Sustained Delivery of Lactoferrin Using Poloxamer Gels for Local Bone Regeneration in a Rat Calvarial Defect Model

Lactoferrin (LF) is a multifunctional milk glycoprotein that promotes bone regeneration. Local delivery of LF at the bone defect site is a promising approach for enhancement of bone regeneration, but efficient systems for sustained local delivery are still largely missing. The aim of this study was to investigate the potential of the poloxamers for sustained delivery of LF to enhance local bone regeneration. The developed LF/poloxamer formulations were liquid at room temperature (20 °C) transforming to a sustained releasing gel depot at body temperature (37 °C). In vitro release studies demonstrated an initial burst release (~50%), followed by slower release of LF for up to 72 h. Poloxamer, with and without LF, increased osteoblast viability at 72 h (p < 0.05) compared to control, and the immune response from THP-1 cells was mild when compared to the suture material. In rat calvarial defects, the LF/poloxamer group had lower bone volume than the controls (p = 0.0435). No difference was observed in tissue mineral density and lower bone defect coverage scores (p = 0.0267) at 12 weeks after surgery. In conclusion, LF/poloxamer formulations support cell viability and do not induce an unfavourable immune response; however, LF delivery via the current formulation of LF200/poloxamer gel did not demonstrate enhanced bone regeneration and was not compatible with the rat calvarial defect model.

Bone Regeneration by Dedifferentiated Fat Cells Using Composite Sponge of Alfa-Tricalcium Phosphate and Gelatin in a Rat Calvarial Defect Model

Mechanical and resorbable scaffolds are in high demand for stem cell-based regenerative medicine, to treat refractory bone defects in craniofacial abnormalities and injuries. Multipotent progenitor cells, such as dedifferentiated fat (DFAT) cells, are prospective sources for regenerative therapies. Herein, we aimed to demonstrate that a composite gelatin sponge (α-TCP/GS) of alfa-tricalcium phosphate (α-TCP) mixed with gelatin scaffolds (GS), with/without DFATs, induced bone regeneration in a rat calvarial defect model in vivo. α-TCP/GS was prepared by mixing α-TCP and 2% GS using vacuum-heated methods. α-TCP/GS samples with/without DFATs were transplanted into the model. After 4 weeks of implantation, the samples were subjected to micro-computed tomography (μ-CT) and histological analysis. α-TCP/GS possessed adequate mechanical strength; α-TCP did not convert to hydroxyapatite upon contact with water, as determined by X-ray diffraction. Moreover, stable α-TCP/GS was formed by electrostatic interactions, and verified based on the infrared peak shifts. μ-CT analyses showed that bone formation was higher in the α-TCP/GS+ DFAT group than in the α-TCP/GS group. Therefore, the implantation of α-TCP/GS comprising DFAT cells enhanced bone regeneration and vascularization, demonstrating the potential for healing critical-sized bone defects.

Efficacy and safety of a novel hemostatic material, BoneStat, compared with Ostene and Bone Wax in a rat calvarial defect model

Hemostasis has critical significance during surgical procedures. Bone Wax has traditionally been commonly used for bone hemostasis despite well-documented undesirable side effects: hindering osteogenesis and induction of inflammatory reactions with consequent increase in infection rates. A later developed formulation, Ostene, offers an alternative to Bone Wax with lesser undesired effects. In this study, BoneStat, a newly developed bone hemostatic formulation comprising water-soluble alkylene oxide co-polymers, was evaluated for water solubility, hemostatic efficacy, ease of handling, bone healing efficacy, and inflammatory reactions compared with Bone Wax and Ostene in a rat calvarial defect model. More than 95% of BoneStat was dissolved in water within 48 h, as was Ostene, but not Bone Wax. The time to hemostasis using BoneStat was significantly faster than with Ostene or Bone Wax. BoneStat also improved ease of handling compared to Ostene or BoneWax. BoneStat- and Ostene-treated groups constantly showed better bone healing than with Bone Wax. The BoneStat and Ostene groups presented no evidence of chronic inflammation reaction contrary to Bone Wax. These results suggest improved hemostasis, ease of handling, non-hindering bone healing, and unnoticeable chronic inflammatory reactions with BoneStat. Thus, Bonestat is a useful and reliable formulation for mechanical hemostasis.

Endochondral Ossification Induced by Cell Transplantation of Endothelial Cells and Bone Marrow Stromal Cells with Copolymer Scaffold Using a Rat Calvarial Defect Model

It has been recently reported that, in a rat calvarial defect model, adding endothelial cells (ECs) to a culture of bone marrow stromal cells (BMSCs) significantly enhanced bone formation. The aim of this study is to further investigate the ossification process of newly formed osteoid and host response to the poly(L-lactide-co-1,5-dioxepan-2-one) [poly(LLA-co-DXO)] scaffolds based on previous research. Several different histological methods and a PCR Array were applied to evaluate newly formed osteoid after 8 weeks after implantation. Histological results showed osteoid formed in rat calvarial defects and endochondral ossification-related genes, such as dentin matrix acidic phosphoprotein 1 (Dmp1) and collagen type II, and alpha 1 (Col2a1) exhibited greater expression in the CO (implantation with BMSC/EC/Scaffold constructs) than the BMSC group (implantation with BMSC/Scaffold constructs) as demonstrated by PCR Array. It was important to notice that cartilage-like tissue formed in the pores of the copolymer scaffolds. In addition, multinucleated giant cells (MNGCs) were observed surrounding the scaffold fragments. It was concluded that the mechanism of ossification might be an endochondral ossification process when the copolymer scaffolds loaded with co-cultured ECs/BMSCs were implanted into rat calvarial defects. MNGCs were induced by the poly(LLA-co-DXO) scaffolds after implantation, and more specific in vivo studies are needed to gain a better understanding of host response to copolymer scaffolds.

Cathelicidin LL37 Promotes Osteogenic Differentiation in vitro and Bone Regeneration in vivo

Different types of biomaterials have been used to repair the defect of bony orbit. However, exposure and infections are still critical risks in clinical application. Biomaterials with characteristics of osteogenesis and antibiosis are needed for bone regeneration. In this study, we aimed to characterize the antimicrobial effects of cathelicidin-LL37 and to assess any impacts on osteogenic activity. Furthermore, we attempted to demonstrate the feasibility of LL37 as a potential strategy in the reconstruction of clinical bone defects. Human adipose-derived mesenchyme stem cells (hADSCs) were cultured with different concentrations of LL37 and the optimum concentration for osteogenesis was selected for further in vitro studies. We then evaluated the antibiotic properties of LL37 at the optimum osteogenic concentration. Finally, we estimated the efficiency of a PSeD/hADSCs/LL37 combined scaffold on reconstructing bone defects in the rat calvarial defect model. The osteogenic ability on hADSCs in vitro was shown to be dependent on the concentration of LL37 and reached a peak at 4 μg/ml. The optimum concentration of LL37 showed good antimicrobial properties against Escherichia coli and Staphylococcus anurans. The combination scaffold of PSeD/hADSCs/LL37 showed superior osteogenic properties compared to the PSeD/hADSCs, PSeD, and control groups scaffolds, indicating a strong bone reconstruction effect in the rat calvarial bone defect model. In Conclusion, LL37 was shown to promote osteogenic differentiation in vitro as well as antibacterial properties. The combination of PSeD/hADSCs/LL37 was advantageous in the rat calvarial defect reconstruction model, showing high potential in clinical bone regeneration.

Amine Modification of Calcium Phosphates by Low-Pressure CH4/N2/He Plasma for Bone Regeneration

Calcium phosphates are promising materials for artificial bone but lack of satisfied osteogenic ability on their surfaces. In the present study, we applied a low-pressure plasma technology to chemically (amine) modify the surface of calcium phosphates (hydroxyapatite or beta-tricalcium phosphate) using a CH4/N2/He plasm gas mixture to improve their osteogenic ability. The CH4/N2/He plasma treatment produced a thin, stable amine-rich carbon polymer on the surface of the calcium phosphates, and enhanced hydrophilicity, deep infiltration of cells into porous calcium phosphates, cell adhesion and osteogenic differentiation on the surface of calcium phosphates. In a rat calvarial defect model, the CH4/N2/He plasma treatment afforded calcium phosphates a significant higher bone regeneration capacity. Together, these results suggest that surface modification of calcium phosphates with CH4/N2/He plasma might improve osteogenic ability of calcium phosphates in vitro and in vivo.

Porous polyetheretherketone microcarriers fabricated via hydroxylation together with cell-derived mineralized extracellular matrix coatings promote cell expansion and bone regeneration

Porous microcarriers have aroused increasing attention recently by facilitating oxygen and nutrient transfer, supporting cell attachment and growth with sufficient cell seeding density. In this study, porous polyetheretherketone (PEEK) microcarriers coated with mineralized extracellular matrix (mECM), known for their chemical, mechanical and biological superiority, were developed for orthopedic applications. Porous PEEK microcarriers were derived from smooth microcarriers using a simple wet-chemistry strategy involving the reduction of carbonyl groups. This treatment simultaneously modified surface topology and chemical composition. Furthermore, the microstructure, protein absorption, cytotoxicity and bioactivity of the obtained porous microcarriers were investigated. The deposition of mECM through repeated recellularization and decellularization on the surface of porous MCs further promoted cell proliferation and osteogenic activity. Additionally, the mECM coated porous microcarriers exhibited excellent bone regeneration in a rat calvarial defect repair model in vivo, suggesting huge potential applications in bone tissue engineering.

An Ultrasonic Osteotomy Device Enhanced Post-osteotomy Bone Healing Beyond That With a Conventional Rotary Device in a Rat Calvarial Model

Ultrasonic osteotomy devices (UODs) have many clinical benefits; for example, they cause little damage to adjacent tissues in oral surgery. However, few reports have focused on bone healing with UODs. This study aimed to compare bone healing after osteotomy with UODs versus rotary osteotomy devices (RODs) in a rat calvarial defect model. Calvarial bone defects were made with a UOD on the right side and an ROD on the left side. Micro-CT analysis revealed that the bone volume was greater with a UOD than with an ROD at 2-3 weeks. In HE-stained sections and micro-CT images, the bone wound gap was closed earlier on the UOD side than on the ROD side. Bone thickness, the quantity of newly formed bone, and the number of osteocytes were greater on the UOD side than on the ROD side. Scanning electron microscopy (SEM) showed that UOD cuts had smoother surfaces than ROD cuts. Osteoblast-like cells harvested from bone chips cut by the UOD had greater proliferative activity than those harvested from ROD-cut bone chips. The use of a UOD may assist bone regeneration, presumably because of osteoblast activation by ultrasonic microvibration and UODs cause less damage to the bone than RODs.