Browsing by Subject "Bone regeneration"
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Item 467 Enhancing Cell Infiltration and Controlled Growth Factor Release for a Customized 3D-Printed Bone Graft Composite(Cambridge University Press, 2024-04-03) Alston, Claudia Benito; Chadwick, Madelyn; Rupani, Saaniya; Moldovan, Nicanor; Barco, Clark; Solorio, Luis; Medicine, School of MedicineOBJECTIVES/GOALS: Annually, 1.5 million global patients receive maxillofacial reconstruction. The gold standard, involving bone particulate, lacks reproducibility. To improve this, we have developed a custom 3D-printable, porous cover-core design. This study optimizes the hydrogel core properties and growth factor (GF) release for enhanced bone regeneration. METHODS/STUDY POPULATION: Different ratios of Methacrylated Gelatin (GelMa), Methacrylated Alginate (AlgMa) and tricalcium phosphate (α²-TCP) were combined to optimize cell viability, GF sequestration and mechanical stability. Material characterization was performed using a rheometer to determine the viscoelastic properties of the blends. Release from disks loaded with FGF-containing PLGA microparticles was quantified with an ELISA kit. Furthermore, scanning electron microscopy (SEM) was conducted to quantify hydrogel porosity. In vitro studies were performed using NIH 3T3 murine fibroblasts in Corning Transwells while immunofluorescent, metabolic and osteogenic studies were performed in 96 well plates to investigate cell infiltration, cell adhesion, viability and differentiation, respectively. RESULTS/ANTICIPATED RESULTS: By adjusting the AlgGelMa ratio, we manipulated matrix properties. GelMa possesses excellent durability and cell adhesion due to intrinsic RGD-binding motifs. AlgMa enhanced swelling by 30%, growth factor sequestration by 50% in 24hrs, and matrix storage modulus without increasing the loss modulus which could cause cell migration away from the hydrogel. Varying the AlgGelMa ratio lowered pH, promoted cell infiltration, and reduced fibronectin accumulation. The addition of β-TCP is anticipated to improve cell differentiation towards an osteogenic lineage due to improved elastic modulus, calcium and phosphate ion concentration improving mineral deposition. DISCUSSION/SIGNIFICANCE: These findings suggest through the use of this composite, early cell infiltration can be increased and promoted due to FGF release, leading to increased osteointegration. Our porous cover-core design ensures efficient clot integration and early cell infiltration, enhancing osteointegration through FGF release.Item AGN1 implant material to treat bone loss: Resorbable implant forms normal bone with and without alendronate in a canine critical size humeral defect model(Elsevier, 2022) Shaul, Jonathan L.; Hill, Ronald S.; Bouxsein, Mary L.; Burr, David B.; Tilton, Annemarie K.; Howe, James G.; Anatomy, Cell Biology and Physiology, School of MedicineBackground: Fractures secondary to osteoporosis, particularly those of the hip and spine, are a major public health concern with high social and economic costs. The Local Osteo-Enhancement Procedure (LOEP) is an approach intended to strengthen skeletal areas that are at the highest risk for fracture due to osteoporosis. LOEP involves the implantation of AGN1, a triphasic, calcium-based, osteoconductive material which is then resorbed and replaced by bone. Since alendronate is the most prescribed osteoporotic treatment, the purpose of this canine study is to determine if the newly formed bone has the same properties as normal bone and whether alendronate treatment impacts AGN1 resorption and replacement with bone. Methods: Sixty skeletally mature male hounds (24-38 kg) were evenly divided between alendronate (0.2 mg/kg/day) and non-alendronate treatment groups. A critical-size core bone defect created in one proximal humerus was implanted with AGN1 while the contralateral non-operated humerus served as a paired control in each animal. Animals were sacrificed 13, 26, and 52 weeks post-operatively (10 per treatment per timepoint). The control and treatment site bone specimens from each animal were examined using radiographic, histomorphometric, and biomechanical techniques. Results between alendronate-treated and non-alendronate-treated animals were compared as groups. Results: AGN1 implant material was consistently resorbed and replaced by bone in all animals. At 52 weeks, only minimal residual implant material could be detected (0.9 ± 2.3% non-alendronate group; 2.2 ± 3.1% alendronate group), and new bone filled the defects in both the non-alendronate and alendronate groups. At 13 and 26 weeks, microCT revealed the newly formed bone in the defects had significantly higher trabecular bone volume and number connectivity than control bone in both groups. Mechanical testing demonstrated that the new bone had ultimate compressive strength and modulus equivalent to control bone as early as 13 weeks post-surgery which was maintained to 52 weeks in both groups. Conclusions: In this canine critical-sized humeral core defect model, AGN1 was progressively replaced by normal bone as evaluated by all outcome measures. Concurrent alendronate therapy did not significantly impact AGN1 resorption or new bone formation. These results demonstrate that AGN1 can be used in conjunction with alendronate in non-osteoporotic animals. Clinical relevance: This study suggests that the AGN1 implant material demonstrates potential for local restoration of bone in critical-size core defects, and that the material is compatible with alendronate drug therapy. Further studies will be required to determine if these results apply to other osteoporosis medications.Item Bone regeneration in novel porous titanium implants(2010) Khouja, Naseeba, 1981-; Chu, Tien-Min Gabriel; Brown, David T.; Platt, Jeffery A., 1958-; Blanchard, Steven B.; Levon, John A.The objective of this study was to evaluate the in vivo performance of the novel press-fit dental implant fabricated via electron beam melting (EBM, Southern Methodist Univ.) and compare it to a commercially-available porous-coated press-fit dental implant (Endopore, Innova Corp.). Twelve cylindrical shaped implants 3 mm in diameter x 5 mm long were made by EBM (Southern Methodist Univ.) using Ti6Al4V ELI alloy. Twelve commercial implants (Endopore, Innova Corp.) of the same geometry were used as controls. Samples were implanted in rabbit tibia and retrieved six weeks postoperatively. Six specimens from each implant type were embedded undecalcified, sectioned, and stained with toluidine blue (Sigma) for histomorphometry analysis. Bone-to-implant contact (BIC) was measured. On the six remaining samples from each implant type, the mechanical properties were evaluated by pushout test on a material testing machine. The samples were loaded at a loading rate of 1 mm/min. The pushout strength was measured and the apparent shear stiffness was calculated. The results were analyzed with a paired-t test. The histology shows osteointegration of surrounding bone with both implant types. Bone was found to grow into the porous space between the beads. Both the Endopore (Innova Corp.) and the EBM (Southern Methodist Univ.) showed similar BIC. The mean BIC for the Endopore (Innova Corp.) and EBM (Southern Methodist Univ.) implant were 35 ± 6% and 32 ± 9%, respectively. It failed to reach statistical significance (p > 0.05). The peak pushout force for Endopore (Innova Corp.) and EBM (Southern Methodist Univ.) implants were 198.80 ± 61.29 N and 243.21 ± 69.75 N, respectively. The apparent shear stiffness between bone and implant for the Endopore (Innova Corp.) and EBM (Southern Methodist Univ.) implants were 577.36 ± 129.99 N/mm; and 584.48 ± 146.63 N/mm, respectively. Neither the peak pushout force nor the apparent shear stiffness of the implants was statistically different between the two groups (p > 0.05). The results suggest that the implants manufactured by EBM (Southern Methodist Univ.) perform equally well as the commercial implant Endopore (Innova Corp.) in this current animal model.Item Effects of Bone Morphogenetic Protein-2 on Neovascularization During Large Bone Defect Regeneration(Mary Ann Liebert, Inc., 2019-12) Pearson, Hope B.; Mason, Devon E.; Kegelman, Christopher D.; Zhao, Liming; Dawahare, James H.; Kacena, Melissa A.; Boerckel, Joel D.; Orthopaedic Surgery, School of MedicineInsufficient blood vessel supply is a primary limiting factor for regenerative approaches to large bone defect repair. Recombinant bone morphogenetic protein-2 (BMP-2) delivery induces robust bone formation and has been observed to enhance neovascularization, but whether the angiogenic effects of BMP-2 are due to direct endothelial cell stimulation or due to indirect paracrine signaling remain unclear. In this study, we evaluated the effects of BMP-2 delivery on vascularized bone regeneration and tested whether BMP-2 induces neovascularization directly or indirectly. We found that delivery of BMP-2 (5 μg) enhanced both bone formation and neovascularization in critically sized (8 mm) rat femoral bone defects; however, BMP-2 did not directly stimulate angiogenesis in vitro. In contrast, conditioned medium from both mesenchymal progenitor cells and osteoblasts induced endothelial cell migration in vitro, suggesting a paracrine mechanism of BMP-2 action. Consistent with this inference, codelivery of BMP-2 with endothelial colony forming cells to a heterotopic site, distant from the skeletal stem cell-rich bone marrow niche, induced ossification but had no effect on neovascularization. Taken together, these data suggest that paracrine activation of osteoprogenitor cells is an important contributor to neovascularization during BMP-2-mediated bone regeneration. Impact Statement In this study, we show that bone morphogenetic protein-2 (BMP-2) robustly induces neovascularization during tissue-engineered large bone defect regeneration, and we found that BMP-2 induced angiogenesis, in part, through paracrine signaling from osteoprogenitor cells.Item Effects of GSK3-β Inhibitors on Wnt Signaling in Zebrafish Fin Regeneration: Chemical Biology(2014-04-11) Brannick, Angelica; Mahin, Jennifer L.; Farrel, Mark; Curtis, Courtney; Sarmah, Swapnalee; Collins, Kayla; Chu, Shaoyou; Sato, Mas; Sanchez-Felix, ManuelIn order to develop beneficial drugs for osteoporosis it is important to understand the molecular mechanisms of bone regeneration and define specific regulatory factors. Zebrafish can regenerate damaged tissues, and they prove to be a good model to study bone growth and repair. Previous research showed that GSK3β inhibitor compound at various concentrations and for different treatment periods effectively stimulated fin regeneration. Conducted experiments identified temporal and spatial fluctuations on individual gene markers after GSK3β inhibitor treatment at various concentrations. Recent analyzed data uses the Lilly Research Labs experimental compound LSN 2105786 at 3 nM and 5 nM to stimulate tissue regeneration to determine whether activating Wnt signaling produces cell proliferation and β-catenin translocation to the nucleus for zebrafish bone regeneration. This research has potential to identify mechanism of bone growth and repair, leading to more suitable drugs for patients suffering with osteoporosis.Item Enhancement of osteoblast activity on nanostructured NiTi/hydroxyapatite coatings on additive manufactured NiTi metal implants by nanosecond pulsed laser sintering(Dove Press, 2018) Deng, Biwei; Bruzzaniti, Angela; Cheng, Gary J.; Biomedical Sciences and Comprehensive Care, School of DentistryBackground: The osteoinductive behaviors of nitinol (NiTi)-based metal implants for bone regeneration are largely dependent on their surface composition and topology. Continuous-mode laser sintering often results in complete melting of the materials and aggregation of particles, which lack control of heat transfer, as well as microstructural changes during sintering of the nanocomposite materials. Methods: In the current study, in situ direct laser deposition was used to additively manufacture three-dimensional NiTi structures from Ni and Ti powders. The mechanical property of NiTi has been shown to be similar to bone. Nanosecond pulsed laser sintering process was then utilized to generate a nanoporous composite surface with NiTi alloy and hydroxyapatite (HA) by ultrafast laser heating and cooling of Ni, Ti, and HA nanoparticles mixtures precoated on the 3D NiTi substrates; HA was added in order to improve the biocompatibility of the alloy. We then studied the underlying mechanism in the formation of NiTi/HA nanocomposite, and the synergistic effect of the sintered HA component and the nanoporous topology of the composite coating. In addition, we examined the activity of bone-forming osteoblasts on the NiTi/HA surfaces. For this, osteoblast cell morphology and various biomarkers were examined to evaluate cellular activity and function. Results: We found that the nanoscale porosity delivered by nanosecond pulsed laser sintering and the HA component positively contributed to osteoblast differentiation, as indicated by an increase in the expression of collagen and alkaline phosphatase, both of which are necessary for osteoblast mineralization. In addition, we observed topological complexities which appeared to boost the activity of osteoblasts, including an increase in actin cytoskeletal structures and adhesion structures. Conclusion: These findings demonstrate that the pulsed laser sintering method is an effective tool to generate biocompatible coatings in complex alloy-composite material systems with desired composition and topology. Our findings also provide a better understanding of the osteoinductive behavior of the sintered nanocomposite coatings for use in orthopedic and bone regeneration applications.Item Megakaryocyte and Osteoblast Interactions Modulate Bone Mass and Hematopoiesis(Mary Ann Liebert, 2018-05-15) Alvarez, Marta B.; Xu, LinLin; Childress, Paul J.; Maupin, Kevin A.; Mohamad, Safa F.; Chitteti, Brahmananda R.; Himes, Evan; Olivos, David J.; Cheng, Ying-Hua; Conway, Simon J.; Srour, Edward F.; Kacena, Melissa A.; Orthopaedic Surgery, School of MedicineEmerging evidence demonstrates that megakaryocytes (MK) play key roles in regulating skeletal homeostasis and hematopoiesis. To test if the loss of MK negatively impacts osteoblastogenesis and hematopoiesis, we generated conditional knockout mice where Mpl, the receptor for the main MK growth factor, thrombopoietin, was deleted specifically in MK (Mplf/f;PF4cre). Unexpectedly, at 12 weeks of age, these mice exhibited a 10-fold increase in platelets, a significant expansion of hematopoietic/mesenchymal precursors, and a remarkable 20-fold increase in femoral midshaft bone volume. We then investigated whether MK support hematopoietic stem cell (HSC) function through the interaction of MK with osteoblasts (OB). LSK cells (Lin-Sca1+CD117+, enriched HSC population) were co-cultured with OB+MK for 1 week (1wk OB+MK+LSK) or OB alone (1wk OB+LSK). A significant increase in colony-forming units was observed with cells from 1wk OB+MK cultures. Competitive repopulation studies demonstrated significantly higher engraftment in mice transplanted with cells from 1wk OB+MK+LSK cultures compared to 1wk OB+LSK or LSK cultured alone for 1 week. Furthermore, single-cell expression analysis of OB cultured±MK revealed adiponectin as the most significantly upregulated MK-induced gene, which is required for optimal long-term hematopoietic reconstitution. Understanding the interactions between MK, OB, and HSC can inform the development of novel treatments to enhance both HSC recovery following myelosuppressive injuries, as well as bone loss diseases, such as osteoporosis.Item No pain, no gain? The effects of pain-promoting neuropeptides and neurotrophins on fracture healing(Elsevier, 2020-02) Sun, Seungyup; Diggins, Nicklaus H.; Gunderson, Zachary J.; Fehrenbacher, Jill C.; White, Fletcher A.; Kacena, Melissa A.; Orthopaedic Surgery, School of MedicineNeuropeptides and neurotrophins are key regulators of peripheral nociceptive nerves and contribute to the induction, sensitization, and maintenance of pain. It is now known that these peptides also regulate non-neuronal tissues, including bone. Here, we review the effects of numerous neuropeptides and neurotrophins on fracture healing. The neuropeptides calcitonin-gene related peptide (CGRP), substance P (SP), vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase-activating peptide (PACAP) have varying effects on osteoclastic and osteoblastic activity. Ultimately, CGRP and SP both accelerate fracture healing, while VIP and PACAP seem to negatively impact healing. Unlike the aforementioned neuropeptides, the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have more uniform effects. Both factors upregulate osteoblastic activity, osteoclastic activity, and, in vivo, stimulate osteogenesis to promote fracture healing. Future research will need to clarify the exact mechanism by which the neuropeptides and neurotrophins influence fracture healing. Specifically, understanding the optimal expression patterns for these proteins in the fracture healing process may lead to therapies that can maximize their bone-healing capabilities and minimize their pain-promoting effects. Finally, further examination of protein-sequestering antibodies and/or small molecule agonists and antagonists may lead to new therapies that can decrease the rate of delayed union/nonunion outcomes and fracture-associated pain.Item Periosteum contains skeletal stem cells with high bone regenerative potential controlled by Periostin(Nature Publishing Group, 2018-02-22) Duchamp de Lageneste, Oriane; Julien, Anaïs; Abou-Khalil, Rana; Frangi, Giulia; Carvalho, Caroline; Cagnard, Nicolas; Cordier, Corinne; Conway, Simon J.; Colnot, Céline; Pediatrics, School of MedicineBone regeneration relies on the activation of skeletal stem cells (SSCs) that still remain poorly characterized. Here, we show that periosteum contains SSCs with high bone regenerative potential compared to bone marrow stromal cells/skeletal stem cells (BMSCs) in mice. Although periosteal cells (PCs) and BMSCs are derived from a common embryonic mesenchymal lineage, postnatally PCs exhibit greater clonogenicity, growth and differentiation capacity than BMSCs. During bone repair, PCs can efficiently contribute to cartilage and bone, and integrate long-term after transplantation. Molecular profiling uncovers genes encoding Periostin and other extracellular matrix molecules associated with the enhanced response to injury of PCs. Periostin gene deletion impairs PC functions and fracture consolidation. Periostin-deficient periosteum cannot reconstitute a pool of PCs after injury demonstrating the presence of SSCs within periosteum and the requirement of Periostin in maintaining this pool. Overall our results highlight the importance of analyzing periosteum and PCs to understand bone phenotypesItem Periostin Deficiency Increases Bone Damage and Impairs Injury Response to Fatigue Loading in Adult Mice(Public Library of Science, 2013-10-22) Bonnet, Nicolas; Gineyts, Evelyne; Ammann, Patrick; Conway, Simon J.; Garnero, Patrick; Ferrari, Serge; Pediatrics, School of MedicineBone damage removal and callus formation in response to fatigue loading are essential to prevent fractures. Periostin (Postn) is a matricellular protein that mediates adaptive response of cortical bone to loading. Whether and how periostin influences damage and the injury response to fatigue remains unknown. We investigated the skeletal response of Postn(-/-) and Postn(+/+) mice after fatigue stimulus by axial compression of their tibia. In Postn(+/+) mice, cracks number and surface (CsNb, CsS) increased 1h after fatigue, with a decrease in strength compared to non-fatigued tibia. At 15 days, CsNb had started to decline, while CtTV and CtBV increased in fatigued vs non-fatigued tibia, reflecting a woven bone response that was present in 75% of the fatigued bones. Cortical porosity and remodelling also prominently increased in the fatigued tibia of Postn(+/+) mice. At 30 days, paralleling a continuous removal of cortical damage, strength of the fatigued tibia was similar to the non-fatigue tibia. In Postn(-/-) mice, cracks were detectable even in the absence of fatigue, while the amount of collagen crosslinks and tissue hardness was decreased compared to Postn(+/+). Fatigue significantly increased CsNb and CsS in Postn(-/-), but was not associated with changes in CtTV and CtBV, as only 16% of the fatigued bones formed some woven bone. Cortical porosity and remodelling did not increase either after fatigue in Postn(-/-), and the level of damage remained high even after 30 days. As a result, strength remained compromised in Postn(-/-) mice. Contrary to Postn(+/+), which osteocytic lacunae showed a change in the degree of anisotropy (DA) after fatigue, Postn(-/-) showed no DA change. Hence periostin appears to influence bone materials properties, damage accumulation and repair, including local modeling/remodeling processes in response to fatigue. These observations suggest that the level of periostin expression could influence the propensity to fatigue fractures.