Design and evaluation of collagen-inspired mineral-hydrogel nanocomposites for bone regeneration.

Bone loss due to trauma and tumors remains a serious clinical concern. Due to limited availability and disease transmission risk with autografts and allografts, calcium phosphate bone fillers and growth factor-based substitute bone grafts are currently used in the clinic. However, substitute grafts lack bone regeneration potential when used without growth factors. When used along with the added growth factors, they lead to unwanted side effects such as uncontrolled bone growth. Collagen-based hydrogel grafts available on the market fail to provide structural guidance to native cells due to high water-solubility and faster degradation. To overcome these limitations, we employed bioinspired material design and fabricated three different hydrogels with structural features similar to native collagen at multiple length-scales. These hydrogels fabricated using polyionic complexation of oppositely charged natural polysaccharides exhibited multi-scale architecture mimicking nanoscale banding pattern, and microscale fibrous structure of native collagen. All three hydrogels promoted biomimetic apatite-like mineral deposition in vitro elucidating crystalline structure on the surface while amorphous calcium phosphate inside the hydrogels resulting in mineral-hydrogel nanocomposites. When evaluated in a non-load bearing critical size mouse calvaria defect model, chitosan - kappa carrageenan mineral-hydrogel nanocomposites enhanced bone regeneration without added growth factors compared to empty defect as well as widely used marketed collagen scaffolds. Histological assessment of the regenerated bone revealed improved healing and tissue remodeling with mineral-hydrogel nanocomposites. Overall, these collagen-inspired mineral-hydrogel nanocomposites showed multi-scale hierarchical structure and can potentially serve as promising bioactive hydrogel to promote bone regeneration. STATEMENT OF SIGNIFICANCE: Hydrogels, especially collagen, are widely used in bone tissue engineering. Collagen fibrils play arguably the most important role during natural bone development. Its multi-scale hierarchical structure to form fibers from fibrils and electrostatic charges enable mineral sequestration, nucleation, and growth. However, bulk collagen hydrogels exhibit limited bone regeneration and are mostly used as carriers for highly potent growth factors such as bone morphogenic protein-2, which increase the risk of uncontrolled bone growth. Thus, there is an unmet clinical need for a collagen-inspired biomaterial that can recreate structural hierarchy, mineral sequestration ability, and stimulate recruitment of host progenitor cells to facilitate bone regeneration. Here, we propose collagen-inspired bioactive mineral-hydrogel nanocomposites as a growth factor-free approach to guide and enhance bone regeneration.

Bottom-Up Self-assembled Hydrogel-Mineral Composites Regenerate Rabbit Ulna Defect without Added Growth Factors.

Hydrogel-based biomaterials have advanced bone tissue engineering approaches in the last decade, through their ability to serve as a carrier for potent growth factor, bone morphogenic protein-2 (BMP-2). However, biophysical properties of hydrogels such as multiscale structural hierarchy and bone extracellular matrix (ECM)-mimetic microarchitecture are underutilized while designing current bone grafts. Incorporation of these properties offers great potential to create a favorable biomimetic microenvironment to harness their regenerative potential. Here, we present our approach to fabricate collagen-inspired bioactive hydrogel scaffolds (referred to as "RegenMatrix") to guide and enhance bone regeneration in a rabbit ulna defect model through the mimicry of multiscale architecture of bone ECM, i.e., native collagen. Specifically, we employed polyelectrolyte complexation to promote bottom-up self-assembly of oppositely charged polysaccharides (chitosan and kappa-carrageenan) at multiple length scales forming fibrils, which further assemble into fibers. The self-assembly and bioinspired scaffold fabrication method resulted in robust cylindrical RegenMatrix with excellent retention of the multiscale architecture and uniform mineral deposition throughout the scaffolds. RegenMatrix, in both nonmineralized and mineralized forms, enhanced bone regeneration in the semiload-bearing ulna defect when compared to the empty defect. RegenMatrix also showed greater histocompatibility without any fibrous tissue formation. Collectively, the RegenMatrix developed in this study has a great potential as a bioactive bone graft without any added growth factors.

Effect of the Periapical "Inflammatory Plug" on Dental Pulp Regeneration: A Histologic In Vivo Study.

INTRODUCTION: In the current study, we investigate the effect of the inflammation occupying the apical foramen-a phenomenon we refer to as "inflammatory plug"-on the regenerative potential of a root canal therapy. METHODS: We performed root canal treatment (RCT) in 12 canine root canals while aseptically instrumenting the apex to a 0.5-mm-wide foramen and obturating the canals with the following materials: collagen sponge, platelet-rich fibrin, and blood clot (no material introduced). RESULTS: We were successful in maintaining the integrity of the periapical tissue in 8 of 12 RCTs. Injury to the periapical tissue occurred during the remaining 4 RCTs, which initiated inflammation accompanied by bone and dentin resorption. Our histologic analyses showed that the resulting inflammatory plug contained abundant M1 macrophages and was associated with an absence of intracanal cellular infiltration. On the contrary, noninflamed samples showed signs of repair, as indicated by the migration of periapical cells throughout the root canal. CONCLUSIONS: We conclude that controlling periapical inflammation is key while attempting to achieve dental pulp regeneration.

Controlling magnesium corrosion and degradation-regulating mineralization using matrix GLA protein.

Magnesium (Mg) alloys are embraced for their biodegradability and biocompatibility. However, Mg degrades spontaneously in the biological environment in vivo and in vitro, triggering deposition of calcium phosphate on the metal. Upon complete metal absorption, minerals remain in the tissue, which could lead to pathogenic calcification. Hence, our aims are to test the feasibility of matrix GLA protein (MGP) to locally inhibit Mg mineralization that is induced by metal alloy degradation. MGP is a small secretory protein that has been shown to inhibit soft tissue calcification. We exposed Mg to MGP, stably transfected into mammalian cells. Results showed that less calcium and phosphorous deposition on the Mg surface when MGP was present relative to when it was not. In the in vivo mouse intramuscular model conducted for 4 and 6 weeks, Mg rods were embedded in collagen scaffolds, seeded with cells overexpressing MGP. Microtomography, electron dispersive x-ray spectroscopy, and histology assessments revealed lower deposited mineral volume around Mg rods from the MGP group. Compared to other groups, higher volume loss after implantation was observed from the MGP group at both time points, indicating a higher corrosion rate without the protective mineral layer. This study is the first to our knowledge to demonstrate that local exposure to a biomolecule, such as MGP, can modulate the corrosion of Mg-based implants. These findings may have important implications for the future design of endovascular stents and orthopedic devices.

In vivo study of self-assembled alkylsilane coated degradable magnesium devices.

Magnesium (Mg) and its alloys are candidate materials for resorbable implantable devices, such as orthopedic devices or cardiovascular stents. Mg has a number advantages, including mechanical properties, light weight, its osteogenic effects and the fact that its degradation products are nontoxic and naturally present in the body. However, production of H2 gas during the corrosion reaction can cause formation of gas pockets at the implantation site, posing a barrier to clinical applications of Mg. It is therefore desirable to develop methods to control corrosion rate and gas pocket formation around the implants. Here we evaluate the potential of self-assembled multilayer alkylsilane (AS) coatings to control Mg device corrosion and formation of gas pockets in vivo and to assess effects of the AS coatings on the surrounding tissues in a subcutaneous mouse model over a 6 weeks' period. The coating significantly slowed down corrosion and gas pocket formation as evidenced by smaller gas pockets around the AS coated implants (ANOVA; p = 0.013) and decrease in the weight loss values (t test; p = 0.07). Importantly, the microCT and profilometry analyses demonstrated that the coating inhibited the pitting corrosion. Specifically, the roughness of the coated samples was ∼30% lower than uncoated specimen (p = 0.02). Histological assessment of the tissues under the implant revealed no inflammation or foreign body reaction. Overall, our results demonstrate the feasibility of use of the seld assembled AS coatings for reduction of gas pocket formation around the resorbable Mg devices. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 342-351, 2019.

Aquaporin 5 Interacts with Fluoride and Possibly Protects against Caries.

Aquaporins (AQP) are water channel proteins and the genes coding for AQP2, AQP5, and AQP6 are clustered in 12q13. Since AQP5 is expressed in serous acinar cells of salivary glands, we investigated its involvement in caries. DNA samples from 1,383 individuals from six groups were studied. Genotypes of eight single nucleotide polymorphisms covering the aquaporin locus were tested for association with caries experience. Interaction with genes involved in enamel formation was tested. The association between enamel microhardness at baseline, after creation of artificial caries lesion, and after exposure to fluoride and the genetic markers in AQP5 was tested. Finally, AQP5 expression in human whole saliva, after exposure to fluoride in a mammary gland cell line, which is known to express AQP5, and in Wistar rats was also verified. Nominal associations were found between caries experience and markers in the AQP5 locus. Since these associations suggested that AQP5 may be inhibited by levels of fluoride in the drinking water that cause fluorosis, we showed that fluoride levels above optimal levels change AQP5 expression in humans, cell lines, and rats. We have shown that AQP5 is involved in the pathogenesis of caries and likely interacts with fluoride.

Poly (glycerol sebacate) elastomer supports osteogenic phenotype for bone engineering applications.

For bone engineering, the optimal scaffolding material and composition has yet to be elucidated. In this study, we investigated poly (glycerol sebacate) (PGS), an elastomer known primarily for its soft tissue regeneration ability, as a suitable substrate to support osteo-precursor cell attachment and function. We synthesized PGS in the form of sheets where MC3T3-E1 cells were seeded in three different densities of 25,000, 50,000 and 100,000 cells mm(-3) and we investigated the cells/scaffold constructs for their cellular proliferation, matrix deposition, maturation, mineralization and their mechanical compression strength at 24 h and two and four weeks. MC3T3-E1 cells proliferated, synthesized a collagenous matrix and expressed osteogenic markers Runx2, bone sialoprotein and osteocalcin according to their initial seeding density on PGS. We conclude that PGS can support the osteoblastic phenotype in vitro and is a promising osteoconductive substrate for bone regeneration research and for future clinical translation.

Platelet rich plasma enhances osteoconductive properties of a hydroxyapatite-ß-tricalcium phosphate scaffold (Skelite) for late healing of critical size rabbit calvarial defects.

The use of platelet rich plasma (PRP) in bone repair remains highly controversial. In this work, we evaluated the effect of lyophilized PRP on bone regeneration when associated with a silicon stabilized hydroxyapatite tricalcium phosphate scaffold in a rabbit calvarial defect (Skelite). Critical defects were created in the calvaria of twenty-four rabbits. The periosteum was removed and the defects were either left empty or filled with allogeneic PRP gel; Skelite particles; Skelite and PRP gel. Four animals were killed after 4 weeks, 10 animals after 8 and 10 after 16 weeks. Specimens were processed for X-ray microtomography (µCT) and for resin embedded histology. µCT analysis revealed significant osteoid-like matrix and new bone deposition in PRP + Skelite group at both 8 and 16 weeks in respect to Skelite alone. Histologically, PRP + Skelite defects were highly cellular with more abundant osteoid deposition and more regular collagen fibres. Moreover, in vitro migration assays confirmed the chemotactic effect of PRP to endothelial and osteoprogenitor cells. We conclude that the addition of PRP influenced the local tissue microenvironment by providing key cryptic factors for regeneration, thereby enhancing progenitor cell recruitment, collagen and bone matrix deposition, and by creating a bridging interface between the scaffold and bone.

A platelet-rich plasma-based membrane as a periosteal substitute with enhanced osteogenic and angiogenic properties: a new concept for bone repair.

The periosteum plays a pivotal role during bone development and repair contributing to bone vascularization and osteoprogenitor cells source. We propose a periosteal substitute engineered using a platelet-rich plasma (PRP) membrane incorporating autologous bone marrow-derived mesenchymal stem cells (PRP/BMSC gel membrane) to be wrapped around an osteoconductive scaffold for regeneration of compromised bone defects. The PRP/BMSC gel membrane was optimized using different compositions for optimal release of vascular endothelial growth factor (VEGF) and platelet derived growth factor-BB (PDGF-BB). Survival and proliferation of cells in the PRP gel membrane with time were confirmed in addition to their osteogenic capacity. Furthermore, to evaluate the possible effects of the PRP/BMSC gel membrane on surrounding progenitor cells in the injury area, we found that the PRP gel membrane products could significantly induce the migration of human endothelial cells in vitro, and increased the expression of bone morphogenetic protein 2 in cultured BMSC. These cells also secreted significant amounts of soluble proangiogenic factors, such as PDGF-BB, VEGF, and interleukin-8 (IL-8). Finally, the functionality of the PRP/BMSC gel membrane periosteal substitute for bone regeneration was tested in vivo both in an ectopic mouse model as well as in a rabbit segmental bone defect model providing evidence of its capacity to biomimic a periosteal response enhancing bone regeneration.