Monolithic Polymers for Cell Cultivation, Differentiation, and Tissue Engineering (2008)

Need a new nose? Biocompatible, highly porous monolithic scaffolds have been prepared by electron-beam-initiated free-radical polymerization and ring-opening metathesis polymerization. These scaffolds can be used for the in vitro construction of equivalents to bone and fatty tissue. Thus, autologous stem cells have been differentiated into both lipoblasts and osteocytes. Tissue engineering,1–6 that is, the in vitro seeding of scaffold materials with living cells and their subsequent implantation into a defect site, has been proposed in the rehabilitation of numerous instances of tissue loss or deficiency. Various strategies have been developed to regenerate bone and other tissues. These include techniques focusing on molecular, cellular, and material developments. At a cellular level, mesenchymal stem cells (MSCs) from various sources have proven to be appropriate for regeneration of bone and other tissues.7–9 Bioreactor systems have been applied to aid the seeding process. An essential prerequisite, however, is the availability of suitable scaffold materials that demonstrate biocompatibility, appropriate biodegradability, mechanical strength, and allow for neovascularization. Failures in first clinical studies are presumably related to insufficiencies of current available scaffold materials, among other things.8, 10 Scaffolds in tissue engineering function as matrixes for delivery of the cells to the defect site. Open structures with large pores are typically used if the new tissue is expected to integrate within the host tissue.7 The morphology of the scaffold is intended to guide the structure of the engineered tissue11 with regard to size, shape, and vascularization.12 The use of biodegradable polymers in tissue engineering is especially attractive.13 Thus, bioresorption or degradation of these materials minimizes the foreign-body response and leads to the formation of a completely natural tissue. The design and tailor-made synthesis of biodegradability polymers with suitable mechanical properties is an attractive challenge in tissue engineering. Our objective was to address it in an interdisciplinary approach with a focus on bone and adipose tissue replacement. In search of suitable scaffolds for tissue engineering, we focused on monolithic supports. Monolithic media represent uniformly structured matrixes with large interpenetrating pores, usually in the low micrometer range. During the last 15 years, polymer-based monolithic supports have been introduced into separation science as well as into organic catalysis and biocatalysis. Major contributions have been made by Fréchet, Ŝvec, and co-workers, who initially developed radical-polymerization-based systems for these purposes.14 Our group contributed to this area by developing an approach based on ring-opening metathesis polymerization (ROMP), which allowed for a one-pot synthesis of functionalized monolithic systems. Using this synthetic protocol, a large variety of monolithic systems have been realized.15–18 Recently, we extended this chemistry to the electron-beam (EB)-triggered synthesis of highly porous monolithic materials.19 The use of monolithic supports for tissue engineering has not been reported to date; instead, sol-gel-derived glasses, foamed or electrospinning-derived polymers, polymer templating, or plotting techniques have been applied.20–23 However, a major advantage of monolithic supports is that they may be created in virtually any form by a simple molding process. This allows for creating entire parts of the body. Generally, to be feasible for applications cell cultivation, differentiation, or tissue engineering, a support has to fulfill certain criteria. First, it needs to possess a highly porous structure with pore diameters around 150 μm. Only pores of such diameters allow for both the substantial ingrowth of the cells and for vascularization at a comparably late stage of cell proliferation. Second, the matrix itself needs to be biocompatible. On the one hand, this requires a surface chemistry that favors cell contact and adhesion. On the other hand, the polymer must not contain or form, in the course of the degradation process, any compounds that exhibit cell toxicity. Furthermore, the matrix should display reasonable mechanical strength and, finally, be biodegradable. The fragments that form in the course of this degradation process should have molecular weights smaller than 40 000 g mol−1 in order be released from the body via the kidneys. With these requirements in mind, we synthesized porous monolithic supports by a process based on electron-beam-initiated free-radical polymerization. 2-Methylidene-4-phenyl-1,3-dioxolane (M1) was used as monomer, and trimethylolpropane triacrylate (CL1) was chosen as a crosslinker. A mixture of toluene (microporogen), 2-propanol, and dodecan-1-ol (macroporogens) was chosen as solvent. The optimal mixture shown in Table S1, entry 11 (see the Supporting Information) was applied.19, 24 The resulting crosslinked copolymer displayed a porous structure with pores up to 50 μm. Its formation and chemical structure is shown in Scheme 1 (left). Since the crosslinker CL1 is an ester and the dioxolane derivative M1 is transformed into an ester,25 the copolymer is a polyester that is expected to be biodegradable. A representative scanning electron microscopy image is shown in Figure 1 (top). Porous monolithic scaffolds derived from EB-initiated free-radical polymerization (top) and ROMP (bottom). The scale bars represent 100 (top), 400 (bottom), and 4 μm (inset, bottom). Monolithic polyester matrix derived from EB-initiated free-radical polymerization (left); ROMP-derived monolithic matrix (right). The specific surface of the monolithic material as determined by N2 adsorption was 12 m2 g−1, revealing a macroporous structure. Storage of the support in a phosphate buffer (pH 7.2, 200 ppm NaN3) for 1 month at 37 °C revealed a volume increase from swelling (+5 %) and a decrease in mass (−5 %). Both findings were indicative of a slow disintegration of the support. It should also be mentioned that the EB-based process simultaneously sterilizes the matrix. For biocompatibility studies and tests of in vitro growth and differentiation on the scaffold, human adipose tissue derived stem cells (ATSCs) were used. ATSCs have advantages from a clinical point of view owing to their abundant availability as compared to bone marrow derived stem cells and are likewise suited for bone and soft tissue (adipose tissue) engineering. Both tissues are important targets for regenerative strategies in the craniofacial region. Cultivation of ATSCs on sterilized polyester-based monolithic supports revealed rapid cell growth on the surface of the materials, indicating biocompatibility. However, almost no ingrowth of the cells into the monolithic structure was observed. Instead, after 20 days the cells lifted off from the support after initial growth, suggesting insufficient adhesion. These findings were attributed to both the hydrophobic character and the surface structure of the support. This conclusion was supported by the high water contact angle (142°) observed with these materials. In an effort to increase cell adhesion and to provide a material that allows for the ingrowth of cells, we used more hydrophilic monomers and turned to a ROMP-based protocol. Thus, by using a mixture of norborn-2-ene (M2) and pentaglycerol bis(7-oxanorborn-5-ene-2-ylcarboxylate) monoacrylate (CL2) in varying amounts of a microporogen (toluene) and a macroporogen (2-propanol), different monolithic structures were realized using [RuCl2(Py2)(IMesH2)(CHPh)] (1, Py=pyridine, IMesH2=1,3-dimesitylimidazolin-2-ylidene) as the initiator and various amounts of pyridine as regulator (Table S2 in the Supporting Information). As has been shown for related systems,26 the presence of pyridine in amounts as low as 70 ppm allows for the tuning of the monolith's structure. It should be mentioned that the acrylate group in CL2 acts as a chain-transfer agent, thus significantly reducing the average chain lengths of the norborn-2-ene derived polymer blocks. Scheme 1 (right) illustrates the process of monolith synthesis; a typical structure with pores around 200 μm is shown in Figure 1. These large pores translate into an interparticle-derived porosity of 77 %, as determined by inverse size exclusion chromatography.27 The protocol used was identified as the one out of 13 (Table S2, Supplementary Information) that yields monoliths with the largest pore diameter. A selection of alternative protocols is also given in Table S2. To avoid any potential toxic effects of catalyst residues, a careful washing of the monolithic structure with a mixture of dimethylsulfoxide, ethyl vinyl ether, and THF was carried out, which allowed for the virtually quantitative removal of the transition-metal-based initiator. Final ruthenium concentrations were below 0.1 ppm (i.e. below the detection limit of inductively coupled plasma optical emission spectroscopy, ICP-OES). N2 adsorption measurements revealed a total surface area of 4 m2 g−1 and thus support the highly macroporous nature of the monolith. Upon storage in a phosphate buffer system, the initial water contact angle of 142° was strongly reduced within 13 weeks. In fact, no reliable values could be determined owing to fast moistening (spreading). This phenomenon is attributed to both the highly hydrophilic character of CL2 and the high propensity towards oxidation (in the allylic position) of the poly(norborn-2-ene) blocks. On average (n=4), the Young's modulus of these materials was 1 MPa, the compressive strength was 7.4 MPa, and the Martens hardness was 0.3 N mm−2. These values were found suitable for soft (adipose) tissue engineering. Polymer degradation was monitored over 240 days. Monoliths became crumbly, and polymer fragments in the range of 3000–28 000 g mol−1 were observed. Importantly, no free acrylic acid was observed. In a next step, the ROMP-derived scaffolds were cultivated with ATSCs. Within 12 days, the number of cells on the surface quadrupled. No additional adhesion factors such as fibronectine, RGD-peptides, or matrix factors (aside from differentiation factors or supplements) commonly used to facilitate or trigger cell growth were necessary. To study cell ingrowth and differentiation on the scaffolds in a three-dimensional setting, cultivation was performed under dynamic conditions in rotating culture containers. Culture media were changed weekly. Importantly, cells grew into the pores and partially showed even full ingrowth and penetration of the scaffold (Figure 2 A). Finally, after growth, these cells were then successfully subjected to both osteogenic and adipocytic differentiation (Figure 2). Differentiation of ATSCs into either adipocytes or osteoblasts was initiated by addition of isobutylmethylxanthine or β-glycerolphosphate and dexamethasone, respectively. Figure 2 B shows adipocytes, as identified by the formation of large univacuolar cells. Figure 2 D, E shows mineralizations by the cells grown within the monolithic material after six weeks. As can be seen, biomineralization was effective with these materials and is believed to be supported by the free carboxylic acid groups formed from the large number of ester moieties. Direct evidence of lineage-specific differentiation of ATSCs grown on monolith scaffolds was further provided by an immunohistochemical demonstration of osteocalcin expression in osteogenic differentiation (Figure 2 F) and S-100 positivity in adipogenic differentiation (Figure 2 C). It is known that the S-100 positivity in the cytoplasm is typical for adipocytes. Histomicrographs of monoliths seeded with adipose tissue derived stem cells after adipogenic (upper row) and osteogenic differentiation (bottom row). Images are vertical cross sections that show the degree of cell ingrowth inside the scaffold. A) Low-power magnification with cell ingrowth between the grayish scaffold material after adipogenic differentiation. Cells with nuclei (colored dark purple) are also visible in the center of the scaffold (HE staining). B) High-power magnification of a region with differentiated, mature univacuolated adipocytes in HE staining and C) a similar region labeled with S-100 antibody (DAB, brown, visible in part in the cytoplasmic border of the vacuolated adipocytes). Asterisks in (B) and (C) denote mature, univacuolar adipocytes. D) Von Kossa stain shows massive mineralizations (arrow, black-brown) after osteogenic differentiation within the monolithic scaffold (counterstaining of cell nuclei with HE). The scaffold material is visible as light-grayish granules on the left-hand side, invaded by cellular ingrowth. E) Region with minor ingrowth and mineralization (counterstain with nuclear fast red). Mineralizations are brown (arrows), cell nuclei are rare in this region (red), and the scaffold material is represented by the light-grayish, birefringent granules. F) Labeling with osteocalcin antibody (visualization with BCIP/NBT, blue, counterstain with nuclear fast red); arrows show mineralized nodules. All scale bars are 100 μm. In summary, we have developed novel polymeric monolith-based scaffolds for use in both cell cultivation and tissue engineering. Current work focuses on enhancing the mechanical strength of the supports and on long-term investigations of biodegradability as well as on in vitro and in vivo biocompatibility. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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