Effect of 'in air' freezing on post-thaw recovery of Callithrix jacchus mesenchymal stromal cells and properties of 3D collagen-hydroxyapatite scaffolds.

Through enabling an efficient supply of cells and tissues in the health sector on demand, cryopreservation is increasingly becoming one of the mainstream technologies in rapid translation and commercialization of regenerative medicine research. Cryopreservation of tissue-engineered constructs (TECs) is an emerging trend that requires the development of practically competitive biobanking technologies. In our previous studies, we demonstrated that conventional slow-freezing using dimethyl sulfoxide (Me2SO) does not provide sufficient protection of mesenchymal stromal cells (MSCs) frozen in 3D collagen-hydroxyapatite scaffolds. After simple modifications to a cryopreservation protocol, we report on significantly improved cryopreservation of TECs. Porous 3D scaffolds were fabricated using freeze-drying of a mineralized collagen suspension and following chemical crosslinking. Amnion-derived MSCs from common marmoset monkey Callithrix jacchus were seeded onto scaffolds in static conditions. Cell-seeded scaffolds were subjected to 24 h pre-treatment with 100 mM sucrose and slow freezing in 10% Me2SO/20% FBS alone or supplemented with 300 mM sucrose. Scaffolds were frozen 'in air' and thawed using a two-step procedure. Diverse analytical methods were used for the interpretation of cryopreservation outcome for both cell-seeded and cell-free scaffolds. In both groups, cells exhibited their typical shape and well-preserved cell-cell and cell-matrix contacts after thawing. Moreover, viability test 24 h post-thaw demonstrated that application of sucrose in the cryoprotective solution preserves a significantly greater portion of sucrose-pretreated cells (more than 80%) in comparison to Me2SO alone (60%). No differences in overall protein structure and porosity of frozen scaffolds were revealed whereas their compressive stress was lower than in the control group. In conclusion, this approach holds promise for the cryopreservation of 'ready-to-use' TECs.

* Central Growth Factor Loaded Depots in Bone Tissue Engineering Scaffolds for Enhanced Cell Attraction.

Tissue engineering, the application of stem and progenitor cells in combination with an engineered extracellular matrix, is a promising strategy for bone regeneration. However, its success is limited by the lack of vascularization after implantation. The concept of in situ tissue engineering envisages the recruitment of cells necessary for tissue regeneration from the host environment foregoing ex vivo cell seeding of the scaffold. In this study, we developed a novel scaffold system for enhanced cell attraction, which is based on biomimetic mineralized collagen scaffolds equipped with a central biopolymer depot loaded with chemotactic agents. In humid milieu, as after implantation, the signaling factors are expected to slowly diffuse out of the central depot forming a gradient that stimulates directed cell migration toward the scaffold center. Heparin, hyaluronic acid, and alginate have been shown to be capable of depot formation. By using vascular endothelial growth factor (VEGF) as model factor, it was demonstrated that the release kinetics can be adjusted by varying the depot composition. While alginate and hyaluronic acid are able to reduce the initial burst and prolong the release of VEGF, the addition of heparin led to a much stronger retention that resulted in an almost linear release over 28 days. The biological activity of released VEGF was proven for all variants using an endothelial cell proliferation assay. Furthermore, migration experiments with endothelial cells revealed a relationship between the degree of VEGF retention and migration distance: cells invaded deepest in scaffolds containing a heparin-based depot indicating that the formation of a steep gradient is crucial for cell attraction. In conclusion, this novel in situ tissue engineering approach, specifically designed to recruit and accommodate endogenous cells upon implantation, appeared highly promising to stimulate cell invasion, which in turn would promote vascularization and finally new bone formation.

Design and Fabrication of Complex Scaffolds for Bone Defect Healing: Combined 3D Plotting of a Calcium Phosphate Cement and a Growth Factor-Loaded Hydrogel.

Additive manufacturing enables the fabrication of scaffolds with defined architecture. Versatile printing technologies such as extrusion-based 3D plotting allow in addition the incorporation of biological components increasing the capability to restore functional tissues. We have recently described the fabrication of calcium phosphate cement (CPC) scaffolds by 3D plotting of an oil-based CPC paste under mild conditions. In the present study, we have developed a strategy for growth factor loading based on multichannel plotting: a biphasic scaffold design was realised combining CPC with VEGF-laden, highly concentrated hydrogel strands. As hydrogel component, alginate and an alginate-gellan gum blend were evaluated; the blend exhibited a more favourable VEGF release profile and was chosen for biphasic scaffold fabrication. After plotting, two-step post-processing was performed for both, hydrogel crosslinking and CPC setting, which was shown to be compatible with both materials. Finally, a scaffold was designed and fabricated which can be applied for testing in a rat critical size femur defect. Optimization of CPC plotting enabled the fabrication of highly resolved structures with strand diameters of only 200 µm. Micro-computed tomography revealed a precise strand arrangement and an interconnected pore space within the biphasic scaffold even in swollen state of the hydrogel strands.

The effect of SDF-1α on low dose BMP-2 mediated bone regeneration by release from heparinized mineralized collagen type I matrix scaffolds in a murine critical size bone defect model.

The treatment of critical size bone defects represents a challenge. The growth factor bone morphogenetic protein 2 (BMP-2) is clinically established but has potentially adverse effects when used at high doses. The aim of this study was to evaluate if stromal derived factor-1 alpha (SDF-1α) and BMP-2 released from heparinized mineralized collagen type I matrix (MCM) scaffolds have a cumulative effect on bone regeneration. MCM scaffolds were functionalized with heparin, loaded with BMP-2 and/or SDF-1α and implanted into a murine critical size femoral bone defect (control group, low dose BMP-2 group, low dose BMP-2 + SDF-1α group, and high dose BMP-2 group). After 6 weeks, both the low dose BMP-2 + SDF-1α group (5.8 ± 0.6 mm³, p = 0.0479) and the high dose BMP-2 group (6.5 ± 0.7 mm³, p = 0.008) had a significantly increased regenerated bone volume compared to the control group (4.2 ± 0.5 mm³). There was a higher healing score in the low dose BMP-2 + SDF-1α group (median grade 8; Q1-Q3 7-9; p = 0.0357) than in the low dose BMP-2 group (7; Q1-Q3 5-9) histologically. This study showed that release of BMP-2 and SDF-1α from heparinized MCM scaffolds allows for the reduction of the applied BMP-2 concentration since SDF-1α seems to enhance the osteoinductive potential of BMP-2. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2126-2134, 2016.

Impact of acetylcholine and nicotine on human osteoclastogenesis in vitro.

Recent studies showed that the non-neuronal cholinergic system (NNCS) is taking part in bone metabolism. Most studies investigated its role in osteoblasts, but up to now, the involvement of the NNCS in human osteoclastogenesis remains relatively unclear. Thus, aim of the present study was to determine whether the application of acetylcholine (ACh, 10(−4) M), nicotine (10(−6) M), mineralized collagen membranes or brain derived neurotrophic factor (BDNF, 40 ng/mL) influences the mRNA regulation of molecular components of the NNCS and the neurotrophin family during osteoclastogenesis. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of young healthy donors (n = 8) and incubated with bone fragments and osteoclast differentiation media for 21 days. All the results are based on the measurement of RNA. Real-time RT-PCR analysis demonstrated a down-regulation of nicotinic acetylcholine receptor (nAChR) subunit α2 and muscarinic acetylcholine receptor (mAChR) M3by osteoclastogenesis while BDNF mRNA expression was not regulated. Application of ACh, nicotine, BDNF or collagen membranes did not affect osteoclastic differentiation.No regulation was detected for nAChR subunit α7, tropomyosin-related kinase receptor B (TrkB), and cholineacetyl transferase (ChAT). Taken together, we assume that the transcriptional level of osteoclastogenesis of healthy young humans is not regulated by BDNF, ACh, and nicotine. Thus, these drugs do not seem to worsen bone degradation and might therefore be suitable as modulators of bone substitution materials if having a positive effect on bone formation.

Heparinization of a biomimetic bone matrix: integration of heparin during matrix synthesis versus adsorptive post surface modification.

This study intended to evaluate a contemporary concept of scaffolding in bone tissue engineering in order to mimic functions of the extracellular matrix. The investigated approach considered the effect of the glycosaminoglycan heparin on structural and biological properties of a synthetic biomimetic bone graft material consisting of mineralized collagen. Two strategies for heparin functionalization were explored in order to receive a three-component bone substitute material. Heparin was either incorporated during matrix synthesis by mixing with collagen prior to simultaneous fibril reassembly and mineralization (in situ) or added to the matrix after fabrication (a posteriori). Both methods resulted in an incorporation of comparable amounts of heparin, though its distribution in the matrix varied as indicated by TOF-SIMS analyses, and a similar modulation of their protein binding properties. Differential scanning calorimetry revealed that the thermal stability and thereby the degree of crosslinking of the heparinized matrices was increased. However, in contrast to the a posteriori modification, the in situ integration of heparin led to considerable changes of morphology and composition of the matrix: a more open network of collagen fibers yielding a more porous surface and a reduced mineral content were observed. Cell culture experiments with human mesenchymal stem cells (hMSC) revealed a strong influence of the mode of heparin functionalization on cellular processes, as demonstrated for proliferation and osteogenic differentiation of hMSC. Our results indicate that not only heparin per se but also the way of its incorporation into a collagenous matrix determines the cell response. In conclusion, the a posteriori modification was beneficial to support adhesion, proliferation and differentiation of hMSC.

Heparin modification of a biomimetic bone matrix for controlled release of VEGF.

Bone regeneration using tissue engineered constructs requires strategies to effectively stimulate vascularization within such a construct that is crucial for its supply and integration with the host tissue. In this work, porous scaffolds of a collagen/hydroxyapatite nanocomposite were modified with heparin to generate biomimetic bone matrices which are able to release angiogenic factors in a controlled manner. Heparin was either integrated during material synthesis (in situ) or added to the scaffolds after their fabrication (post). Both approaches resulted in stable incorporation of heparin into the matrix of mineralized collagen. Investigations of binding and release of the vascular endothelial growth factor (VEGF-A165) loaded onto the scaffolds revealed an enhanced binding capacity as well as a sustained and nearly constant delivery of VEGF as result of both heparin modification methods. The release rate could be controlled by varying the quantity of incorporated heparin and the modification method. Although the biological activity of VEGF released after 7 days from the unmodified scaffolds was reduced in comparison to control VEGF, it was maintained after release from post or even enhanced after release from in situ modified scaffolds. In conclusion, the heparin-modified scaffolds of mineralized collagen exhibited favorable growth factor binding and release properties and may be beneficial to stimulate vascularization.

Quantification of calcium content in bone by using ToF-SIMS--a first approach.

The determination of the spatially resolved calcium distribution and concentration in bone is essential for the assessment of bone quality. It enables the diagnosis and elucidation of bone diseases, the course of bone remodelling and the assessment of bone quality at interfaces to implants. With time-of-flight secondary ion mass spectrometry (ToF-SIMS) the calcium distribution in bone cross sections is mapped semi-quantitatively with a lateral resolution of up to 1 µm. As standards for the calibration of the ToF-SIMS data calcium hydroxyapatite collagen scaffolds with different compositions were synthesized. The standards were characterised by loss of ignition, x-ray diffractometry (XRD) and x-ray photoelectron spectroscopy (XPS). The secondary ion count rate for calcium and the calcium content of the standards show a linear dependence. The obtained calibration curve is used for the quantification of the calcium content in the bone of rats. The calcium concentration within an animal model for osteoporosis induction is monitored. Exemplarily the calcium content of the bones was quantified by XPS for validation of the results. Furthermore a calcium mass image is compared with an XPS image to demonstrate the better lateral resolution of ToF-SIMS which advances the locally resolved quantification of the calcium content.