The Use of Adipose Tissue-Derived Progenitors in Bone Tissue Engineering - a Review

2500 years ago, Hippocrates realized that bone can heal without scaring. The natural healing potential of bone is, however, restricted to small defects. Extended bone defects caused by trauma or during tumor resections still pose a huge problem in orthopedics and cranio-maxillofacial surgery. Bone tissue engineering strategies using stem cells, growth factors, and scaffolds could overcome the problems with the treatment of extended bone defects. In this review, we give a short overview on bone tissue engineering with emphasis on the use of adipose tissue-derived stem cells and small molecules

The bromodomain inhibitor N-methyl pyrrolidone reduced fat accumulation in an ovariectomized rat model

BACKGROUND: Weight gain is one of the consequences of estrogen deficiency and constitutes a major health problem. The present study highlights the effects of N-methyl pyrrolidone (NMP) on adipogenesis in osteoporosis induced by estrogen deficiency in an ovariectomized rat model. RESULTS: Ovariectomy resulted in body weight gain, increased femoral marrow adipocytes, and hypertrophic adipocytes in white adipose tissue, distorted serum leptin, and TNF-α and PPARγ levels. Treatment with NMP normalized these parameters similar to the control group. In vitro, NMP inhibited the differentiation of 3T3-L1 pre-adipocytes and hMSCs, indicating its anti-adipogenic effect. Moreover, PPARγ was significantly reduced with NMP treatment in in vivo and in vitro experiments. NMP inhibited BRD2 and BRD4 binding in an AlphaScreen assay, with an IC50 of 3 and 4 mM, respectively. The effect of NMP was consistent with its role as a bromodomain inhibitor. CONCLUSIONS: Our data indicates that NMP inhibits the adipogenic effect of estrogen deficiency at the level of PPARγ expression by BRD4 inhibition. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13148-016-0209-2) contains supplementary material, which is available to authorized users

Inhibition of osteoclast differentiation and bone resorption by N-methylpyrrolidone

Regulation of RANKL (receptor activator of nuclear factor κB ligand)-induced osteoclast differentiation is of current interest in the development of antiresorptive agents. Osteoclasts are multinucleated cells that play a crucial role in bone resorption. In this study, we investigated the effects of N-methylpyrrolidone (NMP) on the regulation of RANKL-induced osteoclastogenesis. NMP inhibited RANKL-induced tartrate-resistant acid phosphatase activity and the formation of tartrate-resistant acid phosphatase-positive multinucleated cells. The RANKL-induced expression of NFATc1 (nuclear factor of activated T cells, cytoplasmic 1) and c-Fos, which are key transcription factors for osteoclastogenesis, was also reduced by treatment with NMP. Furthermore, NMP induced disruption of the actin rings and decreased the mRNAs of cathepsin K and MMP-9 (matrix metalloproteinase-9), both involved in bone resorption. Taken together, these results suggest that NMP inhibits osteoclast differentiation and attenuates bone resorption. Therefore, NMP could prove useful for the treatment of osteoporosis or other bone diseases associated with excessive bone resorption

N-methyl pyrrolidone as a potent bone morphogenetic protein enhancer for bone tissue regeneration

In medicine N-methylpyrrolidone (NMP) has a long track record as constituent in FDA approved medical devices and thus can be considered as safe and biological inactive small chemical. In the present study we report on the newly discovered pharmaceutical properties of NMP as it enhances bone regeneration in a rabbit calvarial defect model in vivo. At the cellular level, the pharmaceutical effect of NMP was confirmed, in particular, in combination with BMP-2, as NMP increased early and late markers for maturation of preosteoblasts and human bone marrow derived stem cells in vitro. When we used the multipotent cell line C2C12 lacking autologous BMP expression, NMP alone had no effect on alkaline phosphatase activity, a marker for osteogenic transdifferentiation. Nevertheless, in combination with low BMP-2-doses alkaline phosphatase activity was increased more than 8 fold. Thus, the pharmaceutical NMP mode of action is that of an enhancer of BMP activity. The dependency of the effects of NMP on BMP was confirmed in preosteoblasts as noggin, an extracellular BMP-inhibitor, suppressed NMP-induced increase in early markers for osteoblast maturation in vitro. At the molecular level, NMP was shown to have no effect on the binding of BMP-2 to the ectodomain of the high affinity BMP receptor IA. However, NMP further increased the phosphorylation of p38 and Smad1,5,8 induced by BMP-2. Thus, the small chemical NMP enhances BMP activity by increasing the kinase activity of the BMP receptor complex for Smad1,5,8 and p38 and could be employed as a potent drug for bone tissue regeneration and engineering

Osteoconductivity of bone substitutes with filament-based microarchitectures: Influence of directionality, filament dimension, and distance

Additive manufacturing can be applied to produce personalized bone substitutes. At present, the major three-dimensional (3D) printing methodology relies on fila­ment extrusion. In bioprinting, the extruded filament consists mainly of hydrogels, in which growth factors and cells are embedded. In this study, we used a lithogra­phy-based 3D printing methodology to mimic filament-based microarchitectures by varying the filament dimension and the distance between the filaments. In the first set of scaffolds, all filaments were aligned toward bone ingrowth direction. In a second set of scaffolds, which were derived from the identical microarchitecture but tilted by 90°, only 50% of the filaments were in line with the bone ingrowth direction. Testing of all tricalcium phosphate-based constructs for osteoconduction and bone regeneration was performed in a rabbit calvarial defect model. The results revealed that if all filaments are in line with the direction of bone ingrowth, filament size and distance (0.40–1.25 mm) had no significant influence on defect bridging. Howev­er, with 50% of filaments aligned, osteoconductivity declined significantly with an increase in filament dimension and distance. Therefore, for filament-based 3D- or bio-printed bone substitutes, the distance between the filaments should be 0.40 to 0.50 mm irrespective of the direction of bone ingrowth or up to 0.83 mm if perfectly aligned to it

3D-Printed HA-Based Scaffolds for Bone Regeneration: Microporosity, Osteoconduction and Osteoclastic Resorption

Additive manufacturing enables the realization of the macro- and microarchitecture of bone substitutes. The macroarchitecture is determined by the bone defect and its shape makes the implant patient specific. The preset distribution of the 3D-printed material in the macroarchitecture defines the microarchitecture. At the lower scale, the nanoarchitecture of 3D-printed scaffolds is dependent on the post-processing methodology such as the sintering temperature. However, the role of microarchitecture and nanoarchitecture of scaffolds for osteoconduction is still elusive. To address these aspects in more detail, we produced lithography-based osteoconductive scaffolds from hydroxyapatite (HA) of identical macro- and microarchitecture and varied their nanoarchitecture, such as microporosity, by increasing the maximum sintering temperatures from 1100 to 1400 °C. The different scaffold types were characterized for microporosity, compression strength, and nanoarchitecture. The in vivo results, based on a rabbit calvarial defect model showed that bony ingrowth, as a measure of osteoconduction, was independent from scaffold's microporosity. The same applies to in vitro osteoclastic resorbability, since on all tested scaffold types, osteoclasts formed on their surfaces and resorption pits upon exposure to mature osteoclasts were visible. Thus, for wide-open porous HA-based scaffolds, a low degree of microporosity and high mechanical strength yield optimal osteoconduction and creeping substitution. Based on our study, non-unions, the major complication during demanding bone regeneration procedures, could be prevented

Optimizing Filament-Based TCP Scaffold Design for Osteoconduction and Bone Augmentation: Insights from In Vivo Rabbit Models

Additive manufacturing has emerged as a transformative tool in biomedical engineering, offering precise control over scaffold design for bone tissue engineering and regenerative medicine. While much attention has been focused on optimizing pore-based scaffold architectures, filament-based microarchitectures remain relatively understudied, despite the fact that the majority of 3D-printers generate filament-based structures. Here, we investigated the influence of filament characteristics on bone regeneration outcomes using a lithography-based additive manufacturing approach. Three distinct filament-based scaffolds (Fil050, Fil083, and Fil125) identical in macroporosity and transparency, crafted from tri-calcium phosphate (TCP) with varying filament thicknesses and distance, were evaluated in a rabbit model of bone augmentation and non-critical calvarial defect. Additionally, two scaffold types differing in filament directionality (Fil and FilG) were compared to elucidate optimal design parameters. Distance of bone ingrowth and percentage of regenerated area within scaffolds were measured by histomorphometric analysis. Our findings reveal filaments of 0.50 mm as the most effective filament-based scaffold, demonstrating superior bone ingrowth and bony regenerated area compared to larger size filament (i.e., 0.83 mm and 1.25 mm scaffolds). Optimized directionality of filaments can overcome the reduced performance of larger filaments. This study advances our understanding of microarchitecture’s role in bone tissue engineering and holds significant implications for clinical practice, paving the way for the development of highly tailored, patient-specific bone substitutes with enhanced efficacy

Functionalization of Ceramic Scaffolds with Exosomes from Bone Marrow Mesenchymal Stromal Cells for Bone Tissue Engineering

The functionalization of bone substitutes with exosomes appears to be a promising technique to enhance bone tissue formation. This study investigates the potential of exosomes derived from bone marrow mesenchymal stromal cells (BMSCs) to improve bone healing and bone augmentation when incorporated into wide open-porous 3D-printed ceramic Gyroid scaffolds. We demonstrated the multipotent characteristics of BMSCs and characterized the extracted exosomes using nanoparticle tracking analysis and proteomic profiling. Through cell culture experimentation, we demonstrated that BMSC-derived exosomes possess the ability to attract cells and significantly facilitate their differentiation into the osteogenic lineage. Furthermore, we observed that scaffold architecture influences exosome release kinetics, with Gyroid scaffolds exhibiting slower release rates compared to Lattice scaffolds. Nevertheless, in vivo implantation did not show increased bone ingrowth in scaffolds loaded with exosomes, suggesting that the scaffold microarchitecture and material were already optimized for osteoconduction and bone augmentation. These findings highlight the lack of understanding about the optimal delivery of exosomes for osteoconduction and bone augmentation by advanced ceramic scaffolds

TPMS Microarchitectures for Vertical Bone Augmentation and Osteoconduction: An In Vivo Study

Triply periodic minimal surface microarchitectures (TPMS) were developed by mathematicians and evolved in all kingdoms of living organisms. Renowned for their lightweight yet robust attributes, TPMS structures find application in diverse fields, such as the construction of satellites, aircrafts, and electric vehicles. Moreover, these microarchitectures, despite their intricate geometric patterns, demonstrate potential for application as bone substitutes, despite the inherent gothic style of natural bone microarchitecture. Here, we produced three TPMS microarchitectures, D-diamond, G-gyroid, and P-primitive, by 3D printing from hydroxyapatite. We explored their mechanical characterization and, further, implanted them to study their bone augmentation and osteoconduction potential. In terms of strength, the D-diamond and G-gyroid performed significantly better than the P-primitive. In a calvarial defect model and a calvarial bone augmentation model, where osteoconduction is determined as the extent of bony bridging of the defect and bone augmentation as the maximal vertical bone ingrowth, the G-gyroid performed significantly better than the P-primitive. No significant difference in performance was observed between the G-gyroid and D-diamond. Since, in real life, the treatment of bone deficiencies in patients comprises elements of defect bridging and bone augmentation, ceramic scaffolds with D-diamond and G-gyroid microarchitectures appear as the best choice for a TPMS-based scaffold in bone tissue engineering