Sven Knaack

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.

Green bioprinting: Fabrication of photosynthetic algae-laden hydrogel scaffolds for biotechnological and medical applications

Embedding of mammalian cells into hydrogel scaffolds of predesigned architecture by rapid prototyping technologies has been intensively investigated with focus on tissue engineering and organ printing. The study demonstrates that such methods can be extended to cells originating from the plant kingdom. By using 3D plotting, microalgae of the species Chlamydomonas reinhardtii were embedded in 3D alginate‐based scaffolds. The algae survived the plotting process and were able to grow within the hydrogel matrix. Under illumination, the cell number increased as indicated by microscopic analyses and determination of the chlorophyll content which increased 16‐fold within 12 days of cultivation. Photosynthetic activity was evidenced by measurement of oxygen release: within the first 24 h, an oxygen production rate of 0.05 mg L−1 h−1 was detected which rapidly increased during further cultivation (0.25 mg L−1 h−1 between 24 and 48 h). Furthermore, multichannel plotting was applied to combine human cells and microalgae within one scaffold in a spatially organized manner and hence, to establish a patterned coculture system in which the algae are cultivated in close vicinity to human cells. This might encourage the development of new therapeutic concepts based on the delivery of oxygen or secondary metabolites as therapeutic agents by microalgae.

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.

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 MODULATES OSTEOGENIC AND ANGIOGENIC CELL RESPONSE IN VITRO

In this study, the effect of heparin-modified collagen type I/hydroxyapatite (HA) nanocomposites on key processes of bone regeneration - osteogenesis and angiogenesis - was characterised in vitro. Two approaches were applied for heparin modification: it was either integrated during material synthesis (in situ) or added to the porous scaffolds after their fabrication (post). Cultivation of human bone marrow-derived stromal cells (hBMSC), in heparin-modified versus heparin-free scaffolds, revealed a positive effect of the heparin modification on their proliferation and osteogenic differentiation. The amount of heparin rather than the method used for modification influenced the cell response favouring proliferation at smaller amount (30 mg/g collagen) and differentiation at larger amount (150 mg/g collagen). A co-culture of human umbilical vein endothelial cells (HUVEC) and osteogenically induced hBMSC was applied for in vitro angiogenesis studies. Pre-vascular networks have formed in the porous structure of scaffolds which were not modified with heparin or modified with a low amount of heparin (30 mg/g collagen). The modification with higher heparin quantities seemed to inhibit tubule formation. Pre-loading of the scaffolds with VEGF influenced formation and stability of the pre-vascular structures depending on the presence of heparin: In heparin-free scaffolds, induction of tubule formation and sprouting was more pronounced whereas heparin-modified scaffolds seemed to promote stabilisation of the pre-vascular structures. In conclusion, the modification of mineralised collagen with heparin by using both approaches was found to modulate cellular processes essential for bone regeneration; the amount of heparin has been identified to be crucial to direct cell responses.

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.