Moderne Tissue Engineering Konzepte für die Knochendefektheilung: Funktionalisierung von Scaffolds auf Basis von mineralisiertem Kollagen zur Stimulation von Angiogenese und Osteogenese

Der Bedarf an modernen Konzepten der regenerativen Medizin für die Therapie von Knochensubstanzdefekten steigt zunehmend angesichts unserer sich demographisch wandeln-den Gesellschaft und der damit einhergehenden, steigenden Zahl altersrelevanter orthopädisch-unfallchirurgischer Erkrankungen. Die wissenschaftlichen Erkenntnisse der letzten Jahrzehnte erlauben es, grundlegende biologische Prozesse der Knochenregeneration nicht nur besser zu verstehen, sondern diese auch durch gezielte Einflussnahme zu nutzen. Auf Grundlage dieser Erkenntnisse fokussiert die Forschung die Entwicklung moderner bioaktiver Biomaterialien mit dem intrinsischen Potenzial, die körpereigene Geweberegeneration lokal im Defektbereich – in situ – zu stimulieren. Die Stimulation zellulärer Regenerationsmechanismen kann entweder direkt durch Zell-Material-Interaktion induziert werden (in situ Regeneration), oder durch chemotaktische Attraktion von Zellen mit regenerativem Potenzial aus dem umliegenden Gewebe, welche ihrerseits die Geweberegeneration induzieren (in situ Tissue Engineering). Ohne extrakorporale Besiedlung der Scaffolds und deren in vitro-Kultivierung vor der Implantation umgehen diese in situ-Strategien mehrere Limitationen und Herausforderungen des klassischen Tissue Engineering-Konzepts. Dem in situ-Konzept folgend wurden im Rahmen dieser Dissertation zwei Strategien zur gezielten Funktionalisierung eines Knochenersatzmaterials aus mineralisiertem Kollagen unter-sucht: I) Zum einen wurde mineralisiertes Kollagen mit dem osteoanabol wirksamen Erdalkalimetall Strontium modifiziert, um lokal die Osteogenese zu stimulieren. II) Zum anderen wurden poröse Scaffolds aus mineralisiertem Kollagen mit einem zentralen Depot funktionalisiert, welches mit einem Wirkstoffgemisch beladen wurde, welches aus dem Sekretom Hypoxie-konditionierter hBMSC (HCM) generiert wurde. Durch gezielte Attraktion von Zellen mit regenerativem Potenzial und gleichzeitiger Stimulation der Vaskularisierung soll dieses Scaffoldsystem gezielt die Knochendefektheilung induzieren. Für die Strontium-Modifikation wurde während der Scaffoldsynthese das Calcium der Mineralphase sukzessive durch Strontium substituiert und die hergestellten Scaffolds eingehend materialwissenschaftlich charakterisiert (Publikation 2.1; Quade et al., 2018a). Die simultane Fibrillierung und Mineralisierung von Kollagen führte zur Bildung von Nanokompositen, wobei die Mineralphasen von nanokristallinem Hydroxylapatit (Sr0), über schwach kristalline Strontium-reiche Phasen zu einer gemischten Mineralphase (Sr100) aus amorphem Strontiumphosphat und hochkristallinem Strontiumhydroxylapatit verschoben wurde. Freisetzungsversu-che über 28 Tage zeigten, dass die getesteten Varianten Sr50 und Sr100 anhaltend Sr2+-Ionen in einem Konzentrationsbereich freisetzten, in dem sowohl die Knochenneubildung stimuliert, als auch die zelluläre Knochenresorption gehemmt wird. In vitro zeigte sich der osteoanabole Effekt Strontium-modifizierter mineralisierter Kollagenscaffolds durch eine signifikant gesteigerte Proliferation und osteogene Differenzierung von hBMSC. In vivo – als Knochenersatzmaterial im murinen segmentalen FemurdefektModell – zeigten Strontium-modifizierte Scaffolds aus mineralisiertem Kollagen zwar ein tendenziell gesteigertes Knochenvolumen mit erhöhter Osteoblastenzahl, signifikant weniger Osteoklasten und signifikant gesteigerter Vaskularisierung, jedoch war der Effekt verhältnismäßig schwach und allein nicht ausreichend für eine knöcherne Überbrückung des Defektbereiches (Publikation 2.2; Quade et al., 2020a). Durch Kombination von Sr-Modifikation und BMP-2-Funktionalisierung konnte die Qualität des neugebildeten Knochens signifikant gesteigert werden. Um das Konzept des neuartigen Wirkstoffdepot-basierten Scaffoldsystems zu testen, wurde das zentrale Depot zunächst mit dem Modell-Wachstumsfaktor VEGF beladen. Der Einsatz der Biopolymere Alginat, Hyaluronsäure und Heparin als Depotbildner erlaubt die Modulation der Wirkstofffreisetzung. Während die Hydrogele Alginat und Hyaluronsäure dabei als physikalischen Barriere fungieren, ermöglichte die hohe ionische Bindungsaffinität von Heparin und VEGF dessen nahezu lineare Freisetzung über den Versuchszeitraum von 28 Tagen. Im Migrationsversuch bewirkte die retardierte VEGF-Freisetzung und damit die Stabilisierung des Wirkstoffgradienten die gerichtete Migration von HDMEC in den Scaffold. Je verzögerter die VEGF-Freisetzung – und damit je steiler der Wirkstoffgradient – desto tiefer migrierten HDMEC in die Scaffolds (Publikation 2.3; Quade et al., 2017a). Entscheidend für eine effiziente Knochenregeneration ist sowohl die Attraktion von Zellen mit regenerativem Potenzial, als auch die Stimulation der Vaskularisierung, um den Gasaustausch, die Nähstoffversorgung und den Abtransport metabolischer Nebenprodukte der Zel-len im Defektbereich zu gewährleisten. Im Sinne des in situ Tissue Engineering-Konzepts wurde das zentrale Wirkstoffdepot mit einem natürliche Wirkstoffgemisch, welches aus dem Sekretom Hypoxie-konditionierter hBMSC (HCM) gewonnen wurde, beladen (Publikation 2.4; (Quade et al., 2020b). Unter hypoxischen Bedingungen sezernieren hBMSC einen Wirkstoffcocktail, der unter anderem Wachstumsfaktoren, Chemokine, Hormone und Exosomen enthält und ein starkes angiogenes und chemotaktisches Potenzial gegenüber hBMSC zeigt. Um das Wirkstoffdepot möglichst effizient zu beladen, wurde zum einen die Wirkstoffausbeute von HCM durch Anpassung der Herstellungsparameter optimiert. Gemessen am Wachstumsfaktor VEGF konnte so die Ausbeute bis zu 100-fach gesteigert werden. Des Weiteren konnte durch Dialyse, Gefriertrocknung und Resuspension mit dem Depot-bildenden Biopolymer zusätzlich eine bis zu 50-fache Konzentrierung des Wirkstoffgemisches erreicht wer-den ohne Verlust der Bioaktivität. Mit steigender HCM-Konzentration im Depot konnte sowohl eine tiefere Migration von hBMSC, als auch eine Verbesserung der Angiogenese erzielt wer-den. Zusätzlich bewirkte die retardierte HCM-Freisetzung aus Alginat-basierten Depots eine signifikante Steigerung von Länge, Dichte und Einsprosstiefe prävaskulärer Strukturen. Zwar ist die Charakterisierung und standardisierte Herstellung des natürlichen HCM-basierten Wirkstoffgemisches eine Herausforderung, jedoch ist davon auszugehen, dass dessen be-deutendes therapeutisches Potenzial gerade durch die Komplexität der sezernierten Faktoren und deren synergistisches Zusammenspiel bedingt wird. Die Standardisierung der HCM-basierten Wirkstoffherstellung, sowie eine bessere Quantifizierung und Charakterisierung der sezernierten Proteine sollte in zukünftigen Studien forciert werden. Beide Strategien, die im Rahmen dieser Dissertation untersucht wurden, haben das Potenzial, als zellfreie „ready-to-use“-Knochenersatzmaterialien zu erschwinglichen Kosten, bei geringerer regulatorischer Komplexität und mit gleichbleibend hoher Qualität produziert zu wer-den. Während Strontium-modifiziertes mineralisiertes Kollagen allein in vivo nur ein schwaches osteoinduktives Potenzial zeigte, konnte die Qualität und Festigkeit des neugebildeten Knochengewebes in Kombination mit BMP-2 signifikant verbessert werden. Als leicht-osteogenes Biomaterial könnte dieses zur Unterstützung etablierter therapeutischer Konzepte eingesetzt werden – vor allem bei Patienten mit systemischen Knochenerkrankungen wie beispielsweise Osteoporose. Das komplexere Depot-basierte Scaffoldsystem hingegen hat ein großes Potenzial für die klinische Translation. Zum einen kann das Depot je nach Bedarf mit verschiedensten Wirkstoffen beladen werden, deren Freisetzung in Abhängigkeit des Depot-bildenden Biopolymers manipuliert werden kann. Beladen mit dem natürlichen HCM-basierten Wirkstoffgemisch zeigte das Scaffoldsystem ein beeindruckendes chemotaktisches und angiogenes Potenzial. Gegenüber etablierten rhBMP-2-Applikationen, stellt dieser in situ Tissue Engineering-Ansatz damit eine vielversprechende Alternative für die Knochen-defekt-Therapie dar, bei gleichzeitig deutlich reduzierten Kosten und Nebenwirkungen. Zu-künftige in vivo-Studien im Großtiermodell sollten das regenerative Potenzial des Depot-basierten Scaffoldsystems umfassend verifizieren.Our demographically changing society causes a rising number of age-related orthopaedic and trauma surgical diseases. Modern approaches following the concept of regenerative medicine are needed for the therapeutical treatment of bone defects. Scientific findings of the last decades not only allow for a better understanding of fundamental biological processes in the field of bone regeneration, but also to use this knowledge for effective therapeutic concepts. Therefore, research is focusing on the development of modern bioactive biomaterials with the intrinsic potential to locally stimulate the body's own regeneration capacity - in situ. The stimulation of tissue regeneration can either be induced directly by cell-material interaction (in situ regeneration), or by chemotactic attraction of cells with regenerative potential from the surrounding tissue, which would in turn induce local tissue regeneration (in situ tissue engineering). Since these in situ strategies forgo the extracorporeal seeding and in vitro cultivation of scaffolds prior implantation, several limitations and challenges of the classical tissue engineering concept can be circumvented. Within the scope of this dissertation two strategies were investigated. Following the in situ concept, scaffolds based on mineralized collagen were specifically functionalized in order to locally induce bone defect healing: I) On the one hand, mineralised collagen was modified with strontium to locally stimulate osteogenesis. II) On the other hand, porous scaffolds of mineralised collagen were functionalised with a central depot loaded with a cocktail of signalling factors generated from the secretome of hypoxia-conditioned hBMSC (HCM). By specifically attracting cells with regenerative potential and simultaneously stimulating vascularisation, this scaffold-system could actively induce bone defect healing. For the strontium modification, the calcium of the mineral phase was successively substituted by strontium during the scaffold synthesis. The generated scaffolds were characterised in detail from a material science perspective (publication 2.1; Quade et al.et al., 2018a). In all tested approaches simultaneous collagen fibrillation and mineralisation led to the formation of nanocomposites. With rising strontium substitution, the mineral phases shifted from nanocrystalline hydroxylapatite (Sr0), via weakly crystalline strontium-rich phases to a mixed mineral phase of amorphous strontium phosphate and highly crystalline strontium hydroxylapatite (Sr100). Release experiments showed that the scaffold variants Sr50 and Sr100 released Sr2+-ions continuously over 28 days in a range, which is known to exploit the dual effect of strontium by simultaneously promoting proliferation and osteogenic differentiation as well as inhibiting the osteoclastic bone resorption without impairing the osteoclastogenesis. In vitro, the osteoanabolic effect of strontium-modified mineralised collagen scaffolds was demonstrated by significantly increased proliferation and osteogenic differentiation of hBMSC. In vivo - in the murine segmental femoral defect model - strontium-modified scaffolds made of mineralised collagen showed a tendency to increase bone volume with an increased number of osteoblasts, significantly reduced osteoclasts and significantly increased vascularisation. However, the effect was relatively weak and not sufficient to cause a bridging of the defect area (publication 2.2; Quade et al., 2020a). By combining Sr modification and BMP-2 functionalisation, the quality of the newly formed bone was significantly improved. To test the concept of the novel depot-based scaffold system, the central depot was loaded with the model growth factor VEGF. The use of the biopolymers alginate, hyaluronic acid and heparin as depot-forming agents allowed the modulation of drug release. While the hydrogels alginate and hyaluronic acid act as a physical barrier, the high ionic binding affinity of heparin and VEGF facilitated an almost linear VEGF-release over the experimental period of 28 days. In migration experiments, the retarded VEGF release and thus the stabilisation of the VEGF- gradient caused the directed migration of HDMEC into the scaffolds. The slower the VEGF release - and thus the steeper the drug gradient - the deeper HDMEC migrated into the scaffolds (publication 2.3; Quade et al., 2017). Crucial for an efficient bone regeneration is both the attraction of cells with regenerative potential and the stimulation of vascularisation to ensure gas exchange, nutrient supply and removal of metabolic by-products in the defect area. In line with the in situ tissue engineering concept, the central depot was loaded with a natural factor mix obtained from the secretome of hypoxia-conditioned hBMSC (HCM) (publication 2.4; (Quade et al., 2020b). Under hypoxic conditions, hBMSC secrete a cocktail of active substances that contains, among others, growth factors, chemokines, hormones and exosomes. This factor mix shows a strong angiogenic potential and is highly chemo-attractive to hBMSC. In order to load the scaffold depot as efficiently as possible, the signalling factor-yield of HCM was optimised by adjusting the cultivation settings for HCM-generation. Measured by VEGF as a model growth factor, the yield was increased up to 100 times. In addition, dialysis, freeze-drying and resuspension with the depot-forming biopolymer made it possible to achieve another 50-fold concentration without loss of bioactivity. With increasing HCM-concentration in the depot, both a deeper migration of hBMSC and an improvement in angiogenesis could be achieved. In addition, the retarded release of HCM from alginate-based depots resulted in a significant increase in length, density and sprouting depth of prevascular structures. Although the characterisation and standardised production of the natural HCM-based signalling factor cocktail is challenging, it can be assumed that its significant therapeutic potential relies particularly on that complexity of the secreted factors and their synergistic interaction. The standardized production of HCM-derived signalling factor cocktails, as well as a better quantification and characterisation of the secreted proteins should be focused by future studies. Both strategies investigated in this dissertation have the potential to be produced as cell-free 'ready-to-use' bone substitute materials at affordable costs, with less regulatory complexity and with consistently high quality. While strontium-modified mineralised collagen alone showed only a weak osteoinductive potential in vivo, the quality and strength of the newly formed bone tissue was significantly improved in combination with BMP-2. This light-osteogenic biomaterial could be used to support established therapeutic concepts - especially in patients with systemic bone diseases such as osteoporosis. The more complex depot-based scaffold system on the other hand has great potential for clinical translation. Depending on the application, the depot can be loaded with a wide variety of active substances – their release kinetics in turn can be manipulated depending on the depot-forming biopolymer. Loaded with the natural HCM-derived cocktail of signalling molecules, the scaffold system showed an impressive chemotactic and angiogenic potential. Compared to established rhBMP-2 applications, this in situ tissue engineering approach represents a promising alternative for bone defect therapy, at significantly reduced costs and side effects. Future in vivo studies in large animal models should verify the regenerative potential of the herewith developed depot-based scaffold system

Chemotactic and Angiogenic Potential of Mineralized Collagen Scaffolds Functionalized with Naturally Occurring Bioactive Factor Mixtures to Stimulate Bone Regeneration

To develop cost-effective and efficient bone substitutes for improved regeneration of bone defects, heparin-modified mineralized collagen scaffolds were functionalized with concentrated, naturally occurring bioactive factor mixtures derived from adipose tissue, platelet-rich plasma and conditioned medium from a hypoxia-treated human bone marrow-derived mesenchymal stem cell line. Besides the analysis of the release kinetics of functionalized scaffolds, the bioactivity of the released bioactive factors was tested with regard to chemotaxis and angiogenic tube formation. Additionally, functionalized scaffolds were seeded with human bone marrow-derived mesenchymal stromal cells (hBM-MSC) and their osteogenic and angiogenic potential was investigated. The release of bioactive factors from the scaffolds was highest within the first 3 days. Bioactivity of the released factors could be confirmed for all bioactive factor mixtures by successful chemoattraction of hBM-MSC in a transwell assay as well as by the formation of prevascular structures in a 2D co-culture system of hBM-MSC and human umbilical vein endothelial cells. The cells seeded directly onto the functionalized scaffolds were able to express osteogenic markers and form tubular networks. In conclusion, heparin-modified mineralized collagen scaffolds could be successfully functionalized with naturally occurring bioactive factor mixtures promoting cell migration and vascularization

Cell spheroids are as effective as single cells suspensions in the treatment of critical-sized bone defects

Background!#!Due to their multilineage potential and high proliferation rate, mesenchymal stem cells (MSC) indicate a sufficient alternative in regenerative medicine. In comparison to the commonly used 2-dimensional culturing method, culturing cells as spheroids stimulates the cell-cell communication and mimics the in vivo milieu more accurately, resulting in an enhanced regenerative potential. To investigate the osteoregenerative potential of MSC spheroids in comparison to MSC suspensions, cell-loaded fibrin gels were implanted into murine critical-sized femoral bone defects.!##!Methods!#!After harvesting MSCs from 4 healthy human donors and preculturing and immobilizing them in fibrin gel, cells were implanted into 2 mm murine femoral defects and stabilized with an external fixator. Therefore, 26 14- to 15-week-old nu/nu NOD/SCID nude mice were randomized into 2 groups (MSC spheroids, MSC suspensions) and observed for 6 weeks. Subsequently, micro-computed tomography scans were performed to analyze regenerated bone volume and bone mineral density. Additionally, histological analysis, evaluating the number of osteoblasts, osteoclasts and vessels at the defect side, were performed. Statistical analyzation was performed by using the Student's t-test and, the Mann-Whitney test. The level of significance was set at p = 0.05.!##!Results!#!μCT-analysis revealed a significantly higher bone mineral density of the MSC spheroid group compared to the MSC suspension group. However, regenerated bone volume of the defect side was comparable between both groups. Furthermore, no significant differences in histological analysis between both groups could be shown.!##!Conclusion!#!Our in vivo results reveal that the osteo-regenerative potential of MSC spheroids is similar to MSC suspensions

Influence of regioselectively sulfated cellulose on in vitro vascularization of biomimetic bone matrices

Vascularization is essential for the regeneration of bone tissue within composite material. We measured the effect of regioselectively modified cellulose/hemicellulose as an additive for porous scaffolds of collagen/hydroxyapatite nanocomposite on the tubule formation of human vascular endothelial cells. Using a coculture of endothelial cells and fibroblasts, endothelial cells formed a network of tubules within an incubation time of 14 to 24 days. A cellulose sulfate with irregular sulfation pattern along the polysaccharide backbone (13-TACS-01) led to an additional increase in vascular endothelial growth factor (VEGF)-induced tubule formation, as observed in an in vitro angiogenesis assays. In contrast with structurally different heparin, these cellulose sulfates have no apparent affinity to VEGF. Their impact on endothelial function may possibly be due to interactions with cell surface receptors/soluble factors not yet defined

Investigation of strontium transport and strontium quantification in cortical rat bone by time-of-flight secondary ion mass spectrometry

Next-generation bone implants will be functionalized with drugs for stimulating bone growth. Modelling of drug release by such functionalized biomaterials and drug dispersion into bone can be used as predicting tool for biomaterials testing in future. Therefore, the determination of experimental parameters to describe and simulate drug release in bone is essential. Here, we focus on Sr2+ transport and quantification in cortical rat bone. Sr2+ dose-dependently stimulates bone-building osteoblasts and inhibits bone-resorbing osteoclasts. It should be preferentially applied in the case of bone fracture in the context of osteoporotic bone status. Transport properties of cortical rat bone were investigated by dipping experiments of bone sections in aqueous Sr2+ solution followed by time-of-flight secondary ion mass spectrometry (ToF-SIMS) depth profiling. Data evaluation was carried out by fitting a suitable mathematical diffusion equation to the experimental data. An average diffusion coefficient of D = (1.68 ?? 0.57) ?? 10-13 cm2 s-1 for healthy cortical bone was obtained. This value differed only slightly from the value of D = (4.30 ?? 1.43) ?? 10-13 cm2 s-1 for osteoporotic cortical bone. Transmission electron microscopy investigations revealed a comparable nano- and ultrastructure for both types of bone status. Additionally, Sr2+-enriched mineralized collagen standards were prepared for ToF-SIMS quantification of Sr2+ content. The obtained calibration curve was used for Sr2+ quantification in cortical and trabecular bone in real bone sections. The results allow important insights regarding the Sr2+ transport properties in healthy and osteoporotic bone and can ultimately be used to perform a simulation of drug release and mobility in bone

Strontium-modification of porous scaffolds from mineralized collagen for potential use in bone defect therapy

The present study describes the development and characterization of strontium(II)-modified biomimetic scaffolds based on mineralized collagen type I as potential biomaterial for the local treatment of defects in systemically impaired (e.g. osteoporotic) bone. In contrast to already described collagen/hydroxyapatite nano composites calcium was substituted with strontium to the extent of 25, 50, 75 and 100 mol% by substituting the CaCl2-stock solution (0.1 M) with SrCl2 (0.1 M) during the scaffold synthesis. Simultaneous fibrillation and mineralization of collagen led to the formation of collagen-mineral nanocomposites with mineral phases shifting from nanocrystalline hydroxyapatite (Sr0) over poorly crystalline Sr-rich phases towards a mixed mineral phase (Sr100), consisting of an amorphous strontium phosphate (identified as Collin's salt, Sr6H3(PO4)(5) * 2 H2O, CS) and highly crystalline strontium hydroxyapatite (Sr-5(PO4)(3)OH, SrHA). The formed mineral phases were characterized by transmission electron microscopy (TEM) and KAMAN spectroscopy. All collagen/mineral nano composites with graded strontium content were processed to scaffolds exhibiting an interconnected porosity suitable for homogenous cell seeding in vitro. Strontium ions (Sr2+) were released in a sustained manner from the modified scaffolds, with a clear correlation between the released Sr2+ concentration and the degree of Sr substitution. The accumulated specific Sr2+ release over the course of 28 days reached 141.2 mu g (similar to 27 mu g mg(-1)) from Sr50 and 266.1 mu g (similar to 35 mu g mg(-1)) from Sr100, respectively. Under cell culture conditions this led to maximum Sr2+ concentrations of 0.41 mM (Sr50) and 0.73 mM (Sr100) measured on day 1, which declined to 0.08 mM and 0.16 mM, respectively, at day 28. Since Sr2+ concentrations in this range are known to have an osteo-anabolic effect, these scaffolds are promising biomaterials for the clinical treatment of defects in systemically impaired bone