Distribution of bone voids in the thoracolumbar spine in Chinese adults with and without osteoporosis: A cross-sectional multi-center study based on 464 vertebrae.

Bone void is a novel intuitive morphological indicator to assess bone quality but its use in vertebrae has not been described. This cross-sectional and multi-center study aimed to investigate the distribution of bone voids in the thoracolumbar spine in Chinese adults based on quantitative computed tomography (QCT). A bone void was defined as a trabecular net region with extremely low bone mineral density (BMD) (<40 mg/cm3), detected by an algorithm based on phantom-less technology. A total of 464 vertebrae from 152 patients (51.8 ± 13.4 years old) were included. The vertebral trabecular bone was divided into eight sections based on the middle sagittal, coronal, and horizontal planes. Bone void of the whole vertebra and each section were compared between healthy, osteopenia, and osteoporosis groups and between spine levels. Receiver operator characteristic (ROC) curves were plotted and optimum cutoff points of void volume between the groups were obtained. The total void volumes of the whole vertebra were 124.3 ± 221.5 mm3, 1256.7 ± 928.7 mm3, and 5624.6 ± 3217.7 mm3 in healthy, osteopenia, and osteoporosis groups, respectively. The detection rate of vertebrae with bone voids was higher and the normalized void volume was larger in the lumbar than in thoracic vertebrae. L3 presented the largest void (2165.0 ± 3396.0 mm3), while T12 had the smallest void (448.9 ± 699.4 mm3). The bone void was mainly located in the superior-posterior-right section (40.8 %). Additionally, bone void correlated positively with age and increased rapidly after 55 years. The most significant void volume increase was found in the inferior-anterior-right section whereas the least increase was found in the inferior-posterior-left section with aging. The cutoff points were 345.1 mm3 between healthy and osteopenia groups (sensitivity = 0.923, specificity = 0.932) and 1693.4 mm3 between osteopenia and osteoporosis groups (sensitivity = 1.000, specificity = 0.897). In conclusion, this study demonstrated the bone void distribution in vertebrae using clinical QCT data. The findings provide a new perspective for the description of bone quality and showed that bone void could guide clinical practice such as osteoporosis screening.

Circular RNA circStag1 promotes bone regeneration by interacting with HuR.

Postmenopausal osteoporosis is a common bone metabolic disorder characterized by deterioration of the bone microarchitecture, leading to an increased risk of fractures. Recently, circular RNAs (circRNAs) have been demonstrated to play pivotal roles in regulating bone metabolism. However, the underlying functions of circRNAs in bone metabolism in postmenopausal osteoporosis remain obscure. Here, we report that circStag1 is a critical osteoporosis-related circRNA that shows significantly downregulated expression in osteoporotic bone marrow mesenchymal stem cells (BMSCs) and clinical bone tissue samples from patients with osteoporosis. Overexpression of circStag1 significantly promoted the osteogenic capability of BMSCs. Mechanistically, we found that circStag1 interacts with human antigen R (HuR), an RNA-binding protein, and promotes the translocation of HuR into the cytoplasm. A high cytoplasmic level of HuR led to the activation of the Wnt signaling pathway by stabilizing and enhancing low-density lipoprotein receptor-related protein 5/6 (Lrp5/6) and ß-catenin expression, thereby stimulating the osteogenic differentiation of BMSCs. Furthermore, overexpression of circStag1 in vivo by circStag1-loaded adeno-associated virus (circStag1-AAV) promoted new bone formation, thereby preventing bone loss in ovariectomized rats. Collectively, we show that circStag1 plays a pivotal role in promoting the regeneration of bone tissue via HuR/Wnt signaling, which may provide new strategies to prevent bone metabolic disorders such as postmenopausal osteoporosis.

PiRNA-63049 inhibits bone formation through Wnt/ß-catenin signaling pathway.

Bone remodeling is a dynamic process between bone formation mediated by osteoblasts and bone resorption mediated by osteoclasts. Disrupted bone remodeling is a key factor in postmenopausal osteoporosis, a metabolic disorder characterized by deteriorated bone microarchitecture and increased risk of fracture. Recent studies have shown that piwi-binding RNA (piRNA) is involved in the pathogenesis of certain diseases at the post-transcriptional level. Here, we analyzed piRNA-63049 (piR-63049), which may play an essential role in bone remodeling. The expression of piR-63049 significantly increased in both bone tissues and plasma of osteoporotic rats and postmenopausal osteoporotic patients. Overexpressing piR-63049 could inhibit the osteoblastogenesis of bone marrow stromal cells (BMSCs) while knocking down piR-63049 could promote the osteoblastogenesis of BMSCs through the Wnt2b/ß-catenin signaling pathway. Moreover, knocking-down piR-63049 (piR-63049-antagonist) in vivo could attenuate the bone loss in ovariectomized rats by promoting bone formation. Taken together, the current study shows that piR-63049 inhibits bone formation through the Wnt2b/ß-catenin signaling pathway. This novel piRNA may be a potential target to increase bone formation in bone loss disorders such as postmenopausal osteoporosis.

Enhanced bone regeneration via spatiotemporal and controlled delivery of a genetically engineered BMP-2 in a composite Hydrogel.

Scaffolds functionalized with bone morphogenetic protein-2 (BMP-2) have shown great potential for bone regeneration. However, structural instability and the necessity for supra-physiological dose have thus far limited practical applications for BMP-2. Protein modification and site-specific covalent immobilization of BMP-2 to carrier materials might be optimal strategies to overcome these problems. Here, we report a broadly applicable strategy where the polyhistidine tag-T4 Lysozyme (His6-T4L) was genetically fused at the N-terminus of BMP-2 and used as a protein spacer, which on one hand enhanced protein solubility and stability, and on the other hand mediated site-specific covalent anchoring of BMP-2 upon binding to nickel-chelated nitrilotriacetic acid (Ni-NTA) microparticles (denoted as MPs-His6-T4L-BMP2) to further maximize its rescued activity. We also constructed a novel gelatin-based hydrogel that was crosslinked by transglutaminase (TG) and tannic acid (TA). This hydrogel, when incorporated with MPs-His6-T4L-BMP2, displayed excellent in-situ injectability, thermosensitivity, adhesiveness and improved mechanical properties. The effective loading mode led to a controlled and long-term sustained release of His6-T4L-BMP2, thereby resulting in enhancement of bone regeneration in a critical-sized bone defect. We believe that the protein modification strategy proposed here opens up new route not only for BMP-2 applications, but can be used to inform novel uses for other macromolecules.

Rationally designed protein cross-linked hydrogel for bone regeneration via synergistic release of magnesium and zinc ions.

The development of recombinant protein cross-linked injectable hydrogels with good mechanical strength and effective drug loading capacity for bone regeneration is extremely attractive and rarely reported. Here, we report the fabrication of a smart hydrogel delivery system by incorporating a rationally designed T4 lysozyme mutant (T4M) to mediate the localized delivery and synergistic release of Mg2+ and Zn2+ for bone repair. Apart from its intrinsic antibacterial properties, T4M bears abundant free amine groups on its surface to function as effective covalent crosslinkers to strengthen the hydrogel network as well as exhibits specific binding affinity to multivalent cations such as Zn2+. Moreover, the integrin receptor-binding Arg-Gly-Asp (RGD) sequence was introduced onto the C-terminus of T4 lysozyme to improve its cellular affinity and further facilitate rapid tissue regeneration. The final composite hydrogel displays excellent injectability, improved mechanical properties, antibacterial activity, and unique bioactivities. The effective loading of Mg2+/Zn2+ in the hydrogels could mediate the sequential and sustained release of Mg2+ and Zn2+, thereby resulting in synergistic enhancement on bone regeneration through modulation of the MAPK signaling pathway. We believe that the strategy proposed in this paper opens up a new route for developing protein cross-linked smart delivery systems for tissue regeneration.

Precisely Controlled Delivery of Abaloparatide through Injectable Hydrogel to Promote Bone Regeneration.

Side-effects from allograft, limited bone stock, and site morbidity from autograft are the major challenges to traditional bone defect treatments. With the advance of tissue engineering, hydrogel injection therapy is introduced as an alternative treatment. Therapeutic drugs and growth factors can be carried by hydrogels and delivered to patients. Abaloparatide, as an analog of human recombinant parathyroid hormone protein (PTHrp) and an alternative to teriparatide, has been considered as a drug for treating postmenopausal osteoporosis since 2017. Since only limited cases of receiving abaloparatide with polymeric scaffolds have been reported, the effects of abaloparatide on pre-osteoblast MC3T3-E1 are investigated in this study. It is found that in vitro abaloparatide treatment can promote pre-osteoblast MC3T3-E1 cells' viability, differentiation, and mineralization significantly. For the drug delivery system, 3D porous structure of the methacrylated gelatin (GelMA) hydrogel is found effective for prolonging the release of abaloparatide (more than 10 days). Therefore, injectable photo-crosslinked GelMA hydrogel is used in this study to prolong the release of abaloparatide and to promote healing of defected bones in rats. Overall, data collected in this study show no contradiction and imply that Abaloparatide-loaded GelMA hydrogel is effective in stimulating bone regeneration.

Conductive Hydrogen Sulfide-Releasing Hydrogel Encapsulating ADSCs for Myocardial Infarction Treatment.

Hydrogen sulfide (H2S) exhibits extensive protective actions in cardiovascular systems, such as anti-inflammatory and stimulating angiogenesis, but its therapeutic potential is severely discounted by the short half-life and the poorly controlled releasing behavior. Herein, we developed a macromolecular H2S prodrug by grafting 2-aminopyridine-5-thiocarboxamide (a small-molecule H2S donor) on partially oxidized alginate (ALG-CHO) to mimic the slow and continuous release of endogenous H2S. In addition, tetraaniline (a conductive oligomer) and adipose-derived stem cells (ADSCs) were introduced to form a stem cell-loaded conductive H2S-releasing hydrogel through the Schiff base reaction between ALG-CHO and gelatin. The hydrogel exhibited adhesive property to ensure a stable anchoring to the wet and beating hearts. After myocardial injection, longer ADSCs retention period and elevated sulfide concentration in rat myocardium were demonstrated, accompanied by upregulation of cardiac-related mRNA (Cx43, α-SMA, and cTnT) and angiogenic factors (VEGFA and Ang-1) and downregulation of inflammatory factors (tumor necrosis factor-α). Echocardiography and histological analysis strongly demonstrated an increase in the ejection fraction value and smaller infarction size, suggesting a remarkable improvement of the cardiac functions of Sprague-Dawley rats. The ADSC-loaded conductive hydrogen sulfide-releasing hydrogel dramatically promoted the therapeutic effects, offering a promising therapeutic strategy for treating myocardial infarction.

Rebuilding Postinfarcted Cardiac Functions by Injecting TIIA@PDA Nanoparticle-Cross-linked ROS-Sensitive Hydrogels.

Drug-loaded injectable hydrogels have been proven to possess huge potential for applications in tissue engineering. However, increasing the drug loading capacity and regulating the release system to adapt to the microenvironment after myocardial infarction face a huge challenge. In this research, an ROS-sensitive injectable hydrogel strengthened by self-nanodrugs was constructed. A hyperbranched ROS-sensitive macromer (HB-PBAE) with multiacrylate end groups was synthesized through dynamic controlled Michael addition. Meanwhile, a simple protocol based on dopamine polymerization was employed to generate a polydopamine (PDA) layer deposited on the tanshinone IIA (TIIA) nanoparticles (NPs) formed from spontaneous hydrophobic self-assembly. The HB-PBAE reacted with thiolate-modified hyaluronic acid (HA-SH) to form an in situ hydrogel, where TIIA@PDA NPs can be conveniently entrapped through the chemical cross-link between thiolate and quinone groups on PDA, which doubles the modulus of hydrogels. The in vivo degradation behavior of the hydrogels was characterized by MRI, exhibiting a much slower degradation behavior that is markedly different from that of in vitro. Importantly, a significant improvement of cardiac functions was achieved after hydrogel injection in terms of increased ejection fraction and decreased infarction size, accompanied by inhibition of the expression of inflammation factors, such as IL-1ß, IL-6, and TNF-α.

Paintable and Rapidly Bondable Conductive Hydrogels as Therapeutic Cardiac Patches.

In recent years, cardiac patches have been developed for the treatment of myocardial infarction. However, the fixation approaches onto the tissue through suture or phototriggered reaction inevitably cause new tissue damage. Herein, a paintable hydrogel is constructed based on Fe3+ -triggered simultaneous polymerization of covalently linked pyrrole and dopamine in the hyperbranched chains where the in situ formed conductive polypyrrole also uniquely serves to crosslink network. This conductive and adhesive hydrogel can be conveniently painted as a patch onto the heart surface without adverse liquid leakage. The functional patch whose conductivity is equivalent to that of normal myocardium is strongly bonded to the beating heart within 4 weeks, accordingly efficiently boosting the transmission of electrophysiological signals. Eventually, the reconstruction of cardiac function and revascularization of the infarct myocardium are remarkably improved. The translatable suture-free strategy reported in this work is promising to address the human clinical challenges in cardiac tissue engineering.

An injectable conductive hydrogel encapsulating plasmid DNA-eNOs and ADSCs for treating myocardial infarction.

Myocardial infarction (MI) leads to the mass death of cardiomyocytes accompanying with the unfavorable alternation of microenvironment, a fibrosis scar deprived of electrical communications, and the lack of blood supply in the infarcted myocardium. The three factors are inextricably intertwined and thus result in a conservative MI therapy efficacy in clinic. A holistic approach pertinently targeted to these three key points would be favorable to rebuild the heart functions. Here, an injectable conductive hydrogel was constructed via in situ Michael addition reaction between multi-armed conductive crosslinker tetraaniline-polyethylene glycol diacrylate (TA-PEG) and thiolated hyaluronic acid (HA-SH). The resultant soft conductive hydrogel with equivalent myocardial conductivity and anti-fatigue performance was loaded with plasmid DNA encoding eNOs (endothelial nitric oxide synthase) nanocomplexes and adipose derived stem cells (ADSCs) for treating MI. The TA-PEG/HA-SH/ADSCs/Gene hydrogel-based holistic system was injected into the infarcted myocardium of SD rats. We demonstrated an increased expression of eNOs in myocardial tissue the heightening of nitrite concentration, accompanied with upregulation of proangiogenic growth factors and myocardium related mRNA. The results of electrocardiography, cardiogram, and histological analysis convincingly revealed a distinct increase of ejection fraction (EF), shortened QRS interval, smaller infarction size, less fibrosis area, and higher vessel density, indicating a significant improvement of heart functions. This conception of combination approach by a conductive injectable hydrogel loaded with stem cells and gene-encoding eNOs nanoparticles will become a robust therapeutic strategy for the treatment of MI.

A π-π conjugation-containing soft and conductive injectable polymer hydrogel highly efficiently rebuilds cardiac function after myocardial infarction.

Previous studies suggested that a stiffer hydrogel system exhibited a better performance to promote heart function after myocardial infarction (MI). However, the nature of myocardium, a tissue that alternately contracts and relaxes with electrical impulses, leads us to hypothesize that a soft and conductive hydrogel may be in favor of mechanical and electrical signals transmission to enhance heart function after MI. In this work, π-π conjugation interaction was first employed to produce a soft injectable hydrogel with conductive property. Melamine with π-π conjugation ring was used as a core to synthesize a multi-armed crosslinker PEGDA700-Melamine (PEG-MEL), which could crosslink with thiol-modified hyaluronic acid (HA-SH) to form an injectable hydrogel rapidly. By incorporating graphene oxide (GO), the injectable PEG-MEL/HA-SH/GO hydrogel exhibited a soft (G' = 25 Pa) and anti-fatigue mechanical property and conductive property (G = 2.84 × 10-4 S/cm). The hydrogel encapsulating adipose tissue-derived stromal cells (ADSCs) was injected into MI area of rats. The significant increase in α-Smooth Muscle Actin (α-SMA) and Connexin 43 (Cx43) expression confirmed that the gel efficiently promoted the transmission of mechanical and electrical signals. Meanwhile, a significant improvement of heart functions, such as distinct increase of ejection fraction (EF), smaller infarction size, less fibrosis area, and higher vessel density, was achieved.