Novel Oligomeric Biodegradable Crosslinkers For Hybrid Biomaterial Fabrication For Regenerative Purposes

INTRODUCTION Increasing age of population is a great success of numerous breakthroughs in life science and improved health care. For a child born in 2015, for example, an average global life expectancy of meanwhile 71.4 years is assumed which increased by around 8% in the last decade [1]. In accordance with enhanced life expectancy, however, age-related health problems continuously rise. In this regard, the gap between patients awaiting transplantation and appropriate donors consequently will get larger in the future [2]. To this end, there is a need for new strategies in regenerative medicine [3]. Biomaterial matrices were developed to foster tissue regeneration by mimicking the key characteristics of the extracellular matrix (ECM) [4]. Modern biomaterial research focuses on 3D scaffolds, which can be adequately adapted toward specific requirements of the target tissue [5]. In this regard, flexible material platforms are wanted, whose properties can be adjusted over a wide range and independently of each other [6]. In this context, the macromer-based material concept is promising due to the high flexibility of macromers in chemical design and processability [7]. Macromers are reactive oligo- or polymeric molecules which act as monomers and can therefore be polymerized/cross-linked into a polymeric network [8]. The key principle of this approach is the synthesis of chemically well-defined structures which allows for a more precise control over the resulting properties of the cross-linked polymeric network when compared to conventional polymers. For example, macromer chemistry can be adjusted in terms of chemical macromer composition, valence, content of cross-linkable functionalities and molecular weight. The versatility of macromer-derived materials greatly increases when different macromer types are combined which potentially enables precise material tunability on multiple levels. The design flexibility of macromer-based networks motivated the investigation of two different macromer-based material concepts with regard to macromer processability and material adjustability. The following objectives were proposed: 1) To synthesize two sets of biodegradable, multi-valent macromers by using free-radical polymerization and ring-opening polymerization combined with established activation strategies. The synthesis setups will be tuned toward high macromer yields which will be required for processing into biomaterials with relevant sizes. 2) To physico-chemically characterize oligomeric macromers with regard to chemical composition, molecular weight and reactivity in order to yield well-defined macromer structures. NMR spectroscopy, gel permeation chromatography (GPC) and wet chemistry will be applied. 3) To characterize macromer processability into covalently cross-linked hybrid matrices. This work will focus on a soft macromer-cross-linked gelatin-derived hydrogel system for versatile biomedical applications as well as a rigid macromer/sol-gel glass hybrid material for hard tissue regeneration. Sets of different formulations will be investigated in order to characterize the range of macromer processability and to establish structure-property relationships. 4) To investigate strategies for the adjustment of material porosity. Besides the adaption via cross-linking density, porogen-leaching and 3D-printing approaches will be followed in order to introduce macroporosity and to enable a decoupling of porosity and chemical (nano)structure of the cross-linked network. 5) To determine key material properties relevant for regenerative applications, including mechanical properties by compression tests and oscillation rheology, in vitro matrix degradability, as well as material cytocompatibility in indirect and direct contact experiments. 6) To identify strategies for covalent functionalization of the hybrid materials. Post-fabrication functionalization via specifically introduced chemical functionalities is favored as it enables effective material decoration (almost) independent of the physico-chemical matrix properties. SUMMARY OF DISSERTATION The first material concept was based on anhydride-containing macromers which can be processed into hydrogel matrices by covalent cross-linking of amine-bearing macromolecules, such as gelatin [9–11]. The innovative aspect of this work was to decouple material functionalization from the physico-chemical properties of the cross-linked hydrogel network. To this end, a second chemical functionality was introduced which remained reactive in the hydrogel state and was therefore available for covalent post-fabrication functionalization strategies. Specifically, dual-functional macromers were synthesized by free-radical polymerization of maleic anhydride (MA) with diacetone acrylamide (DAAm) and pentaerythritol diacrylate monostearate (PEDAS) to yield oligo(PEDAS-co-DAAm-co-MA) (oPDMA) [12]. Amphiphilic oligomers (molecular weight (Mn) < 7.5 kDa) with anhydride contents of 7-20% were obtained. Fractions of chemically intact anhydrides of around 70% enables effective cross-linking with low molecular-weight gelatinous peptides (Collagel® type B, 11 kDa). Rigid two-component hydrogels (elastic modulus (E) = 4-13 kPa) with adjustable composition and physicochemical properties were formed. Reactivity of the incorporated methyl ketone functionality toward hydrazides and hydrazines was shown on the macromer level and in the cross-linked hydrogel by different strategies. Firstly, pre-fabricated hydrogels were successfully reinforced by secondary cross-linking with adipic acid dihydrazide (ADH). Secondly, pH-dependent immobilization of 2,4-dinitrophenylhydrazine (DNPH) to acid-soluble macromer derivatives as well as cross-linked oPDMA/COL matrices was demonstrated. Thirdly, reversible immobilization of a fluorescent hydrazide (AFH) was shown which was controlled by hydrogel ketone content, hydrazide ligand concentration and medium pH. This triple-tunability of hydrazide immobilization holds promise for adjustable and cost-effective hydrogel modification. Lastly, proof-of-concept experiments with hydrazido-functionalized hyaluronan (ATTO-hyHA) demonstrated the potential for covalent post-fabrication hydrogel decoration with ECM components. Hydrogel cytocompatibility was demonstrated and the introduction of DAAm into the hydrogel system resulted in superior cell material interactions when compared with previously established analogous ketone-free gels [13]. Limited ability of cells to migrate into deeper regions of these macromer-cross-linked gelatin-based gels further motivated the investigation of two different strategies to enhance hydrogel porosity [10,14]. On the one hand, the introduction of macropores was attempted by hydrogel fabrication in presence of poly(ethylene glycol) (Mn = 8000 Da, P8k). This polymer acted as porogen by phase separation during hydrogel formation. It was found that P8k was effectively extracted from the cross-linked matrix, while physico-chemical hydrogel properties remained unchanged. The second approach aimed at increasing mesh size of the cross-linked network by using hydrogel building blocks with increased molecular weights. In particular, high molecular-weight gelatin (160 Bloom, G160) was cross-linked by macromers with low MA content. Homogeneous and mechanically stable hydrogels were obtained and physico-chemical properties were determined. Successful optimization of hydrogel porosity was functionally shown by enhanced cell migration and improved release profile of incorporated nanoparticles [15]. In the second macromer-based material, hydrolytically degradable multi-armed macromers were covalently introduced into a tetraethoxysilane(TEOS)-derived silica sol in order to address the insufficient degradability of glass-based materials [16]. In detail, oligo(D,L-lactide) units were introduced into three- (TMPEO, Tx) and four-armed (PETEO, Px) ethoxylated alcohols by ring-opening polymerization, followed by activation with 3-isocyanatopropyltriethoxysilane (ICPTES) to yield TxLAy-Si and PxLAy-Si macromers [17,18]. A series of 18 oligomers (Mn: 1100-3200 Da) with different degrees of ethoxylation and varying length of oligoester units was synthesized. Applicability of a previously established indirect rapid prototyping method enabled fabrication of macromer/sol-gel-glass-derived class II hybrid scaffolds with controlled porosity [19]. Successful processability of a total of 85 different hybrid scaffold formulations allowed for identification of relevant structure-property relationships. In vitro degradation was analyzed over 12 months and a continuous linear weight loss (0.2-0.5 wt%/d) was detected which was controlled by oligo(lactide) content and matrix hydrophilicity. Compressive strength (2-30 MPa) and compressive modulus (44-716 MPa) were determined and total content, oligo(ethylene oxide) content, oligo(lactide) content and molecular weight of the oligomeric cross-linkers as well as material porosity were identified as the main factors determining hybrid mechanics by multiple linear regression. Cell migration into the entire scaffold pore network was indicated in cell culture experiments with human adipose tissue-derived stem cells (hASC) and continuous proliferation over 14 days was found. Overall, two macromer-based material platforms were established in which material versatility was realized by three main principles: I) synthesis of macromers with different chemical composition, II) combination of macromers with a second oligomeric building block, and III) flexible processability of these dual-component hybrid formulations into porous scaffold materials. Precise adjustability of material properties as demonstrated in both concepts offers potential for application of these hybrid materials for a wide range of regenerative purposes. REFERENCES (1) World Health Statistics of the WHO. http://www.who.int/gho/publications/world_health_statistics/en/ 2017. (2) OPTN/UNOS Public Comment. https://optn.transplant.hrsa.gov/ 2017. (3) Puppi, D.; Chiellini, F.; Piras, a. M. M.; Chiellini, E. Prog. Polym. Sci. 2010, 35 (4), 403–440. (4) Patterson, J.; Martino, M. M.; Hubbell, J. A. Mater. Today 2010, 13 (1–2), 14–22. (5) Picke, A.-K.; Salbach-Hirsch, J.; Hintze, V.; Rother, S.; Rauner, M.; Kascholke, C.; Möller, S.; Bernhardt, R.; Rammelt, S.; Pisabarro, M. T.; Ruiz-Gómez, G.; Schnabelrauch, M.; Schulz-Siegmund, M.; Hacker, M. C.; Scharnweber, D.; Hofbauer, C.; Hofbauer, L. C. Biomaterials 2016, 96, 11–23. (6) Loth, R.; Loth, T.; Schwabe, K.; Bernhardt, R.; Schulz-Siegmund, M.; Hacker, M. C. Acta Biomater. 2015, 26, 82–96. (7) DeForest, C. A.; Anseth, K. S. Nat. Chem. 2011, 3 (12), 925–931. (8) Nic, M.; Jirát, J.; Košata, B.; Jenkins, A.; McNaught, A.; Wilkinson, A. IUPAC, Research Triangle Park, NC 2014. (9) Loth, T.; Hennig, R.; Kascholke, C.; Hötzel, R.; Hacker, M. C. React. Funct. Polym. 2013, 73 (11), 1480–1492. (10) Loth, T.; Hötzel, R.; Kascholke, C.; Anderegg, U.; Schulz-Siegmund, M.; Hacker, M. C. Biomacromolecules 2014, 15 (6), 2104–2118. (11) Kohn, C.; Klemens, J. M.; Kascholke, C.; Murthy, N. S.; Kohn, J.; Brandenburger, M.; Hacker, M. C. Biomater. Sci. 2016, 4, 1605–1621. (12) Kascholke, C.; Loth, T.; Kohn-Polster, C.; Möller, S.; Bellstedt, P.; Schulz-Siegmund, M.; Schnabelrauch, M.; Hacker, M. C. Biomacromolecules 2017, 18 (3), 683–694. (13) Sülflow, K.; Schneider, M.; Loth, T.; Kascholke, C.; Schulz-Siegmund, M.; Hacker, M. C.; Simon, J.-C.; Savkovic, V. J. Biomed. Mater. Res. A 2016, 104 (12), 3115–3126. (14) Loth, T. Diss. Univ. Leipzig, Fak. für Biowissenschaften, Pharm. und Psychol. 2016. (15) Schwabe, K.; Ewe, A.; Kohn, C.; Loth, T.; Aigner, A.; Hacker, M. C.; Schulz-Siegmund, M. Int. J. Pharm. 2017, 526 (1–2), 178–187. (16) Rahaman, M. N.; Day, D. E.; Sonny Bal, B.; Fu, Q.; Jung, S. B.; Bonewald, L. F.; Tomsia, A. P. Acta Biomater. 2011, 7 (6), 2355–2373. (17) Schulze, P.; Flath, T.; Dörfler, H.-M.; Schulz-Siegmund, M.; Hacker, M.; Hendrikx, S.; Kascholke, C.; Gressenbuch, M.; Schumann, D. Ger. Pat. No. DE102014224654A1 2016. (18) Kascholke, C.; Hendrikx, S.; Flath, T.; Kuzmenka, D.; Dörfler, H.-M.; Schumann, D.; Gressenbuch, M.; Schulze, F. P.; Schulz-Siegmund, M.; Hacker, M. C. Acta Biomater. 2017, 63, 336–349. (19) Hendrikx, S.; Kascholke, C.; Flath, T.; Schumann, D.; Gressenbuch, M.; Schulze, P.; Hacker, M. C.; Schulz-Siegmund, M. Acta Biomater. 2016, 35, 318–329

Increased pore size of scaffolds improves coating efficiency with sulfated hyaluronan and mineralization capacity of osteoblasts

Background: Delayed bone regeneration of fractures in osteoporosis patients or of critical-size bone defects after tumor resection are a major medical and socio-economic challenge. Therefore, the development of more effective and osteoinductive biomaterials is crucial. Methods: We examined the osteogenic potential of macroporous scaffolds with varying pore sizes after biofunctionalization with a collagen/high-sulfated hyaluronan (sHA3) coating in vitro. The three-dimensional scaffolds were made up from a biodegradable three-armed lactic acid-based macromer (TriLA) by cross-polymerization. Templating with solid lipid particles that melt during fabrication generates a continuous pore network. Human mesenchymal stem cells (hMSC) cultivated on the functionalized scaffolds in vitro were investigated for cell viability, production of alkaline phosphatase (ALP) and bone matrix formation. Statistical analysis was performed using student's t-test or two-way ANOVA. Results: We succeeded in generating scaffolds that feature a significantly higher average pore size and a broader distribution of individual pore sizes (HiPo) by modifying composition and relative amount of lipid particles, macromer concentration and temperature for cross-polymerization during scaffold fabrication. Overall porosity was retained, while the scaffolds showed a 25% decrease in compressive modulus compared to the initial TriLA scaffolds with a lower pore size (LoPo). These HiPo scaffolds were more readily coated as shown by higher amounts of immobilized collagen (+ 44%) and sHA3 (+ 25%) compared to LoPo scaffolds. In vitro, culture of hMSCs on collagen and/or sHA3-coated HiPo scaffolds demonstrated unaltered cell viability. Furthermore, the production of ALP, an early marker of osteogenesis (+ 3-fold), and formation of new bone matrix (+ 2.5-fold) was enhanced by the functionalization with sHA3 of both scaffold types. Nevertheless, effects were more pronounced on HiPo scaffolds about 112%. Conclusion: In summary, we showed that the improvement of scaffold pore sizes enhanced the coating efficiency with collagen and sHA3, which had a significant positive effect on bone formation markers, underlining the promise of using this material approach for in vivo studies. © 2019 The Author(s)

Novel Oligomeric Biodegradable Crosslinkers For Hybrid Biomaterial Fabrication For Regenerative Purposes

INTRODUCTION Increasing age of population is a great success of numerous breakthroughs in life science and improved health care. For a child born in 2015, for example, an average global life expectancy of meanwhile 71.4 years is assumed which increased by around 8% in the last decade [1]. In accordance with enhanced life expectancy, however, age-related health problems continuously rise. In this regard, the gap between patients awaiting transplantation and appropriate donors consequently will get larger in the future [2]. To this end, there is a need for new strategies in regenerative medicine [3]. Biomaterial matrices were developed to foster tissue regeneration by mimicking the key characteristics of the extracellular matrix (ECM) [4]. Modern biomaterial research focuses on 3D scaffolds, which can be adequately adapted toward specific requirements of the target tissue [5]. In this regard, flexible material platforms are wanted, whose properties can be adjusted over a wide range and independently of each other [6]. In this context, the macromer-based material concept is promising due to the high flexibility of macromers in chemical design and processability [7]. Macromers are reactive oligo- or polymeric molecules which act as monomers and can therefore be polymerized/cross-linked into a polymeric network [8]. The key principle of this approach is the synthesis of chemically well-defined structures which allows for a more precise control over the resulting properties of the cross-linked polymeric network when compared to conventional polymers. For example, macromer chemistry can be adjusted in terms of chemical macromer composition, valence, content of cross-linkable functionalities and molecular weight. The versatility of macromer-derived materials greatly increases when different macromer types are combined which potentially enables precise material tunability on multiple levels. The design flexibility of macromer-based networks motivated the investigation of two different macromer-based material concepts with regard to macromer processability and material adjustability. The following objectives were proposed: 1) To synthesize two sets of biodegradable, multi-valent macromers by using free-radical polymerization and ring-opening polymerization combined with established activation strategies. The synthesis setups will be tuned toward high macromer yields which will be required for processing into biomaterials with relevant sizes. 2) To physico-chemically characterize oligomeric macromers with regard to chemical composition, molecular weight and reactivity in order to yield well-defined macromer structures. NMR spectroscopy, gel permeation chromatography (GPC) and wet chemistry will be applied. 3) To characterize macromer processability into covalently cross-linked hybrid matrices. This work will focus on a soft macromer-cross-linked gelatin-derived hydrogel system for versatile biomedical applications as well as a rigid macromer/sol-gel glass hybrid material for hard tissue regeneration. Sets of different formulations will be investigated in order to characterize the range of macromer processability and to establish structure-property relationships. 4) To investigate strategies for the adjustment of material porosity. Besides the adaption via cross-linking density, porogen-leaching and 3D-printing approaches will be followed in order to introduce macroporosity and to enable a decoupling of porosity and chemical (nano)structure of the cross-linked network. 5) To determine key material properties relevant for regenerative applications, including mechanical properties by compression tests and oscillation rheology, in vitro matrix degradability, as well as material cytocompatibility in indirect and direct contact experiments. 6) To identify strategies for covalent functionalization of the hybrid materials. Post-fabrication functionalization via specifically introduced chemical functionalities is favored as it enables effective material decoration (almost) independent of the physico-chemical matrix properties. SUMMARY OF DISSERTATION The first material concept was based on anhydride-containing macromers which can be processed into hydrogel matrices by covalent cross-linking of amine-bearing macromolecules, such as gelatin [9–11]. The innovative aspect of this work was to decouple material functionalization from the physico-chemical properties of the cross-linked hydrogel network. To this end, a second chemical functionality was introduced which remained reactive in the hydrogel state and was therefore available for covalent post-fabrication functionalization strategies. Specifically, dual-functional macromers were synthesized by free-radical polymerization of maleic anhydride (MA) with diacetone acrylamide (DAAm) and pentaerythritol diacrylate monostearate (PEDAS) to yield oligo(PEDAS-co-DAAm-co-MA) (oPDMA) [12]. Amphiphilic oligomers (molecular weight (Mn) < 7.5 kDa) with anhydride contents of 7-20% were obtained. Fractions of chemically intact anhydrides of around 70% enables effective cross-linking with low molecular-weight gelatinous peptides (Collagel® type B, 11 kDa). Rigid two-component hydrogels (elastic modulus (E) = 4-13 kPa) with adjustable composition and physicochemical properties were formed. Reactivity of the incorporated methyl ketone functionality toward hydrazides and hydrazines was shown on the macromer level and in the cross-linked hydrogel by different strategies. Firstly, pre-fabricated hydrogels were successfully reinforced by secondary cross-linking with adipic acid dihydrazide (ADH). Secondly, pH-dependent immobilization of 2,4-dinitrophenylhydrazine (DNPH) to acid-soluble macromer derivatives as well as cross-linked oPDMA/COL matrices was demonstrated. Thirdly, reversible immobilization of a fluorescent hydrazide (AFH) was shown which was controlled by hydrogel ketone content, hydrazide ligand concentration and medium pH. This triple-tunability of hydrazide immobilization holds promise for adjustable and cost-effective hydrogel modification. Lastly, proof-of-concept experiments with hydrazido-functionalized hyaluronan (ATTO-hyHA) demonstrated the potential for covalent post-fabrication hydrogel decoration with ECM components. Hydrogel cytocompatibility was demonstrated and the introduction of DAAm into the hydrogel system resulted in superior cell material interactions when compared with previously established analogous ketone-free gels [13]. Limited ability of cells to migrate into deeper regions of these macromer-cross-linked gelatin-based gels further motivated the investigation of two different strategies to enhance hydrogel porosity [10,14]. On the one hand, the introduction of macropores was attempted by hydrogel fabrication in presence of poly(ethylene glycol) (Mn = 8000 Da, P8k). This polymer acted as porogen by phase separation during hydrogel formation. It was found that P8k was effectively extracted from the cross-linked matrix, while physico-chemical hydrogel properties remained unchanged. The second approach aimed at increasing mesh size of the cross-linked network by using hydrogel building blocks with increased molecular weights. In particular, high molecular-weight gelatin (160 Bloom, G160) was cross-linked by macromers with low MA content. Homogeneous and mechanically stable hydrogels were obtained and physico-chemical properties were determined. Successful optimization of hydrogel porosity was functionally shown by enhanced cell migration and improved release profile of incorporated nanoparticles [15]. In the second macromer-based material, hydrolytically degradable multi-armed macromers were covalently introduced into a tetraethoxysilane(TEOS)-derived silica sol in order to address the insufficient degradability of glass-based materials [16]. In detail, oligo(D,L-lactide) units were introduced into three- (TMPEO, Tx) and four-armed (PETEO, Px) ethoxylated alcohols by ring-opening polymerization, followed by activation with 3-isocyanatopropyltriethoxysilane (ICPTES) to yield TxLAy-Si and PxLAy-Si macromers [17,18]. A series of 18 oligomers (Mn: 1100-3200 Da) with different degrees of ethoxylation and varying length of oligoester units was synthesized. Applicability of a previously established indirect rapid prototyping method enabled fabrication of macromer/sol-gel-glass-derived class II hybrid scaffolds with controlled porosity [19]. Successful processability of a total of 85 different hybrid scaffold formulations allowed for identification of relevant structure-property relationships. In vitro degradation was analyzed over 12 months and a continuous linear weight loss (0.2-0.5 wt%/d) was detected which was controlled by oligo(lactide) content and matrix hydrophilicity. Compressive strength (2-30 MPa) and compressive modulus (44-716 MPa) were determined and total content, oligo(ethylene oxide) content, oligo(lactide) content and molecular weight of the oligomeric cross-linkers as well as material porosity were identified as the main factors determining hybrid mechanics by multiple linear regression. Cell migration into the entire scaffold pore network was indicated in cell culture experiments with human adipose tissue-derived stem cells (hASC) and continuous proliferation over 14 days was found. Overall, two macromer-based material platforms were established in which material versatility was realized by three main principles: I) synthesis of macromers with different chemical composition, II) combination of macromers with a second oligomeric building block, and III) flexible processability of these dual-component hybrid formulations into porous scaffold materials. Precise adjustability of material properties as demonstrated in both concepts offers potential for application of these hybrid materials for a wide range of regenerative purposes. REFERENCES (1) World Health Statistics of the WHO. http://www.who.int/gho/publications/world_health_statistics/en/ 2017. (2) OPTN/UNOS Public Comment. https://optn.transplant.hrsa.gov/ 2017. (3) Puppi, D.; Chiellini, F.; Piras, a. M. M.; Chiellini, E. Prog. Polym. Sci. 2010, 35 (4), 403–440. (4) Patterson, J.; Martino, M. M.; Hubbell, J. A. Mater. Today 2010, 13 (1–2), 14–22. (5) Picke, A.-K.; Salbach-Hirsch, J.; Hintze, V.; Rother, S.; Rauner, M.; Kascholke, C.; Möller, S.; Bernhardt, R.; Rammelt, S.; Pisabarro, M. T.; Ruiz-Gómez, G.; Schnabelrauch, M.; Schulz-Siegmund, M.; Hacker, M. C.; Scharnweber, D.; Hofbauer, C.; Hofbauer, L. C. Biomaterials 2016, 96, 11–23. (6) Loth, R.; Loth, T.; Schwabe, K.; Bernhardt, R.; Schulz-Siegmund, M.; Hacker, M. C. Acta Biomater. 2015, 26, 82–96. (7) DeForest, C. A.; Anseth, K. S. Nat. Chem. 2011, 3 (12), 925–931. (8) Nic, M.; Jirát, J.; Košata, B.; Jenkins, A.; McNaught, A.; Wilkinson, A. IUPAC, Research Triangle Park, NC 2014. (9) Loth, T.; Hennig, R.; Kascholke, C.; Hötzel, R.; Hacker, M. C. React. Funct. Polym. 2013, 73 (11), 1480–1492. (10) Loth, T.; Hötzel, R.; Kascholke, C.; Anderegg, U.; Schulz-Siegmund, M.; Hacker, M. C. Biomacromolecules 2014, 15 (6), 2104–2118. (11) Kohn, C.; Klemens, J. M.; Kascholke, C.; Murthy, N. S.; Kohn, J.; Brandenburger, M.; Hacker, M. C. Biomater. Sci. 2016, 4, 1605–1621. (12) Kascholke, C.; Loth, T.; Kohn-Polster, C.; Möller, S.; Bellstedt, P.; Schulz-Siegmund, M.; Schnabelrauch, M.; Hacker, M. C. Biomacromolecules 2017, 18 (3), 683–694. (13) Sülflow, K.; Schneider, M.; Loth, T.; Kascholke, C.; Schulz-Siegmund, M.; Hacker, M. C.; Simon, J.-C.; Savkovic, V. J. Biomed. Mater. Res. A 2016, 104 (12), 3115–3126. (14) Loth, T. Diss. Univ. Leipzig, Fak. für Biowissenschaften, Pharm. und Psychol. 2016. (15) Schwabe, K.; Ewe, A.; Kohn, C.; Loth, T.; Aigner, A.; Hacker, M. C.; Schulz-Siegmund, M. Int. J. Pharm. 2017, 526 (1–2), 178–187. (16) Rahaman, M. N.; Day, D. E.; Sonny Bal, B.; Fu, Q.; Jung, S. B.; Bonewald, L. F.; Tomsia, A. P. Acta Biomater. 2011, 7 (6), 2355–2373. (17) Schulze, P.; Flath, T.; Dörfler, H.-M.; Schulz-Siegmund, M.; Hacker, M.; Hendrikx, S.; Kascholke, C.; Gressenbuch, M.; Schumann, D. Ger. Pat. No. DE102014224654A1 2016. (18) Kascholke, C.; Hendrikx, S.; Flath, T.; Kuzmenka, D.; Dörfler, H.-M.; Schumann, D.; Gressenbuch, M.; Schulze, F. P.; Schulz-Siegmund, M.; Hacker, M. C. Acta Biomater. 2017, 63, 336–349. (19) Hendrikx, S.; Kascholke, C.; Flath, T.; Schumann, D.; Gressenbuch, M.; Schulze, P.; Hacker, M. C.; Schulz-Siegmund, M. Acta Biomater. 2016, 35, 318–329

Novel Oligomeric Biodegradable Crosslinkers For Hybrid Biomaterial Fabrication For Regenerative Purposes

INTRODUCTION Increasing age of population is a great success of numerous breakthroughs in life science and improved health care. For a child born in 2015, for example, an average global life expectancy of meanwhile 71.4 years is assumed which increased by around 8% in the last decade [1]. In accordance with enhanced life expectancy, however, age-related health problems continuously rise. In this regard, the gap between patients awaiting transplantation and appropriate donors consequently will get larger in the future [2]. To this end, there is a need for new strategies in regenerative medicine [3]. Biomaterial matrices were developed to foster tissue regeneration by mimicking the key characteristics of the extracellular matrix (ECM) [4]. Modern biomaterial research focuses on 3D scaffolds, which can be adequately adapted toward specific requirements of the target tissue [5]. In this regard, flexible material platforms are wanted, whose properties can be adjusted over a wide range and independently of each other [6]. In this context, the macromer-based material concept is promising due to the high flexibility of macromers in chemical design and processability [7]. Macromers are reactive oligo- or polymeric molecules which act as monomers and can therefore be polymerized/cross-linked into a polymeric network [8]. The key principle of this approach is the synthesis of chemically well-defined structures which allows for a more precise control over the resulting properties of the cross-linked polymeric network when compared to conventional polymers. For example, macromer chemistry can be adjusted in terms of chemical macromer composition, valence, content of cross-linkable functionalities and molecular weight. The versatility of macromer-derived materials greatly increases when different macromer types are combined which potentially enables precise material tunability on multiple levels. The design flexibility of macromer-based networks motivated the investigation of two different macromer-based material concepts with regard to macromer processability and material adjustability. The following objectives were proposed: 1) To synthesize two sets of biodegradable, multi-valent macromers by using free-radical polymerization and ring-opening polymerization combined with established activation strategies. The synthesis setups will be tuned toward high macromer yields which will be required for processing into biomaterials with relevant sizes. 2) To physico-chemically characterize oligomeric macromers with regard to chemical composition, molecular weight and reactivity in order to yield well-defined macromer structures. NMR spectroscopy, gel permeation chromatography (GPC) and wet chemistry will be applied. 3) To characterize macromer processability into covalently cross-linked hybrid matrices. This work will focus on a soft macromer-cross-linked gelatin-derived hydrogel system for versatile biomedical applications as well as a rigid macromer/sol-gel glass hybrid material for hard tissue regeneration. Sets of different formulations will be investigated in order to characterize the range of macromer processability and to establish structure-property relationships. 4) To investigate strategies for the adjustment of material porosity. Besides the adaption via cross-linking density, porogen-leaching and 3D-printing approaches will be followed in order to introduce macroporosity and to enable a decoupling of porosity and chemical (nano)structure of the cross-linked network. 5) To determine key material properties relevant for regenerative applications, including mechanical properties by compression tests and oscillation rheology, in vitro matrix degradability, as well as material cytocompatibility in indirect and direct contact experiments. 6) To identify strategies for covalent functionalization of the hybrid materials. Post-fabrication functionalization via specifically introduced chemical functionalities is favored as it enables effective material decoration (almost) independent of the physico-chemical matrix properties. SUMMARY OF DISSERTATION The first material concept was based on anhydride-containing macromers which can be processed into hydrogel matrices by covalent cross-linking of amine-bearing macromolecules, such as gelatin [9–11]. The innovative aspect of this work was to decouple material functionalization from the physico-chemical properties of the cross-linked hydrogel network. To this end, a second chemical functionality was introduced which remained reactive in the hydrogel state and was therefore available for covalent post-fabrication functionalization strategies. Specifically, dual-functional macromers were synthesized by free-radical polymerization of maleic anhydride (MA) with diacetone acrylamide (DAAm) and pentaerythritol diacrylate monostearate (PEDAS) to yield oligo(PEDAS-co-DAAm-co-MA) (oPDMA) [12]. Amphiphilic oligomers (molecular weight (Mn) < 7.5 kDa) with anhydride contents of 7-20% were obtained. Fractions of chemically intact anhydrides of around 70% enables effective cross-linking with low molecular-weight gelatinous peptides (Collagel® type B, 11 kDa). Rigid two-component hydrogels (elastic modulus (E) = 4-13 kPa) with adjustable composition and physicochemical properties were formed. Reactivity of the incorporated methyl ketone functionality toward hydrazides and hydrazines was shown on the macromer level and in the cross-linked hydrogel by different strategies. Firstly, pre-fabricated hydrogels were successfully reinforced by secondary cross-linking with adipic acid dihydrazide (ADH). Secondly, pH-dependent immobilization of 2,4-dinitrophenylhydrazine (DNPH) to acid-soluble macromer derivatives as well as cross-linked oPDMA/COL matrices was demonstrated. Thirdly, reversible immobilization of a fluorescent hydrazide (AFH) was shown which was controlled by hydrogel ketone content, hydrazide ligand concentration and medium pH. This triple-tunability of hydrazide immobilization holds promise for adjustable and cost-effective hydrogel modification. Lastly, proof-of-concept experiments with hydrazido-functionalized hyaluronan (ATTO-hyHA) demonstrated the potential for covalent post-fabrication hydrogel decoration with ECM components. Hydrogel cytocompatibility was demonstrated and the introduction of DAAm into the hydrogel system resulted in superior cell material interactions when compared with previously established analogous ketone-free gels [13]. Limited ability of cells to migrate into deeper regions of these macromer-cross-linked gelatin-based gels further motivated the investigation of two different strategies to enhance hydrogel porosity [10,14]. On the one hand, the introduction of macropores was attempted by hydrogel fabrication in presence of poly(ethylene glycol) (Mn = 8000 Da, P8k). This polymer acted as porogen by phase separation during hydrogel formation. It was found that P8k was effectively extracted from the cross-linked matrix, while physico-chemical hydrogel properties remained unchanged. The second approach aimed at increasing mesh size of the cross-linked network by using hydrogel building blocks with increased molecular weights. In particular, high molecular-weight gelatin (160 Bloom, G160) was cross-linked by macromers with low MA content. Homogeneous and mechanically stable hydrogels were obtained and physico-chemical properties were determined. Successful optimization of hydrogel porosity was functionally shown by enhanced cell migration and improved release profile of incorporated nanoparticles [15]. In the second macromer-based material, hydrolytically degradable multi-armed macromers were covalently introduced into a tetraethoxysilane(TEOS)-derived silica sol in order to address the insufficient degradability of glass-based materials [16]. In detail, oligo(D,L-lactide) units were introduced into three- (TMPEO, Tx) and four-armed (PETEO, Px) ethoxylated alcohols by ring-opening polymerization, followed by activation with 3-isocyanatopropyltriethoxysilane (ICPTES) to yield TxLAy-Si and PxLAy-Si macromers [17,18]. A series of 18 oligomers (Mn: 1100-3200 Da) with different degrees of ethoxylation and varying length of oligoester units was synthesized. Applicability of a previously established indirect rapid prototyping method enabled fabrication of macromer/sol-gel-glass-derived class II hybrid scaffolds with controlled porosity [19]. Successful processability of a total of 85 different hybrid scaffold formulations allowed for identification of relevant structure-property relationships. In vitro degradation was analyzed over 12 months and a continuous linear weight loss (0.2-0.5 wt%/d) was detected which was controlled by oligo(lactide) content and matrix hydrophilicity. Compressive strength (2-30 MPa) and compressive modulus (44-716 MPa) were determined and total content, oligo(ethylene oxide) content, oligo(lactide) content and molecular weight of the oligomeric cross-linkers as well as material porosity were identified as the main factors determining hybrid mechanics by multiple linear regression. Cell migration into the entire scaffold pore network was indicated in cell culture experiments with human adipose tissue-derived stem cells (hASC) and continuous proliferation over 14 days was found. Overall, two macromer-based material platforms were established in which material versatility was realized by three main principles: I) synthesis of macromers with different chemical composition, II) combination of macromers with a second oligomeric building block, and III) flexible processability of these dual-component hybrid formulations into porous scaffold materials. Precise adjustability of material properties as demonstrated in both concepts offers potential for application of these hybrid materials for a wide range of regenerative purposes. REFERENCES (1) World Health Statistics of the WHO. http://www.who.int/gho/publications/world_health_statistics/en/ 2017. (2) OPTN/UNOS Public Comment. https://optn.transplant.hrsa.gov/ 2017. (3) Puppi, D.; Chiellini, F.; Piras, a. M. M.; Chiellini, E. Prog. Polym. Sci. 2010, 35 (4), 403–440. (4) Patterson, J.; Martino, M. M.; Hubbell, J. A. Mater. Today 2010, 13 (1–2), 14–22. (5) Picke, A.-K.; Salbach-Hirsch, J.; Hintze, V.; Rother, S.; Rauner, M.; Kascholke, C.; Möller, S.; Bernhardt, R.; Rammelt, S.; Pisabarro, M. T.; Ruiz-Gómez, G.; Schnabelrauch, M.; Schulz-Siegmund, M.; Hacker, M. C.; Scharnweber, D.; Hofbauer, C.; Hofbauer, L. C. Biomaterials 2016, 96, 11–23. (6) Loth, R.; Loth, T.; Schwabe, K.; Bernhardt, R.; Schulz-Siegmund, M.; Hacker, M. C. Acta Biomater. 2015, 26, 82–96. (7) DeForest, C. A.; Anseth, K. S. Nat. Chem. 2011, 3 (12), 925–931. (8) Nic, M.; Jirát, J.; Košata, B.; Jenkins, A.; McNaught, A.; Wilkinson, A. IUPAC, Research Triangle Park, NC 2014. (9) Loth, T.; Hennig, R.; Kascholke, C.; Hötzel, R.; Hacker, M. C. React. Funct. Polym. 2013, 73 (11), 1480–1492. (10) Loth, T.; Hötzel, R.; Kascholke, C.; Anderegg, U.; Schulz-Siegmund, M.; Hacker, M. C. Biomacromolecules 2014, 15 (6), 2104–2118. (11) Kohn, C.; Klemens, J. M.; Kascholke, C.; Murthy, N. S.; Kohn, J.; Brandenburger, M.; Hacker, M. C. Biomater. Sci. 2016, 4, 1605–1621. (12) Kascholke, C.; Loth, T.; Kohn-Polster, C.; Möller, S.; Bellstedt, P.; Schulz-Siegmund, M.; Schnabelrauch, M.; Hacker, M. C. Biomacromolecules 2017, 18 (3), 683–694. (13) Sülflow, K.; Schneider, M.; Loth, T.; Kascholke, C.; Schulz-Siegmund, M.; Hacker, M. C.; Simon, J.-C.; Savkovic, V. J. Biomed. Mater. Res. A 2016, 104 (12), 3115–3126. (14) Loth, T. Diss. Univ. Leipzig, Fak. für Biowissenschaften, Pharm. und Psychol. 2016. (15) Schwabe, K.; Ewe, A.; Kohn, C.; Loth, T.; Aigner, A.; Hacker, M. C.; Schulz-Siegmund, M. Int. J. Pharm. 2017, 526 (1–2), 178–187. (16) Rahaman, M. N.; Day, D. E.; Sonny Bal, B.; Fu, Q.; Jung, S. B.; Bonewald, L. F.; Tomsia, A. P. Acta Biomater. 2011, 7 (6), 2355–2373. (17) Schulze, P.; Flath, T.; Dörfler, H.-M.; Schulz-Siegmund, M.; Hacker, M.; Hendrikx, S.; Kascholke, C.; Gressenbuch, M.; Schumann, D. Ger. Pat. No. DE102014224654A1 2016. (18) Kascholke, C.; Hendrikx, S.; Flath, T.; Kuzmenka, D.; Dörfler, H.-M.; Schumann, D.; Gressenbuch, M.; Schulze, F. P.; Schulz-Siegmund, M.; Hacker, M. C. Acta Biomater. 2017, 63, 336–349. (19) Hendrikx, S.; Kascholke, C.; Flath, T.; Schumann, D.; Gressenbuch, M.; Schulze, P.; Hacker, M. C.; Schulz-Siegmund, M. Acta Biomater. 2016, 35, 318–329

Reactive and stimuli-responsive maleic anhydride containing macromers–multi-functional cross-linkers and building blocks for hydrogel fabrication

Macromers with functional groups that allow for chemical derivatization, polymerization reactions or impart specific physico-chemical properties are functional building blocks for polymeric systems used in different biomedical applications. With this motivation, a series of oligomeric macromers was synthesized by free radical polymerization of maleic anhydride (MA) with N-isopropylacrylamide (NiPAAm) and pentaerythritol diacrylate monostearate (PEDAS). This chemical design provides anhydride groups for effective reactivity of the macromers with amines and other nucleophiles, copolymerized NiPAAm for temperature responsiveness and lipophilic stearate domains for increased hydrogel stability. Macromers were synthesized with different MA co-monomer feeds and oligomeric molecules (Mn below 5000 Da) were obtained with MA contents between 7% and 27% as determined by titration. The fraction of chemically intact anhydrides was calculated to range from 75% to 80%. The ability of the macromers to cross-link di- or oligovalent amines as a function of MA content was investigated rheologically. It was also demonstrated that monovalent amines, e.g. aminofluorescein, could be grafted to the macromer chain utilizing only a fraction of the anhydride functionalities. The derivatized macromers could still participate in cross-linking reactions due to the remaining anhydrides. Temperature sensitivity was shown for aqueous solutions of macromers with fully dissociated anhydride groups. The solutions were additionally responsive to changes in calcium ion concentration and pH. Extracts from macromer cross-linked polyether hydrogels showed no toxicity on L929 fibroblasts. The macromers have perspective as biocompatible cross-linkers for hydrogel fabrication from various biomacromolecules with the opportunity to decorate the gels with monoamine molecules that alter the biological or physico-chemical properties

Gelatin-Based Biomaterial Engineering with Anhydride-Containing Oligomeric Cross-Linkers

Chemically cross-linked gelatin hydrogels are versatile cell-adhesive hydrogel materials that have been established for a variety of biomedical applications. The most prominent cross-linker is glutaraldehyde, which, however, has been described to cause compatibility problems and loss of microscopic but relevant structural features. A recently developed oligomeric cross-linker that contains anhydride functionalities was evaluated as cross-linker for the fabrication of gelatin-based hydrogels and microparticles. In a fast curing reaction, hydrogels composed of gelatin and oligomeric cross-linker were fabricated with good conversion over a wide concentration range of constituents and with cross-linkers of different anhydride contents. Hydrogel properties, such as dry weight and mechanics, could be controlled by hydrogel composition and rheological properties correlated to elastic moduli from 1 to 10 kPa. The gels were shown to be cytocompatible and promoted cell adhesion. In soft formulations, cells migrated into the hydrogel bulk. Gelatin microparticles prepared by a standard water-in-oil emulsion technique were also treated with the novel oligomers, and cross-linking degrees matching those obtained with glutaraldehyde were obtained. At the same time, fewer interparticular cross-links were observed. Fluorescein-derivatized cross-linkers yielded labeled microparticles in a concentration-dependent manner. The oligomeric cross-linkers are presented as an efficient and possibly more functional and compatible alternative to glutaraldehyde. The engineered hydrogel materials hold potential for various biomedical applications

Dual-Functional Hydrazide-Reactive and Anhydride-Containing Oligomeric Hydrogel Building Blocks

Biomimetic hydrogels are advanced biomaterials that have been developed following different synthetic routes. Covalent postfabrication functionalization is a promising strategy to achieve efficient matrix modification decoupled of general material properties. To this end, dual-functional macromers were synthesized by free radical polymerization of maleic anhydride with diacetone acrylamide (<i>N</i>-(1,1-dimethyl-3-oxobutyl)­acrylamide) and pentaerythritol diacrylate monostearate. Amphiphilic oligomers (<i>M</i>_n < 7.5 kDa) with anhydride contents of 7–20% offered cross-linking reactivity to yield rigid hydrogels with gelatinous peptides (<i>E</i> = 4–13 kPa) and good cell adhesion properties. Mildly reactive methyl ketones as second functionality remained intact during hydrogel formation and potential of covalent matrix modification was shown using hydrazide and hydrazine model compounds. Successful secondary dihydrazide cross-linking was demonstrated by an increase of hydrogel stiffness (>40%). Efficient hydrazide/hydrazine immobilization depending on solution pH, hydrogel ketone content as well as ligand concentration for bioconjugation was shown and reversibility of hydrazone formation was indicated by physiologically relevant hydrazide release over 7 days. Proof-of-concept experiments with hydrazido-functionalized hyaluronan demonstrated potential for covalent aECM immobilization. The presented dual-functional macromers have perspective as reactive hydrogel building blocks for various biomedical applications