Alginate-hydrogel versus alginate-solid system. Efficacy in bone regeneration in osteoporosis.

Rosuvastatin/calcium carbonate co-precipitated nanoparticles: A novel synergistic approach enhancing local bone regeneration in osteoporotic rat model

BackgroundMesenchymal stem cells (MSCs) can be differentiated into an osteoblastic lineage in the presence of growth factors (GFs). Platelet-rich plasma (PRP), which can be easily isolated from whole blood, contains a large amount of GFs, and, therefore, promotes bone growth and regeneration. The main goal of this work was to develop and investigate the effect of a new sandwich-like bone scaffold which combines a nano-calcium sulfate (nCS) disc along with PRP fibrin gel (nCS/PRP) with BMP2-modified MSCs on bone repair and regeneration in rat critical-sized calvarial defects.MethodsWe evaluated the cytotoxicity, osteogenic differentiation and mineralization effect of PRP extract on BMP2-modified MSCs and constructed a sandwich-like nCS/PRP scaffold (mimicking the nano-calcium matrix of bone and carrying multi GFs in the PRP) containing BMP2-modified MSCs. The capacity of this multifunctional bone regeneration system in promoting bone repair was assessed in vivo in a rat critical-sized (8 mm) calvarial bone defect model.ResultsWe developed an optimized nCS/PRP sandwich-like scaffold. Scanning electron microscopy (SEM) results showed that nCS/PRP are polyporous with an average pore diameter of 70–80 μm and the cells can survive in the nCS/PRP scaffold. PRP extract dramatically stimulated proliferation and differentiation of BMP2-modified MSCs in vitro. Our in vivo results showed that the combination of BMP2-modified MSCs and nCS/PRP scaffold dramatically increased new bone regeneration compared with the groups without PRP and/or BMP2.ConclusionsnCS/PRP scaffolds containing BMP2-modified MSCs successfully promotes bone regeneration in critical-sized bone defects. This system could ultimately enable clinicians to better reconstruct the craniofacial bone and avoid donor site morbidity for critical-sized bone defects.

Introduction : Osteoporosis stems from misbalance between bone forming and bone resorption, which lead to increased risks of bone fractures. In recent years, stem cell therapy introduced as a promising strategy for bone regeneration in osteoporosis due to their bone regeneration potential. However, stem cells require different stimulator to accelerate bone regeneration and repair processes. Previous Studies showed that Vitamin K2, as an osteoprotective factor, promote and inhibit proliferation and activity osteoblast and osteoclasts cell line, respectively. We aimed to elucidate effect vitamin K2 on dental pulp stem cells (DPSCs) proliferation and their differentiation into osteoblast, and evaluation effect of this vitamin on the process of differentiating peripheral blood mononuclear cells (PBMNCs) into osteoclast and the activity of these cells. Material and Methods: DPSCs and PBMNCs were used for induction towards the osteoblast and osteoclasts, respectively in the presence of various concentrations of vitamin K2. Cell viability was assessed by MTT assay. Osteogenesis assayed by alizarin red S staining and osteogenic gene expression as well as osteoclastogenesis by tartrate-resistant acid phosphatase (TRAP) staining, Annexin V/PI assay, pit formation and NF-κB gene expression. Results: Our data showed that vitamin K2 at a concentration of 10µM increased both proliferation and osteogenesis activities of DPSCs and also increased the incidence of apoptosis in TRAP-positive cells as well as decrees in an expression of NF-κB and pit formation. Conclusion: These results suggest that simultaneous use of vitamin K2 and DPSCs can be a purpose of stem cell therapy in osteoporosis and conducting further pre-clinical studies.

Bone regeneration is impaired in patients with osteoporosis. Previous studies have shown that periostin (Postn) shows great potential in bone regeneration treatments. However, the role of Postn in bone marrow mesenchymal stem cells (BMMSCs) remains to be elucidated. In this study, we isolated BMMSCs from ovariectomized rats (OVX-BMMSCs) and normal rats. Then, the expression levels of Postn and osteogenesis in OVX-BMMSCs were detected by alizarin red and alkaline phosphatase substrate staining, qPCR, and western blotting. We found that the levels of Postn in OVX-BMMSCs were significantly reduced. Furthermore, Postn overexpression in OVX-BMMSCs using recombinant lentivirus could improve the expression of alkaline phosphatase, runt-related transcription factor 2, and osteocalcin and reduce the expression of sclerostin. Besides, micro-computed tomography analysis, hematoxylin-eosin, and Masson's staining showed that the healing of the alveolar bone defect in osteoporotic rats could be promoted using Postn-modified OVX-BMMSC sheets. In conclusion, Postn-modified OVX-BMMSCs might restore the osteogenic capacity and promote alveolar bone regeneration, which may serve as a new therapeutic approach for bone regeneration in osteoporosis.

Alendronate loaded graphene oxide functionalized collagen sponge for the dual effects of osteogenesis and anti-osteoclastogenesis in osteoporotic rats

In the field of bone tissue engineering, which is being developed for the ideal restoration of bone defects, researchers are exploring the improvement of the bone regeneration efficacy of scaffolds through various approaches involving osteoconductive, osteoinductive, and angiogenic factors. In the current trend of research, there is also a suggestion that the topological factors of recent scaffolds may influence the attachment, migration, proliferation, and differentiation of bone cells. Building upon experimental confirmation of the effect of scaffold conformity with the defect site on enhanced bone regeneration in previous studies, we conducted this research to experimentally investigate the relationship between contact area with the defect site and bone regeneration efficacy. The results demonstrated that as the contact area of the scaffold increased, not only did the resistance to bone tissue growth increase, more significant bone regeneration also occurred, as evidenced through histological analysis and micro-CT analysis. This research confirms that the contact area between the scaffold and the defect site is a critical variable affecting bone regeneration efficacy, emphasizing its importance when designing customized scaffolds. This finding holds promising implications for future studies and applications in the field.

Event Abstract Back to Event Macroporous microribbon-based hydrogels enhance stem cell survival and bone regeneration in a mouse critical-size cranial defect model Li-Hsin Han1, Bogi Conrad2, Michael Chung3, Lorenzo Deveza1, 4, Xinyi Jiang1, Andrew Wang5, Manish Butte5, Michael Longaker3, Derrick Wan3 and Fan Yang1, 4 1 Stanford University, Orthopaedic Surgery and Bioengineering, United States 2 Stanford, Stem Cells and Regenerative Medicine, United States 3 Stanford University, Plastic Surgery, United States 4 Stanford University, Bioengineering, United States 5 Stanford University, Pediatrics, United States Introduction: Stem cell-based therapy hold great promise for regenerating lost tissues such as bone, and synthetic grafting biomaterials, such as hydrogels, have been developed to provide structural support and promote desirable cell fates and tissue formation. However, the efficacy of previously developed scaffolds for bone repair are often limited by poor cell survival, non-uniform cell distribution, or the lack of macroporosity that is desirable to promote cell migration and new matrix deposition. To overcome the above limitations, we recently developed microribbon (μRB)-like, crosslinkable elastomers as scaffold building blocks, which can encapsulate cells in 3D while simultaneously forming a highly macroporous scaffold[1]. Our previous studies showed that μRBs facilitate uniform cell encapsulation, allow formation of 3D scaffolds with tunable macroporosity, and support rapid cell spreading and proliferation in vitro. We hypothesized that macroporous μRB-based scaffolds would promote stem cell engraftment and survival after transplantation, and enhance host tissue ingrowth and promote bone regeneration. The goal of the current study was to evaluate the potential of gelatin-based μRB scaffolds for in vivo bone repair using a critical-sized, mouse cranial defect model. Materials and Methods: Photocrosslinkable gelatin mRBs were synthesized by wet-spinning as we previously reported[1]. Prior to use, mRBs were rehydrated in phosphate-buffered saline and mixed with trypsinized GFP/Luc+ mouse adipose-derived stem cells (ADSCs). To fabricate macroporous scaffolds, molded mRBs were photocrosslinked (365 nm, 5 min, 4 mW/cm2) in the presence of a photoinitiator. The resulting scaffolds were characterized by unconfined compression testing and scanning electron microscopy for morphology. The stiffness of individual μRBs was measured using atomic force microscopy. To compare the efficacy of mRB-based scaffolds for supporting bone repair with that of existing hydrogel platforms, hydrogels of methacrylated gelatin were used as control groups. To assess the ability of mRB-based scaffolds to promote ADSC survival and bone regeneration in vivo, mouse critical sized cranial defects (4 mm in diameter) were treated with the following five groups including: (1) μRB+ADSC, (2) hydrogel (HG) +ADSC, (3) μRB only, and (4) HG only, (5) untreated defect (neg ctrl). All groups were monitored for up to 6 weeks, and outcomes were evaluated using bioluminescence imaging, micro-CT, and histology. Results and Discussion: Photocrosslinkable gelatin-based μRBs were synthesized by wet-spinning as previously reported[1] (Fig. 1). The resulting μRBs are 40-70 m in width and injectable through 16G needle, and allows direct encapsulation of ADSCs in 3D (Fig. 1). Upon photocrosslinking, the microribbons formed a highly macroporous scaffold that supported cell spreading and proliferation (Fig. 1). Results from a critical-sized, mouse cranial defect model demonstrated that microribbon-based scaffolds significantly enhanced the survival and proliferation of transplanted adipose-derived stromal cells compared with conventional hydrogels (Fig. 2). Macroporosity among microribbons facilitated mineralized bone formation (as shown by microCT) and host tissue ingrowth (histology) (Fig. 3). The observed enhancement of cell survival and proliferation further promoted the paracrine-signaling effects of adipose-derived stromal cells for stimulating endogenous bone regeneration. Conclusions: Here we validate the efficacy of photocrosslinkable μRBs as injectable macroporous scaffolds for stem cell delivery and tissue regeneration. Compared to conventional nanoporous hydrogels, our μRB hydrogels are macroporous, which led to enhanced stem cell survival and engraftment, accelerated vascularization and endogenous bone repair. We anticipate such microribbon-based scaffolds will provide a novel injectable matrices for stem cell-based therapies to enhance tissue regeneration outcomes by substantially improving cell survival, vascularization and new extracellular matrix deposition. The authors would like to acknowledge the following funding support including NIH R01DE024772 (F.Y), California Institute for Regenerative Medicine (Grant #TR3-05569) (F. Y.), National Science Foundation CAREER award(CBET-1351289, F.Y.) Stanford Chem-H Institute Biomaterials Seed Grant (F. Y.), Stanford Coulter Translational Grant, and Stanford Child Health Research Institute Faculty Scholar Award (F. Y.)

Low-density lipoprotein receptor-related protein 5 (LRP5) plays a vital role in bone formation and regeneration. In this study, we developed an injectable and sustained-release composite loading LRP5 which could gelatinize in situ. The sustained release of the composite and its efficacy in bone regeneration were evaluated. Sodium alginate, collagen, hydroxyapatite, and LRP5 formed the composite LRP5-Alg/Col/HA. It was found that the initial setting time and final setting time of LRP5-Alg/Col/HA containing 4% alginate were suitable for surgical operation. When the composite was loaded with 40 μg/mL LRP5, LRP5-Alg/Col/HA did not exhibit a burst-release behavior and could sustainably release LRP5 up to 21 days. Up to 18 days, LRP5 released from LRP5-Alg/Col/HA still present the binding activity with DKK1 (Wnt signaling pathway antagonist) and could increase the downstream β-catenin mRNA in bone marrow mesenchymal stem cells. Moreover, LRP5-Alg/Col/HA was found to significantly increase bone mineral density in the defect area after 6 weeks’ implantation of LRP5-Alg/Col/HA into the rats’ calvarial defect area. H&E staining detection demonstrated that LRP5-Alg/Col/HA could mediate the formation of a new bone tissue. Therefore, we concluded that Alg/Col/HA was a suitable sustained-release carrier for LRP5 and LRP5-Alg/Col/HA had a significant effect on repairing bone defects and could be a good bone regeneration material.

The purpose of this study was to test several modifications of the polymethylmethacrylate (PMMA) bone cement by incorporating osteoconductive and biodegradable materials for enhancing bone regeneration capacity in an osteoporotic rat model. Three bio-composites (PHT-1 [80% PMMA, 16% HA, 4% β-TCP], PHT-2 [70% PMMA, 24% HA, 6% β-TCP], and PHT-3 [30% PMMA, 56% HA, 14% β-TCP]) were prepared using different concentrations of PMMA, hydroxyapatite (HA), and β-tricalcium phosphate (β-TCP). Their morphological structure was then examined using a scanning electron microscope (SEM) and mechanical properties were determined using a MTS 858 Bionics test machine (MTS, Minneapolis, MN, USA). For in vivo studies, 35 female Wister rats (250 g, 12 weeks of age) were prepared and divided into five groups including a sham group (control), an ovariectomy-induced osteoporosis group (OVX), an OVX with pure PMMA group (PMMA), an OVX with PHT-2 group (PHT-2), and an OVX with PHT-3 group (PHT-3). In vivo bone regeneration efficacy was assessed using micro-CT and histological analysis after injecting the prepared bone cement into the tibial defects of osteoporotic rats. SEM investigation showed that the PHT-3 sample had the highest porosity and roughness among all samples. In comparison to other samples, the PHT-3 exhibited favorable mechanical properties for use in vertebroplasty procedures. Micro-CT and histological analysis of OVX-induced osteoporotic rats revealed that PHT-3 was more effective in regenerating bone and restoring bone density than other samples. This study suggests that the PHT-3 bio-composite can be a promising candidate for treating osteoporosis-related vertebral fractures.

Neuro-bone tissue engineering: Multiple potential translational strategies between nerve and bone

Small extracellular vesicles derived from hypoxic mesenchymal stem cells promote vascularized bone regeneration through the miR-210-3p/EFNA3/PI3K pathway

Healing of critical-sized bone defects (CSDs) remains a significant challenge in clinical treatment. Piezoelectric materials, which play a prominent role in the bioelectricity of bone homeostasis, have garnered increasing attention in bone regeneration. Under physiological conditions in bone tissue, the relative sliding of collagen fibers after mechanical deformation generates electrical signals, which form the intrinsic structural basis for the natural piezoelectric properties of blood vessels and bone tissue. In this study, we developed a collagen-decalcified bone matrix gel composite to mimic the piezoelectric mechanism of bone tissue and further incorporated barium titanate piezoelectric nanoparticles to enhance piezoelectricity, creating a collagen-decalcified bone matrix gel-barium titanate scaffold (COL/DBM/BT). This scaffold provides piezoelectric and osteoinductive properties to stimulate bone repair. Under ultrasound activation, in vitro and in vivo experiments revealed that the COL/DBM/BT piezoelectric scaffold significantly enhanced the migration, proliferation, adhesion, and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), as well as the migration, adhesion, and vascularization of human umbilical vein endothelial cells (HUVECs). Notably, significant bone regeneration was observed in critical-sized mandibular bone defects in vivo. In summary, the ultrasound-assisted COL/DBM/BT scaffold creates a highly piezoelectric and osteoinductive microenvironment, promoting more efficient bone repair.

Craniofacial bone defects are considered difficult to treat in osteoporosis individuals. Strontium (Sr) has been proven therapeutically beneficial for osteoporotic patients by multiple studies. Systemically administered, strontium may cause unfavorable adverse effects, but local application is promising. In this study, mesoporous silica nanoparticles (MSN) were used as nanocarriers of strontium, embedded into the hydrogel scaffold along with adipose-derived stem cells under local administration to evaluate its bone regenerating potential in osteoporotic rat model. Mesoporous silica nanoparticles (MSN) and Sr-doped MSN (MSN-Sr) were synthesized using a cetyltrimethylammonium bromide-templated method with tetraethyl orthosilicate. Nanoparticles were characterized by inductively coupled plasma mass spectrometry, transmission electron microscope, dynamic light scattering nano-analyzer, and X-ray photoelectron spectrometer. Gelatin-based cryogels containing these nanoparticles were fabricated through crosslinking and freeze-drying, and evaluated for physicochemical properties, porosity, degradation, thermal stability, and swelling. Rat adipose-derived stem cells (ASCs) were seeded onto cryogels for osteogenic analysis by scanning electron microscope, field emission scanning electron microscope/energy dispersive spectrometer, and quantitative polymerase chain reaction (qPCR). In vivo, ASC-cryogels were implanted in nude mice and osteoporotic rats, with bone regeneration evaluated by micro-CT, histology, and immunohistochemistry. The release profile of Sr-doped MSN was verified, and its pore volume and structure were evaluated. After MSNs were embedded into hydrogel, the physiochemistry characteristics including porosity, thermal stability, degradation, and swelling ability showed optimal results. For in vivo study in subdermal layer of nude mice, MSN-Sr cryogel scaffolds cultured with adipose-derived mesenchymal stem cell showed increase expression of osteogenic-related mRNA, including type I collegen, catenin beta 1, and Runx2. MSN-Sr embedded cryogels (G-MSN-Sr) were placed in bone defect of osteoporosis rats. Surface area of the bone defects showed significant decrease in the G-MSN-Sr group compared to pure gelatin gel group and MSN doped cryogel group. Histological analysis of bone formation using H&E, ALP, Masson's Trichrome, and Osteocyte stains further validated the bone regeneration. MSN is a feasible nanocarrier for the delivery of Sr ion. Hydrogel scaffolds embedded with strontium doped MSN and mesenchymal stem cells showed promising bone regenerating potential in osteoporotic rat model.

ObjectivesTo compare new bone formation in mandibular critical-sized bone defects (CSBDs) in healthy, diabetic, osteoporotic, and diabetic-osteoporotic rats filled with bioceramics (BCs) with or without bone marrow mesenchymal stem cells (BMSCs). MethodsA total of 64 adult female Sprague-Dawley rats were randomized into four groups (n = 16 per group): Group 1 healthy, Group 2 diabetic, Group 3 osteoporotic, and Group 4 diabetic-osteoporotic rats. Streptozotocin was used to induce type 1 diabetes in Group 2 and 4, while bilateral ovariectomy was used to induce osteoporosis in Group 3 and 4. The central portion of the rat mandibular symphysis was used as a physiological CSBD. In each group, eight defects were filled with BC (hydroxypatatite 60% and β-tricalcium phosphate 40%) alone and eight with BMSCs cultured on BC. The animals were sacrificed at 4 and 8 weeks, and the mandibles were processed for micro-computed tomography to analyze radiological union and bone mineral density (BMD); histological analysis of the bone union; and immunohistochemical analysis, which included immunoreactivity of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2). ResultsIn all groups (healthy, diabetics, osteoporotics, and diabetics-osteoporotics), the CSBDs filled with BC + BMSCs showed greater radiological bone union, BMD, histological bone union, and more VEGF and BMP-2 positivity, in comparison with CSBDs treated with BC alone (at 4 and 8 weeks). ConclusionsApplication of BMSCs cultured on BCs improves bone regeneration in CSBDs compared with application of BCs alone in healthy, diabetic, osteoporotic, and diabetic-osteoporotic rats.

This review considers the potential use of bone morphogenetic protein-2 (BMP-2) for bone regeneration in osteoporosis. Studies on the effect of BMP-2 on bone regeneration in vitro and in vivo are analysed. The results of clinical application of osteoinducers and possible complications are described.