-1 specifically overexpressed in lymphocytes were ovariectomized and compared with each other and with WT sham mice, we found that BMI-1 overexpression in lymphocytes clearly reversed all alterations induced by ovariectomy. Results from this study indicate that estrogen deficiency downregulates BMI-1 and subsequently increases ROS, T cell activation, and RANKL production in T cells, thus enhancing osteoclastogenesis and accelerating bone loss. This study clarifies a novel mechanism regulating estrogen deficiency-induced bone loss. © 2016 American Society for Bone and Mineral Research.

Overexpression of Bmi1 in Lymphocytes Stimulates Skeletogenesis by Improving the Osteogenic Microenvironment.

To investigate whether overexpression of Bmi1 in lymphocytes can stimulate skeletogenesis by improving the osteogenic microenvironment, we examined the skeletal phenotype of EµBmi1 transgenic mice with overexpression of Bmi1 in lymphocytes. The size of the skeleton, trabecular bone volume and osteoblast number, indices of proliferation and differentiation of bone marrow mesenchymal stem cells (BM-MSCs) were increased significantly, ROS levels were reduced and antioxidative capacity was enhanced in EµBmi1 mice compared to WT mice. In PTHrP1-84 knockin (Pthrp(KI/KI)) mice, the expression levels of Bmi1 are reduced and potentially can mediate the premature osteoporosis observed. We therefore generated a Pthrp(KI/KI) mice overexpressing Bmi1 in lymphocytes and compared them with Pthrp(KI/KI) and WT littermates. Overexpression of Bmi1 in Pthrp(KI/KI) mice resulted in a longer lifespan, increased body weight and improvement in skeletal growth and parameters of osteoblastic bone formation with reduced ROS levels and DNA damage response parameters. Our results demonstrate that overexpression of Bmi1 in lymphocytes can stimulate osteogenesis in vivo and partially rescue defects in skeletal growth and osteogenesis in Pthrp(KI/KI) mice. These studies therefore indicate that overexpression of Bmi1 in lymphocytes can stimulate skeletogenesis by inhibiting oxidative stress and improving the osteogenic microenvironment.

A novel silk fibroin nanofibrous membrane for guided bone regeneration: a study in rat calvarial defects.

A novel membrane for guided bone regeneration (GBR), constituting silk fibroin (SF) nanofiber from native silk nanofibril solution, was prepared by electrospinning process. Another barrier membrane, a collagen-type membrane (Bio-Gide®), was used as a comparative sample. Twelve healthy male Sprague-Dawley rats were used in this study. Bilateral round defects were created in the calvarial bone. The bone regenerative efficacy was evaluated in rat calvarial defects. Animals were killed at 4 and 12 weeks. Bone regeneration was analyzed using micro-computed tomography and histological analysis. The SF nanofibrous membrane showed superior results with regard to mechanical tensile properties. At 4 weeks, the bone volume and collagen I positive areas in the SF group were greater than in the Bio-Gide group. At 12 weeks, the defect had completely healed with new bone in both the groups. In conclusion, the SF nanofibrous membranes showed satisfactory mechanical stability, good biocompatibility, slow degradability, and improved new bone regeneration without any adverse inflammatory reactions. Considering the low cost and low risk of disease transmission, the SF nanofibrous membrane is a potential candidate for GBR therapy compared with the widely used collagen membranes.

Endogenous parathyroid hormone-related protein compensates for the absence of parathyroid hormone in promoting bone accrual in vivo in a model of bone marrow ablation.

To assess the effect of hypoparathyroidism on osteogenesis and bone turnover in vivo, bone marrow ablation (BMXs) were performed in tibias of 8-week-old wild-type and parathyroid hormone-null (PTH(-/-)) mice and newly formed bone tissue was analyzed from 5 days to 3 weeks after BMX. At 1 week after BMX, trabecular bone volume, osteoblast numbers, alkaline phosphatase-positive areas, type I collagen-positive areas, PTH receptor-positive areas, calcium sensing receptor-positive areas, and expression of bone formation-related genes were all decreased significantly in the diaphyseal regions of bones of PTH(-/-) mice compared to wild-type mice. In contrast, by 2 weeks after BMX, all parameters related to osteoblastic bone accrual were increased significantly in PTH(-/-) mice. At 5 days after BMX, active tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts had appeared in wild-type mice but were undetectable in PTH(-/-) mice, Both the ratio of mRNA levels of receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) and TRAP-positive osteoclast surface were still reduced in PTH(-/-) mice at 1 week but were increased by 2 weeks after BMX. The expression levels of parathyroid hormone-related protein (PTHrP) at both mRNA and protein levels were upregulated significantly at 1 week and more dramatically at 2 weeks after BMX in PTH(-/-) mice. To determine whether the increased newly formed bones in PTH(-/-) mice at 2 weeks after BMX resulted from the compensatory action of PTHrP, PTH(-/-) PTHrP(+/-) mice were generated and newly formed bone tissue was compared in these mice with PTH(-/-) and wild-type mice at 2 weeks after BMX. All parameters related to osteoblastic bone formation and osteoclastic bone resorption were reduced significantly in PTH(-/-) PTHrP(+/-) mice compared to PTH(-/-) mice. These results demonstrate that PTH deficiency itself impairs osteogenesis, osteoclastogenesis, and osteoclastic bone resorption, whereas subsequent upregulation of PTHrP in osteogenic cells compensates by increasing bone accrual.