Rh(III)-Catalyzed Successive C-H Activations of 2-Phenyl-3H-indoles and Cyclization Cascades to Construct Highly Fused Indole Heteropolycycles.

Rh(III)-catalyzed successive C-H activations of 2-phenyl-3H-indoles and cyclization cascades with diazo compounds were developed to construct highly fused indole heteropolycycles with a broad range of substrates and good yields. In particular, this transformation included two successive C-H activations and unusual [3+3] and [4+2] sequential cyclization cascades, in which the diazo compound played a different role in the two cyclization processes, while simultaneously forming a highly fused polycyclic indole scaffold with a new quaternary carbon center.

Reversed windshield-wiper effect leads to failure of cement-augmented pedicle screw: Biomechanical mechanism analysis by finite element experiment.

The failure mode of cement-augmented pedicle screw (CAPS) was different from common pedicle screw. No biomechanical study of this failure mode named as "reversed windshield-wiper effect" was reported. To investigate the mechanisms underlying this failure mode, a series of finite element models of CAPS and PS were modified on L4 osseous model. Nine models were created according to the cement volume at 0.5 mL interval (range: 1-5 mL). Pullout load and cranio-caudal loads were applied on the screws. Stress and instantaneous rotation center (IRC) of the vertebra were observed. Under cranio-caudal load, the stress concentrated on the screw tip and pedicle region. The maximal stress (MS) at the screw tip region was +2.143 MPa higher than pedicle region. With cement volume increasing, the maximal stress (MS) at the screw tip region decreased dramatically, while MS at pedicle region was not obviously affected. As dose increased to 1.5 mL, the MS at pedicle region became higher than screw tip region and the maximal stress difference was observed at 3.5 mL. IRC of the vertebra located at the facet joint region in PS model. While IRC in CAPS models shifted anteriorly closer to the vertebral body with the increasing of cement volume. Under axial pull-out load, the maximal stress (MS) of cancellous bone in CAPS models was 29.53-50.04% lower than that 2.228 MPa in PS model. MS in the screw-bone interface did not change significantly with cement volume increasing. Therefore, the possible mechanism is that anterior shift of IRC and the negative difference value of MS between screw tip and pedicle region due to cement augmentation, leading to the screw rotate around the cement-screw complex as the fulcrum point.

Role of Posterior Longitudinal Ligament Complex in Spinal Deformity Secondary to Surgical Resection of the Intradural Tumor.

OBJECTIVE: In most cases, complete resection of the intradural tumor is accompanied by long-term neurological complications. Postoperative spinal deformity is the most common complication after surgical resection of intradural tumors, and posterior longitudinal ligament complex (PLC) plays an important role in postoperative spinal deformity. In this study, we investigated the role of PLC in spinal deformity after the surgical treatment of intradural tumors. METHODS: We analyzed the data of 218 consecutive patients who underwent intradural tumor resection from 2000 to 2018 in this retrospective study. Before 2010, patients underwent laminoplasty without maintaining the integrity of PLC (laminoplasty group, n = 155). After 2010, patients performed single-port laminoplasty to maintain the integrity of PLC (laminoplasty retain posterior ligament complex group, n = 63). The score of quality of life, painful cortex, spinal cord movement, progressive kyphosis or scoliosis, perioperative morbidity, and neurological results were analyzed in the laminoplasty group and laminoplasty retain posterior ligament complex group. The distributed variable was shown as mean ± standard deviation and an independent t-test or one-way analysis of variance was calculated. RESULTS: There are 155 patients (71.1%) included in the laminoplasty group, and 63 patients (28.9%) in the laminoplasty retain posterior ligament complex group. The average age of patients was 42 ± 2.3 years, and the average modified McCormick score was 2. There were 158 (72.4%) patients with intramedullary tumors and 115 (52.7%) patients with extramedullary tumors. The length of hospital stays (8 days vs. 6 days; p = 0.023) and discharge to inpatient rehabilitation (48.4% vs. 26.9%; p = 0.012) were significantly lower in the laminoplasty retain posterior ligament complex group than the laminoplasty group. There was no significant difference in the risk of progressive deformity between the two groups at 18 months after surgery (relative risk 0.12; 95% confidence interval [CI] 0.43-1.25; p = 0.258) and at 20 months after surgery (relative risk 0.24; 95% CI 0.21-2.1). CONCLUSION: Laminoplasty retains posterior ligament complex showed no impact on the spinal deformities compared with laminoplasty, but significantly improved the postoperative spinal activity, alleviated pain symptoms, and reduced hospital recovery time.

Association of population migration with air quality: Role of city attributes in China during COVID-19 pandemic (2019-2021).

Atmospheric pollution studies have linked diminished human activity during the COVID-19 pandemic to improve air quality. This study was conducted during January to March (2019-2021) in 332 cities in China to examine the association between population migration and air quality, and examined the role of three city attributes (pollution level, city scale, and lockdown status) in this effect. This study assessed six air pollutants, namely CO, NO2, O3, PM10, PM2.5, and SO2, and measured meteorological data, with-in city migration (WCM) index, and inter-city migration (ICM) index. A linear mixed-effects model with an autoregressive distributed lag model was fitted to estimate the effect of the percent change in migration on air pollution, adjusting for potential confounding factors. In summary, lower migration was associated with decreased air pollution (other than O3). Pollution change in susceptibility is more likely to occur in NO2 decrease and O3 increase, but unsusceptibility is more likely to occur in CO and SO2, to city attributes from low migration. Cities that are less air polluted and population-dense may benefit more from decreasing PM10 and PM2.5. The associations between population migration and air pollution were stronger in cities with stringent traffic restrictions than in cities with no lockdowns. Based on city attributes, an insignificant difference was observed between the effects of ICM and WCM on air pollution. Findings from this study may gain knowledge about the potential interaction between migration and city attributes, which may help decision-makers adopt air-quality policies with city-specific targets and paths to pursue similar air quality improvements for public health but at a much lower economic cost than lockdowns.

Modelling regional ecological security pattern and restoration priorities after long-term intensive open-pit coal mining.

Long-term intensive open-pit mining can have huge impacts on ecosystems and the services they provide, affecting the integrity of ecosystem structures, functions and process and thus the "ecological security" of a whole mining region. The indirect and direct impacts of mining are spatially and temporally complex and therefore ecological security patterns need to be considered. However, to date there has been little research focusing on ecological security patterns in mining regions. This study aims to model and map ecological security and restoration priorities in an intensive open-cut coal mining region accounting for spatio-temporal changes of multiple ecosystem services. Four ecosystem services including habitat quality, carbon sequestration, water yield, and sediment retention were assessed and mapped in 1990, 2000, 2010, and 2020. Next ecological security patterns and restoration priorities were modelled and characterized using the Self-Organizing Feature Map to identify ecological sources, and circuit theory in Linkage Mapper to characterize connectivity and flows. The results showed that habitat quality, carbon sequestration, and water yield services were most affected by vegetation cover changes due to mining exploitation, while sediment retention was influenced by multiple natural factors, especially topography. Ecological sources, corridors, pinch-points and barriers showed various changing trends due to mined and built-up land expansion over time. Ecological source area declined from 1355.08km2 in 2000 to 584.92 km2 in 2020, while the number of corridors increased from 17 in 2000 to 25 in 2010 and then decreased to 21 in 2020. Although restoration has been conducted on some mine sites, regional-scale restoration needs greater consideration. This study provides decision-makers and stakeholders with a method for assessing regional-scale ecological security and restoration in a holistic and systematic way moving beyond a single mine, which is critical for balancing ecological security protection with minerals production in intensive mining regions.

Intraarticular bone grafting in atlantoaxial facet joints via a posterior approach: nonstructural or structural-a minimum 24-month follow-up.

OBJECTIVE: To investigate the necessity of nonstructural or structural intraarticular bone grafting in atlantoaxial facet joints via a posterior approach and the influence by the presence of basilar invagination (BI). METHODS: From November 2016 to October 2018, patients who underwent posterior atlantoaxial or occipitocervical arthrodesis surgery at one institute were retrospectively reviewed. Operation records, preoperative and postoperative clinical status, and radiological films were analyzed. RESULTS: Thirty-three patients (19 without BI, 14 with BI) underwent posterior facet joint release followed by intraarticular bone grafting were enrolled finally. Twenty-four nonstructural (15 without BI, 9 with BI) and 9 structural (4 without BI, 5 with BI) grafting were performed. The average follow-up was 32.15±6.73 months (24-47 months). Among them, 1 (3.03%) implant failure occurred, and 32 (96.97%) achieved satisfactory neurological outcomes, including 28 (84.85%) complete and 4 (12.12%) acceptable reductions with complete fusion within 6 months. For patients without BI, structural and nonstructural grafting showed no significant difference in terms of reduction maintenance (100% vs 73.33%, p = 0.530), while for those with BI, structural grafting significantly increased the postoperative height of the joint space (5.67±1.22 mm vs 3.43±1.78 mm, p = 0.002) and maintained it much better than nonstructural grafting (88.89% vs 20.00%, p = 0.023), contributing notably to BI correction. CONCLUSION: Intraarticular structural bone grafting in atlantoaxial facet joints has the advantage of maintaining anterior column height in the case of lateral mass collapse or when BI correction is needed; otherwise, nonstructural bone grafting is enough.

Urban road greenbelt configuration: The perspective of PM2.5 removal and air quality regulation.

The establishment of the road green belt (RGB) is an effective means to reduce particle matter (PM2.5) emissions from road traffic. This study tested the ability of 23 common tree species in Shenzhen to reduce PM2.5 concentrations using field investigations and wind tunnel tests. The association between leaf microstructure and individual reduction ability was also analyzed. Finally, the impact of three RGB configurations (i.e., arbor, shrub, arbor + shrub) on road PM2.5 dispersion and deposition was simulated using the ENVI-met three-dimensional aerodynamic model, based on which an optimal RGB configuration was proposed. There were three key findings of the tests. First, the wind speed was the main factor affecting the PM2.5 concentration (54.2%), followed by vehicle flow (27.7%), temperature (14.2%), and time factor (7.6%). Second, the range of dry deposition velocity (Vd) was 0.04-6.4 m/s, and the dominant dust-retaining plant species were the evergreen trees, Ficus microcarpa and Ficus altissima, and the evergreen shrubs, Codiaeum variegatum and Fagraea ceilanica. A higher proportion of grooves or larger stomata would increase the probability that the blade would capture PM2.5. Third, the shrub RGB demonstrated the best performance in terms of pollutant dispersion; its PM2.5 concentration at the respiratory height (RH, 1.5 m) on the pedestrian crossing was 15-20% lower than the other RGB configurations. In terms of pollutant deposition, the arbor + shrub composite RGB was two-fold better than the other RGB configurations. Moreover, it was more advantageous to plant shrub RGBs in street canyons to achieve a balance between the lowest concentration and the largest deposition of PM2.5 pollutants. The findings of this study will facilitate the RGB configurations with good dust retention ability.

Corrigendum to 'Phosphorylation inhibition of protein-tyrosine phosphatase 1B tyrosine-152 induces bone regeneration coupled with angiogenesis for bone tissue engineering' [Bioactive Mater. Vol. 6, Issue 7, Pages 2039-2057].

[This corrects the article DOI: 10.1016/j.bioactmat.2020.12.025.].

Laminin alpha 4 promotes bone regeneration by facilitating cell adhesion and vascularization.

Selective cell retention (SCR) has been widely used as a bone tissue engineering technique for the real-time fabrication of bone grafts. The greater the number of mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) retained in the scaffold, the better the osteoinductive and angiogenic properties of the scaffold's microenvironment. Improved bioscaffold properties in turn lead to improved bone graft survival, bone regeneration, and angiogenesis. Laminin plays a key role in cell-matrix adhesion, cell proliferation, and differentiation. We designed a collagen-binding domain (CBD) containing the core functional amino acid sequences of laminin α4 (CBD-LN peptide) to supplement the functional surface of a collagen-based decalcified bone matrix (DBM) scaffold. This scaffold promoted MSCs and EPCs early cell adhesion through up-regulating the expression of integrin α5ß1 and integrin αvß3 respectively, thus accelerated the following cell spreading, proliferation, and differentiation. Interestingly, it promoted the retention of MSCs (CD90+/CD105+ cells) and EPCs (CD31+ cells) in the scaffold following the use of clinical SCR technology. Furthermore, the DBM/CBD-LN scaffold induced the formation of type H vessels through the activation of the HIF-1α signaling pathway. The DBM/CBD-LN scaffold displayed rapid bone formation and angiogenesis in vivo, suggesting that it might be used as a new biomaterial in bone tissue engineering. STATEMENT OF SIGNIFICANCE: Selective cell retention technology (SCR) has been utilized in clinical settings to manufacture bioactive bone grafts. Specifically, demineralized bone matrix (DBM) is a widely-used SCR clinical biomaterial but it displays poor adhesion performance and angiogenic activity. In this work, we designed a collagen-binding domain (CBD) containing the core functional amino acid sequences of laminin α4 to supplement the functional surface of a collagen-based DBM scaffold. This bioscaffold promoted SCR-mediated MSCs and EPCs early cell adhesion, thus accelerated the following cell spreading, proliferation, and differentiation. Our results indicate this bioscaffold greatly induced osteogenesis and angiogenesis in vivo. In general, this bioscaffold has a good prospect for SCR application and may provide highly bioactive bone implant in clinical environment.

Phosphorylation inhibition of protein-tyrosine phosphatase 1B tyrosine-152 induces bone regeneration coupled with angiogenesis for bone tissue engineering.

A close relationship has been reported to exist between cadherin-mediated cell-cell adhesion and integrin-mediated cell mobility, and protein tyrosine phosphatase 1B (PTP1B) may be involved in maintaining this homeostasis. The stable residence of mesenchymal stem cells (MSCs) and endothelial cells (ECs) in their niches is closely related to the regulation of PTP1B. However, the exact role of the departure of MSCs and ECs from their niches during bone regeneration is largely unknown. Here, we show that the phosphorylation state of PTP1B tyrosine-152 (Y152) plays a central role in initiating the departure of these cells from their niches and their subsequent recruitment to bone defects. Based on our previous design of a PTP1B Y152 region-mimicking peptide (152RM) that significantly inhibits the phosphorylation of PTP1B Y152, further investigations revealed that 152RM enhanced cell migration partly via integrin αvß3 and promoted MSCs osteogenic differentiation partly by inhibiting ATF3. Moreover, 152RM induced type H vessels formation by activating Notch signaling. Demineralized bone matrix (DBM) scaffolds were fabricated with mesoporous silica nanoparticles (MSNs), and 152RM was then loaded onto them by electrostatic adsorption. The DBM-MSN/152RM scaffolds were demonstrated to induce bone formation and type H vessels expansion in vivo. In conclusion, our data reveal that 152RM contributes to bone formation by coupling osteogenesis with angiogenesis, which may offer a potential therapeutic strategy for bone defects.

[Effect of demineralized bone matrix modified by laminin α4 chain functional peptide on H-type angiogenesis and osteogenesis to promote bone defect repair].

OBJECTIVE: Based on the cell-extracellular matrix adhesion theory in selective cell retention (SCR) technology, demineralized bone matrix (DBM) modified by simplified polypeptide surface was designed to promote both bone regeneration and angiogenesis. METHODS: Functional peptide of α4 chains of laminin protein (LNα4), cyclic RGDfK (cRGD), and collagen-binding domain (CBD) peptides were selected. CBD-LNα4-cRGD peptide was synthesized in solid phase and modified on DBM to construct DBM/CBD-LNα4-cRGD scaffold (DBM/LN). Firstly, scanning electron microscope and laser scanning confocal microscope were used to examine the characteristics and stability of the modified scaffold. Then, the adhesion, proliferation, and tube formation properties of CBD-LNα4-cRGD peptide on endothelial progenitor cells (EPCs) were detected, respectively. Western blot method was used to verify the molecular mechanism affecting EPCs. Finally, 24 10-week-old male C57 mice were used to establish a 2-mm-length defect of femoral bone model. DBM/LN and DBM scaffolds after SCR treatment were used to repair bone defects in DBM/LN group ( n=12) and DBM group ( n=12), respectively. At 8 weeks after operation, the angiogenesis and bone regeneration ability of DBM/LN scaffolds were evaluated by X-ray film, Micro-CT, angiography, histology, and immunofluorescence staining [CD31, endomucin (Emcn), Ki67]. RESULTS: Material related tests showed that the surface of DBM/LN scaffold was rougher than DBM scaffold, but the pore diameter did not change significantly ( t=0.218, P=0.835). After SCR treatment, DBM/LN scaffold was still stable and effective. Compared with DBM scaffold, DBM/LN scaffold could adhere to more EPCs after the surface modification of CBD-LNα4-cRGD ( P<0.05), and the proliferation rate and tube formation ability increased. Western blot analysis showed that the relative expressions of VEGF, phosphorylated FAK (p-FAK), and phosphorylated ERK1/2 (p-ERK1/2) proteins were higher in DBM/LN than in DBM ( P<0.05). In the femoral bone defect model of mice, it was found that mice implanted with DBM/LN scaffold had stronger angiogenesis and bone regeneration capacity ( P<0.05), and the number of CD31 hiEmcn hi cells increased significantly ( P<0.05). CONCLUSION: DBM/LN scaffold can promote the adhesion of EPCs. Importantly, it can significantly promote the generation of H-type vessels and realize the effective coupling between angiogenesis and bone regeneration in bone defect repair.

Assessment of the association between paraspinal muscle degeneration and quality of life in patients with degenerative lumbar scoliosis.

The present study aimed to determine the characteristics of multifidus, erector spinae and psoas major degeneration in elderly patients with degenerative lumbar scoliosis (DLS) and the correlation between asymmetric changes and patient quality of life. A total of 49 patients with lumbar scoliosis (DLS group) and 38 healthy individuals (control group) were prospectively examined. The functional cross-sectional area, cross-sectional area difference index (CDI) and fat infiltration rate (FIR) of the multifidus, erector spinae and psoas major at the apical vertebral level were measured using MRI. The visual analogue scale (VAS) score, Oswestry Disability Index (ODI) and 36-item Short Form Health Survey (SF-36) score were used to evaluate patient quality of life. Correlations between the degree of asymmetric muscular degeneration and quality of life were analysed. The CDI of the multifidus, erector spinal and psoas major was higher in the DLS group compared with that in the control group. The CDI of the multifidus was found to be positively associated with the Cobb angle of lumbar scoliosis. Similar results were obtained for fat infiltration between the two groups. In addition, the CDI and FIR difference index of the multifidus was positively correlated with the VAS score and ODI but negatively correlated with the SF-36 score. The quality of life significantly decreased with increasing asymmetric atrophy and fat infiltration in the multifidus. Thus, strategies to enhance the function of the multifidus may have a positive impact on quality of life (Chinese Clinical Trial Registry, registration date, 2018.11.12; registration no. ChiCTR1800019459.).

Inhibition of protein-tyrosine phosphatase 1B phosphorylation enhances early adhesion of mesenchymal stem cells to facilitate fabrication of tissue-engineered bone.

Enhancement of cell-matrix adhesion is preferable and crucial in various fields of tissue engineering. Integrins are important receptors that facilitate cell-matrix adhesion, mediated by intracellular molecules and crosstalk with the cadherin adhesion pathway, which mainly facilitates cell-cell adhesion. Protein-tyrosine phosphatase 1B (PTP1B) has emerged as a pivot in the crosstalk between the cadherin adhesion pathway and the integrin adhesion pathway. The phosphorylation state of PTP1B tyrosine-152 (Y152) plays a central role in balancing the two different cell adhesion forms. In this study, a PTP1B Y152 region mimicking (152RM) peptide was designed to decrease the phosphorylation of PTP1B Y152 via competitive inhibition. As a result, the dissociation of cadherin complexes and the release of PTP1B from cadherin had sharply increased, and Src, an important intracellular component of integrin, was activated, indicating that the cadherin adhesion pathway was inhibited, whereas the integrin adhesion pathway was enhanced. Moreover, upon treatment with the 152RM peptide, we observed that the early adhesion of human bone marrow-derived mesenchymal stem cells (MSCs) was accelerated and the anchoring of MSCs on the surface of integrin ligands was enhanced by an enhanced matrix adhesion ability of MSCs themselves. Importantly, the 152RM peptide significantly promoted the adhesion efficiency of MSCs in the selective cell retention technology, which fabricates instant bone implants in clinical settings, to stimulate osteogenesis in vivo.

Multiple integrin ligands provide a highly adhesive and osteoinductive surface that improves selective cell retention technology.

Among various bone tissue engineering strategies, selective cell retention (SCR) technology has been used as a practical clinical method for bone graft manufacturing in real time. The more mesenchymal stem cells (MSCs) are retained, the better the osteoinductive microenvironment provided by the scaffold, which in turn promotes the osteogenesis of the SCR-fabricated bone grafts. Integrin receptors are crucial to cell-matrix adhesion and signal transduction. We designed a collagen-binding domain (CBD)-containing IKVAV-cRGD peptide (CBD-IKVAV-cRGD peptide) to complement the collagen-based demineralized bone matrix (DBM) with a functionalized surface containing multiple integrin ligands, which correspond to the highly expressed integrin subtypes on MSCs. This DBM/CBD-IKVAV-cRGD composite exhibited superior in vitro adhesion capacity to cultured MSCs, as determined by oscillatory cell adhesion assay, centrifugal cell adhesion assay and mimetic SCR. Moreover, it promoted the retention of MSC-like CD271+ cells and MSC-like CD90+/CD105+ cells in the clinical SCR method. Furthermore, the DBM/CBD-IKVAV-cRGD composite induced robust MSC osteogenesis, coupled with the activation of the downstream FAK-ERK1/2 signaling pathway of integrins. The SCR-prepared DBM/CBD-IKVAV-cRGD composite displayed superior in vivo osteogenesis, indicating that it may be potentially utilized as a biomaterial in SCR-mediated bone transplantation. STATEMENT OF SIGNIFICANCE: Selective cell retention technology (SCR) has been utilized in clinical settings to manufacture bioactive bone grafts. Specifically, demineralized bone matrix (DBM) is a widely-used SCR clinical biomaterial but it displays poor adhesion performance and osteoinduction. Improvements of the DBM that promote cell adhesion and osteoinduction will benefit SCR-prepared implants. In this work, we developed a novel peptide that complements the DBM with a functionalized surface of multiple integrin ligands, which are corresponding to integrin subtypes available on human bone marrow-derived mesenchymal stem cells (MSCs). Our results indicate this novel functionalized bioscaffold greatly increases SCR-mediated MSC adhesion and in vivo osteogenesis. Overall, this novel material has promising SCR applications and may likely provide highly bioactive bone implants in clinical settings.

Alendronate induces osteoclast precursor apoptosis via peroxisomal dysfunction mediated ER stress.

Nitrogen-containing bisphosphonates including alendronate (ALN) are the current first line antiresorptive drug in treating osteoporosis. In our study, we found that ALN administration impaired the secretion of platelet derived growth factor-BB (PDGF-BB), the most important angiogenic cytokines produced by preosteoclast (POC), in both sham and ovariectomized (OVX) mice. To further understand this phenomenon, we induced bone marrow macrophages (BMMs) to POCs in vitro and detected the effects of ALN particularly in POCs. The proapoptotic effect of ALN in POCs was confirmed by flow cytometry. On the molecular level, we found that farnesyl diphosphate synthase (FDPS) inhibition of ALN led to peroxisomal dysfunction and up regulation of cytoprotective protein glucose-regulated protein (GRP) 78. Peroxisomal dysfunction further induced endoplasmic reticulum (ER) stress in POCs and finally resulted in cell apoptosis marked by reduced expression of B-cell lymphoma 2 (Bcl-2) and increased expressions of CCAAT/enhancer binding protein homologous protein (CHOP), Bcl2 associated X (Bax), and cleaved caspase-3. We concluded that ALN has no selectivity in inhibiting POC and mature osteoclast. For POCs, ALN inhibition of FDPS leads to peroxisomal dysfunction, which further mediates ER stress and finally causes cell apoptosis. Considering that decreased angiogenesis is also an important issue in treating osteoporosis, how to preserve pro-angiogenic POCs while depleting mature osteoclasts is a problem worthy to be solved.

IGFBP3 deposited in the human umbilical cord mesenchymal stem cell-secreted extracellular matrix promotes bone formation.

The extracellular matrix (ECM) contains rich biological cues for cell recruitment, proliferationm, and even differentiation. The osteoinductive potential of scaffolds could be enhanced through human bone marrow mesenchymal stem cell (hBMSC) directly depositing ECM on surface of scaffolds. However, the role and mechanism of human umbilical cord mesenchymal stem cells (hUCMSC)-secreted ECM in bone formation remain unknown. We tested the osteoinductive properties of a hUCMSC-secreted ECM construct (hUCMSC-ECM) in a large femur defect of a severe combined immunodeficiency (SCID) mouse model. The hUCMSC-ECM improved the colonization of endogenous MSCs and bone regeneration, similar to the hUCMSC-seeded scaffold and superior to the scaffold substrate. Besides, the hUCMSC-ECM enhanced the promigratory molecular expressions of the homing cells, including CCR2 and TßRI. Furthermore, the hUCMSC-ECM increased the number of migrated MSCs by nearly 3.3 ± 0.1-fold, relative to the scaffold substrate. As the most abundant cytokine deposited in the hUCMSC-ECM, insulin-like growth factor binding protein 3 (IGFBP3) promoted hBMSC migration in the TßRI/II- and CCR2-dependent mechanisms. The hUCMSC-ECM integrating shRNA-mediated silencing of Igfbp3 that down-regulated IGFBP3 expression by approximately 60%, reduced the number of migrated hBMSCs by 47%. In vivo, the hUCMSC-ECM recruited 10-fold more endogenous MSCs to initiate bone formation compared to the scaffold substrate. The knock-down of Igfbp3 in the hUCMSC-ECM inhibited nearly 60% of MSC homing and bone regeneration capacity. This research demonstrates that IGFBP3 is an important MSC homing molecule and the therapeutic potential of hUCMSC-ECM in bone regeneration is enhanced by improving MSC homing in an IGFBP3-dependent mechanism.

TGFß3 recruits endogenous mesenchymal stem cells to initiate bone regeneration.

BACKGROUND: The recruitment of a sufficient number of endogenous mesenchymal stem cells (MSCs) is the first stage of in-situ tissue regeneration. Transforming growth factor beta-3 (TGFß3) could recruit stem or progenitor cells and endothelial cells to participate in tissue regeneration. However, the mechanism of TGFß3 recruiting MSCs toward bone regeneration has remained obscure. METHODS: We estimated the promigratory property of TGFß3 on human bone marrow MSCs (hBMSCs) cocultured with the vascular cells (human umbilical artery smooth muscle cells or human umbilical vein endothelial cells) or not by Transwell assay. After the addition of the inhibitor (SB431542) or Smad3 siRNA, the levels of MCP1 and SDF1 in coculture medium were tested by ELISA kit, and then the migratory signaling pathway of hBMSCs induced by TGFß3 was investigated by western blot analysis. In vivo, a 2-mm FVB/N mouse femur defect model was used to evaluate chemokine secretion, endogenous cell homing, and bone regeneration induced by scaffolds loading 1 µg TGFß3 through qPCR, immunofluorescent staining, immunohistochemical analysis, and Micro-CT, compared to the vehicle group. RESULTS: TGFß3 (25 ng/ml) directly showed a nearly 40% increase in migrated hBMSCs via the TGFß signaling pathway, compared to the vehicle treatment. Then, in the coculture system of hBMSCs and vascular cells, TGFß3 further upregulated nearly 3-fold MCP1 secretion from vascular cells in a Smad3-dependent manner, to indirectly enhance nearly more than 50% of migrated hBMSCs. In vivo, TGFß3 delivery improved MCP1 expression by nearly 7.9-fold, recruited approximately 2.0-fold CD31+ vascular cells and 2.0-fold Sca-1+ PDGFR-α+ MSCs, and achieved 2.5-fold bone volume fraction (BV/TV) and 2.0-fold bone mineral density, relative to TGFß3-free delivery. CONCLUSIONS: TGFß3, as a MSC homing molecule, recruited MSCs to initiate bone formation in the direct-dependent and indirect-dependent mechanisms. This may shed light on the improvement of MSC homing in bone regeneration.

A nano-scaled and multi-layered recombinant fibronectin/cadherin chimera composite selectively concentrates osteogenesis-related cells and factors to aid bone repair.

Easily accessible and effective bone grafts are in urgent need in clinic. The selective cell retention (SCR) strategy, by which osteogenesis-related cells and factors are enriched from bone marrow into bio-scaffolds, holds great promise. However, the retention efficacy is limited by the relatively low densities of osteogenesis-related cells and factors in marrow; in addition, a lack of satisfactory surface modifiers for scaffolds further exacerbates the dilemma. To address this issue, a multi-layered construct consisting of a recombinant fibronectin/cadherin chimera was established via a layer-by-layer self-assembly technique (LBL-rFN/CDH) and used to modify demineralised bone matrix (DBM) scaffolds. The modification was proven stable and effective. By the mechanisms of physical interception and more importantly, chemical recognition (fibronectin/integrins), the LBL-rFN/CDH modification significantly improved the retention efficacy and selectivity for osteogenesis-related cells, e.g., monocytes, mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs), and bioactive factors, e.g., bFGF, BMP-2 and SDF-1α. Moreover, the resulting composite (designated as DBM-LBL-rFN/CDH) not only exhibited a strong MSC-recruiting capacity after SCR, but also provided favourable microenvironments for the proliferation and osteogenic differentiation of MSCs. Eventually, bone repair was evidently improved. Collectively, DBM-LBL-rFN/CDH presented a suitable biomaterial for SCR and a promising solution for tremendous need for bone grafts. STATEMENT OF SIGNIFICANCE: There is an urgent need for effective bone grafts. With the potential of integrating osteogenicity, osteoinductivity and osteoconductivity, selective cell retention (SCR) technology brings hope for developing ideal grafts. However, it is constrained by low efficacy and selectivity. Thus, we modified demineralized bone matrix with nano-scaled and multi-layered recombinant fibronectin/cadherin chimera (DBM-rFN/CDH-LBL), and evaluate its effects on SCR and bone repair. DBM-rFN/CDH-LBL significantly improved the efficacy and selectivity of SCR via physical interception and chemical recognition. The post-enriched DBM-rFN/CDH-LBL provided favourable microenvironments to facilitate the migration, proliferation and osteogenic differentiation of MSCs, thus accelerating bone repair. Conclusively, DBM-rFN/CDH-LBL presents a novel biomaterial with advantages including high cost-effectiveness, more convenience for storage and transport and can be rapidly constructed intraoperatively.