Surface Functionalization of Poly(l-lactide-co-glycolide) Membranes with RGD-Grafted Poly(2-oxazoline) for Periodontal Tissue Engineering.

Bone tissue defects resulting from periodontal disease are often treated using guided tissue regeneration (GTR). The barrier membranes utilized here should prevent soft tissue infiltration into the bony defect and simultaneously support bone regeneration. In this study, we designed a degradable poly(l-lactide-co-glycolide) (PLGA) membrane that was surface-modified with cell adhesive arginine-glycine-aspartic acid (RGD) motifs. For a novel method of membrane manufacture, the RGD motifs were coupled with the non-ionic amphiphilic polymer poly(2-oxazoline) (POx). The RGD-containing membranes were then prepared by solvent casting of PLGA, POx coupled with RGD (POx_RGD), and poly(ethylene glycol) (PEG) solution in methylene chloride (DCM), followed by DCM evaporation and PEG leaching. Successful coupling of RGD to POx was confirmed spectroscopically by Raman, Fourier transform infrared in attenuated reflection mode (FTIR-ATR), and X-ray photoelectron (XPS) spectroscopy, while successful immobilization of POx_RGD on the membrane surface was confirmed by XPS and FTIR-ATR. The resulting membranes had an asymmetric microstructure, as shown by scanning electron microscopy (SEM), where the glass-cured surface was more porous and had a higher surface area then the air-cured surface. The higher porosity should support bone tissue regeneration, while the air-cured side is more suited to preventing soft tissue infiltration. The behavior of osteoblast-like cells on PLGA membranes modified with POx_RGD was compared to cell behavior on PLGA foil, non-modified PLGA membranes, or PLGA membranes modified only with POx. For this, MG-63 cells were cultured for 4, 24, and 96 h on the membranes and analyzed by metabolic activity tests, live/dead staining, and fluorescent staining of actin fibers. The results showed bone cell adhesion, proliferation, and viability to be the highest on membranes modified with POx_RGD, making them possible candidates for GTR applications in periodontology and in bone tissue engineering.

Covalent linkage of sulfated hyaluronan to the collagen scaffold Mucograft® enhances scaffold stability and reduces proinflammatory macrophage activation in vivo.

Sulfated glycosaminoglycans (sGAG) show interaction with biological mediator proteins. Although collagen-based biomaterials are widely used in clinics, their combination with high-sulfated hyaluronan (sHA3) is unexplored. This study aims to functionalize a collagen-based scaffold (Mucograft®) with sHA3 via electrostatic (sHA3/PBS) or covalent binding to collagen fibrils (sHA3+EDC/NHS). Crosslinking without sHA3 was used as a control (EDC/NHS Ctrl). The properties of the sHA3-functionalized materials were characterized. In vitro growth factor and cytokine release after culturing with liquid platelet-rich fibrin was performed by means of ELISA. The cellular reaction to the biomaterials was analyzed in a subcutaneous rat model. The study revealed that covalent linking of sHA3 to collagen allowed only a marginal release of sHA3 over 28 days in contrast to electrostatically bound sHA3. sHA3+EDC/NHS scaffolds showed reduced vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGF-ß1) and enhanced interleukin-8 (IL-8) and epithelial growth factor (EGF) release in vitro compared to the other scaffolds. Both sHA3/PBS and EDC/NHS Ctrl scaffolds showed a high proinflammatory reaction (M1: CD-68+/CCR7+) and induced multinucleated giant cell (MNGC) formation in vivo. Only sHA3+EDC/NHS scaffolds reduced the proinflammatory macrophage M1 response and did not induce MNGC formation during the 30 days. SHA3+EDC/NHS scaffolds had a stable structure in vivo and showed sufficient integration into the implantation region after 30 days, whereas EDC/NHS Ctrl scaffolds underwent marked disintegration and lost their initial structure. In summary, functionalized collagen (sHA3+EDC/NHS) modulates the inflammatory response and is a promising biomaterial as a stable scaffold for full-thickness skin regeneration in the future.

Chemically modified glycosaminoglycan derivatives as building blocks for biomaterial coatings and hydrogels.

Tissue regeneration is regulated by the cellular microenvironment, e.g. the extracellular matrix. Here, sulfated glycosaminoglycans (GAG), are of vital importance interacting with mediator proteins and influencing their biological activity. Hence, they are promising candidates for controlling tissue regeneration. This review addresses recent achievements regarding chemically modified GAG as well as collagen/GAG-based coatings and hydrogels including (i) chemical functionalization strategies for native GAG, (ii) GAG-based biomaterial strategies for controlling cellular responses, (iii) (bio)chemical methods for characterization and iv) protein interaction profiles and attained tissue regeneration in vitro and in vivo. The potential of GAG for bioinspired, functional biomaterials is highlighted.

Hyaluronan/Collagen Hydrogels with Sulfated Glycosaminoglycans Maintain VEGF165 Activity and Fine-Tune Endothelial Cell Response.

In order to restore the regeneration capacity of large-size vascularized tissue defects, innovative biomaterial concepts are required. Vascular endothelial growth factor (VEGF165) is a key factor of angiogenesis interacting with sulfated glycosaminoglycans (sGAG) within the extracellular matrix. As this interplay mainly controls and directs the biological activity of VEGF165, we used chemically modified sGAG derivatives to evaluate the structural requirements of sGAG for controlling and tuning VEGF165 function and to translate these findings into the design of biomaterials. The in-depth analysis of this interaction by surface plasmon resonance and ELISA studies in combination with molecular modeling stressed the relevance of the substitution position, degree of sulfation, and carbohydrate backbone of GAG. Acrylated hyaluronan (HA-AC)/collagen (coll)-based hydrogels containing cross-linked acrylated, sulfated hyaluronan (sHA-AC) derivatives with different substitution patterns or an acrylated chondroitin sulfate (CS-AC) derivative function as multivalent carbohydrate-based scaffolds for VEGF165 delivery with multiple tuning capacities. Depending on the substitution pattern of sGAG, the release of biologically active VEGF165 was retarded in a defined manner compared to pure HA/coll gels, which further controlled the VEGF165-induced stimulation of endothelial cell proliferation and extended morphology of cells. This indicates that sGAG can act as modulators of protein interaction profiles of HA/coll hydrogels. In addition, sHA-AC-containing gels with and even without VEGF165 strongly stimulate endothelial cell proliferation compared to gels containing only CS-AC or HA-AC. Thus, HA/coll-based hydrogels containing cross-linked sHA-AC are biomimetic materials able to directly influence endothelial cells in vitro, which might translate into an improved healing of injured vascularized tissues.

The influence of different artificial extracellular matrix implant coatings on the regeneration of a critical size femur defect in rats.

Resorbable biomaterials based on artificial extracellular matrices (aECM) represent promising scaffolds for the treatment of large bone defects. Here, we investigated various glycosaminoglycan (GAG) derivatives of varying sulfation degree with respect to their influence on in vivo bone healing. The materials used in this study consisted of GAG-coated degradable polycaprolactone-co-lactide (PCL). Critical size femur defects in rats were filled with autologous bone serving as positive control or the respective coated or uncoated PCL scaffolds. After 2 and 12 weeks, progress in the healing process was investigated by analyzing the new bone matrix formation, the collagen content and hydroxyapatite formation by using micro-computed tomography (µCT), biomechanical testing, nuclear magnetic resonance spectroscopy (NMR) and histology. The sulfated GAG coating contributed substantially to bone regeneration, increased collagen synthesis and initiated mineralization of the organic matrix. Most substantial collagen production was detected in scaffolds coated with chondroitin sulfate. Scaffolds coated with hypersulfated hyaluronan induced formation of new bone volume comparable to what was observed in the positive control. GAG differing in the sugar backbone and degree of sulfation modulate the healing process at different times, eventually leading to improved bone healing.

Sodium alendronate loaded poly(l-lactide- co-glycolide) microparticles immobilized on ceramic scaffolds for local treatment of bone defects.

Bone tissue regeneration in critical-size defects is possible after implantation of a 3D scaffold and can be additionally enhanced once the scaffold is enriched with drugs or other factors supporting bone remodelling and healing. Sodium alendronate (Aln), a widely used anti-osteoporosis drug, exhibits strong inhibitory effect on bone resorption performed by osteoclasts. Thus, we propose a new approach for the treatment of bone defects in craniofacial region combining biocompatible titanium dioxide scaffolds and poly(l-lactide-co-glycolide) microparticles (MPs) loaded with Aln. The MPs were effectively attached to the surface of the scaffolds' pore walls by human recombinant collagen. Drug release from the scaffolds was characterized by initial burst (24 ± 6% of the drug released within first 24 h) followed by a sustained release phase (on average 5 µg of Aln released per day from Day 3 to Day 18). In vitro tests evidenced that Aln at concentrations of 5 and 2.5 µg/ml was not cytotoxic for MG-63 osteoblast-like cells (viability between 81 ± 6% and 98 ± 3% of control), but it prevented RANKL-induced formation of osteoclast-like cells from macrophages derived from peripheral blood mononuclear cells, as shown by reduced fusion capability and decreased tartrate-resistant acid phosphatase 5b activity (56 ± 5% reduction in comparison to control after 8 days of culture). Results show that it is feasible to design the scaffolds providing required doses of Aln inhibiting osteoclastogenesis, reducing osteoclast activity, but not affecting osteoblast functions, which may be beneficial in the treatment of critical-size bone tissue defects.

Success and side effects of different treatment options in the low current attack of bacterial biofilms on titanium implants.

The long-term success of peri-implantitis treatments is generally insufficient. Attacking the bacteria on the titanium implant surface using electrochemical polarization could be an alternative approach. In this study an E. coli biofilm in phosphate buffered saline was treated with low current densities (0.25 to 2 mA/cm2) using anodic, cathodic, or combined polarization regimes, either alone or with the antiseptic chlorhexidine. The antibacterial effect was assessed using LIVE/DEAD® staining and through quantification of viable bacteria, sample surfaces were characterized pre- and post-treatment with electrochemical impedance spectroscopy. All polarization treatments had an antibacterial effect that increased with current density, with at least 1 mA/cm2 necessary to reduce colony forming units by four orders of magnitude. Cathodic treatment was slightly superior to anodic treatment, and there was no difference between alternating polarization and single-type polarization. Neither treatment resulted in a significant detachment of bacteria, but combination with chlorhexidine improved the antibacterial effect synergistically. The use of chloride containing electrolytes is not recommended in this context. The low current densities used here were not sufficient to generate adequate bactericidal chlorine reactive species, but first signs of pitting corrosion were already detected for anodic polarization at 1 mA/cm2.

Dual Action of Sulfated Hyaluronan on Angiogenic Processes in Relation to Vascular Endothelial Growth Factor-A.

Pathological healing characterized by abnormal angiogenesis presents a serious burden to patients' quality of life requiring innovative treatment strategies. Glycosaminoglycans (GAG) are important regulators of angiogenic processes. This experimental and computational study revealed how sulfated GAG derivatives (sGAG) influence the interplay of vascular endothelial growth factor (VEGF)165 and its heparin-binding domain (HBD) with the signaling receptor VEGFR-2 up to atomic detail. There was profound evidence for a HBD-GAG-HBD stacking configuration. Here, the sGAG act as a "molecular glue" leading to recognition modes in which sGAG interact with two VEGF165-HBDs. A 3D angiogenesis model demonstrated the dual regulatory role of high-sulfated derivatives on the biological activity of endothelial cells. While GAG alone promote sprouting, they downregulate VEGF165-mediated signaling and, thereby, elicit VEGF165-independent and -dependent effects. These findings provide novel insights into the modulatory potential of sGAG derivatives on angiogenic processes and point towards their prospective application in treating abnormal angiogenesis.

Biomaterials in repairing rat femoral defects: In vivo insights from small animal positron emission tomography/computed tomography (PET/CT) studies.

Biomaterials coated with artificial extracellular matrices (aECM) are intended to support the healing of critical size bone defects. This pilot study investigated (i) the feasibility of dual-tracer PET/CT imaging for functional characterization of biomaterial-assisted bone healing in a rat femoral defect model and (ii) the bone healing ability of polycaprolactone-co-lactide (PCL) scaffolds, coated with various aECM consisting of collagen type I (Col) and glycosaminoglycans (GAGs) such as chondroitin sulfate (CS) or polysulfated hyaluronan (sHA3). [18F]FDG and [18F]fluoride PET 4 and 8 weeks after implantation of aECM-coated PCL scaffolds, which provide an in vivo measure of cellular activation and bone mineralization, respectively, combined with CT imaging (in vivo/ex vivo) and histological/immunohistochemical investigations (ex vivo) showed that coating with CS in particular is beneficial for bone healing. The possible involvement of COX-2 and TGase 2, key enzymes of inflammation and ECM remodeling, in these processes offers starting points for targeted adjuvant therapy in the course of various bone healing phases. Our investigations show the feasibility of the selected dual-tracer approach for PET/CT imaging. In principle, this approach can be extended by further PET tracers for the functional characterization of physiological processes such as hypoxia/reperfusion or selected molecular players.

Recapitulating bone development events in a customised bioreactor through interplay of oxygen tension, medium pH, and systematic differentiation approaches.

Bone development and homeostasis are intricate processes that require co-existence and dynamic interactions among multiple cell types. However, controlled dynamic niches that derive and support stable propagation of these cells from single stem cell source is not sustainable in conventional culturing vessels. In bioreactor cultures that support dynamic niches, the limited source and stability of growth factors are often a major limiting factor for long-term in vitro cultures. Hence, alternative growth factor-free differentiation approaches are designed and their efficacy to achieve different osteochondral cell types is investigated. Briefly, a dynamic niche is achieved by varying medium pH, oxygen tension (pO2 ) distribution in bioreactor, initiating chondrogenic differentiation with chondroitin sulphate A (CSA), and implementing systematic differentiation regimes. In this study, we demonstrated that CSA is a potent chondrogenic inducer, specifically in combination with acidic medium and low pO2 . Further, endochondral ossification is recapitulated through a systematic chondrogenic-osteogenic (ch-os) differentiation regime, and multiple osteochondral cell types are derived. Chondrogenic hypertrophy was also enhanced specifically in high pO2 regions. Consequently, mineralised constructs with higher structural integrity, volume, and tailored dimensions are achieved. In contrast, a continuous osteogenic differentiation regime (os-os) has derived compact and dense constructs, whereas a continuous chondrogenic differentiation regime (ch-ch) has attenuated construct mineralisation and impaired development. In conclusion, a growth factor-free differentiation approach is achieved through interplay of pO2 , medium pH, and systematic differentiation regimes. The controlled dynamic niches have recapitulated endochondral ossification and can potentially be exploited to derive larger bone constructs with near physiological properties.

Hyaluronan/collagen hydrogel matrices containing high-sulfated hyaluronan microgels for regulating transforming growth factor-ß1.

Hyaluronan (HA)-based microgels generated in a microfluidic approach, containing an artificial extracellular matrix composed of collagen and high-sulfated hyaluronan (sHA3), were incorporated into a HA/collagen-based hydrogel matrix. This significantly enhanced the retention of noncrosslinked sHA3 within the gels enabling controlled sHA3 presentation. Gels containing sHA3 bound higher amounts of transforming growth factor-ß1 (TGF-ß1) compared to pure HA/collagen hydrogels. Moreover, the presence of sHA3-containing microgels improved the TGF-ß1 retention within the hydrogels. These findings are promising for developing innovative biomaterials with adjustable sHA3 release and growth factor interaction profiles to foster skin repair, e.g., by rebalancing dysregulated TGF-ß1 levels.

Sulfated hyaluronan-containing artificial extracellular matrices promote proliferation of keratinocytes and melanotic phenotype of melanocytes from the outer root sheath of hair follicles.

The aim of this work was to establish improved cultivation conditions for human keratinocytes (HUKORS) and melanocytes (HUMORS) from the outer root sheath (ORS) of human hair follicles for purposes of generating an epidermal graft. To this end, the cells were cultivated on artificial extracellular matrix coatings composed of collagen (COLL) and hyaluronan (HA) with varying sulfation degrees. HUKORS and HUMORS were characterized based on their morphology and proliferation, marker gene expression, protein expression and melanin content in melanocytes. Depending on the sulfation degree, the matrices provided a favorable proliferation environment for HUKORS and improved the balance between proliferation and the exertion of melanotic phenotype (gene expression and melanin content) in HUMORS. Based on the increased gene expression of microphthalmia-associated transcription factor, as well as the downstream-affected melanotic genes premelanosome protein and tyrosinase in HUMORS cultivated on collagen matrices with high-sulfated HA, we assume that sulfated HA enhanced melanotic phenotype either by directly binding the CD44 receptor or by concentrating signaling mediators on site. Being a promising cultivation environment for both HUKORS and HUMORS, collagen matrices with sulfated HA have a potential of significantly improving the development of ORS-based epidermal grafts. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1640-1653, 2019.

Immobilization of Denosumab on Titanium Affects Osteoclastogenesis of Human Peripheral Blood Monocytes.

Immobilization of proteins has been examined to improve implant surfaces. In this study, titanium surfaces were modified with nanofunctionalized denosumab (cDMAB), a human monoclonal anti-RANKL IgG. Noncoding DNA oligonucleotides (ODN) served as linker molecules between titanium and DMAB. Binding and release experiments demonstrated a high binding capacity of cDMAB and continuous release. Human peripheral mononuclear blood cells (PBMCs) were cultured in the presence of RANKL/MCSF for 28 days and differentiated into osteoclasts. Adding soluble DMAB to the medium inhibited osteoclast differentiation. On nanofunctionalized titanium specimens, the osteoclast-specific TRAP5b protein was monitored and showed a significantly decreased amount on cDMAB-titanium in PBMCs + RANKL/MCSF. PBMCs on cDMAB-titanium also changed SEM cell morphology. In conclusion, the results indicate that cDMAB reduces osteoclast formation and has the potential to reduce osteoclastogenesis on titanium surfaces.

Synergistic effect of bimodal pore distribution and artificial extracellular matrices in polymeric scaffolds on osteogenic differentiation of human mesenchymal stem cells.

The main objective of this study was to enhance the biological performance of resorbable polymeric scaffolds for bone tissue engineering. Specifically, we focused on both microstructure and surface modification of the scaffolds to augment adhesion, proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSC). Moreover, a new cell seeding method assuring 90% seeding efficiency on the scaffolds was developed. Poly(l­lactide­co­glycolide) (PLGA) scaffolds with monomodal and bimodal pore distribution were produced by solvent casting/phase separation followed by porogen leaching and modified with artificial extracellular matrices (aECM) consisting of collagen type I and high sulphated hyaluronan (sHya). The application of two porogens resulted in bimodal pore distribution within the PLGA scaffolds as shown by scanning electron microscopy and microcomputer tomography. Two types of pores with diameters 400-600 µm and 2-20 µm were obtained. The scaffolds were successfully coated with a homogenous layer of aECM as shown by Sirius red and toluidine blue staining. In vitro study showed that presence of bimodal pore distribution in combination with collagen/sHya did not significantly influence hMSC proliferation and early osteogenic differentiation compared to scaffolds with monomodal pore distribution. However, it enhanced mineralization as well as the expression of Runt-related transcription factor 2, osteopontin and bone sialoprotein II. As a result PLGA scaffolds with bimodal pore distribution modified with collagen/sHya can be considered as prospective material promoting bone regeneration.

Hyaluronan/collagen hydrogels containing sulfated hyaluronan improve wound healing by sustained release of heparin-binding EGF-like growth factor.

Functional biomaterials that are able to bind, stabilize and release bioactive proteins in a defined manner are required for the controlled delivery of such to the desired place of action, stimulating wound healing in health-compromised patients. Glycosaminoglycans (GAG) represent a very promising group of components since they may be functionally engineered and are well tolerated by the recipient tissues due to their relative immunological inertness. Ligands of the Epidermal Growth Factor (EGF) receptor (EGFR) activate keratinocytes and dermal fibroblasts and, thus, contribute to skin wound healing. Heparin-binding EGF-like growth factor (HB-EGF) bound to GAG in biomaterials (e.g. hydrogels) might serve as a reservoir that induces prolonged activation of the EGF receptor and to recover disturbed wound healing. Based on previous findings, the capacity of hyaluronan (HA) and its sulfated derivatives (sHA) to bind and release HB-EGF from HA/collagen-based hydrogels was investigated. Docking and molecular dynamics analysis of a molecular model of HB-EGF led to the identification of residues in the heparin-binding domain of the protein being essential for the recognition of GAG derivatives. Furthermore, molecular modeling and surface plasmon resonance (SPR) analyses demonstrated that sulfation of HA increases binding strength to HB-EGF thus providing a rationale for the development of sHA-containing hydrogels. In line with computational observations and in agreement with SPR results, gels containing sHA displayed a retarded HB-EGF release in vitro compared to pure HA/collagen gels. Hydrogels containing HA and collagen or a mixture with sHA were shown to bind and release bioactive HB-EGF over at least 72 h, which induced keratinocyte migration, EGFR-signaling and HGF expression in dermal fibroblasts. Importantly, hydrogels containing sHA strongly increased the effectivity of HB-EGF in inducing epithelial tip growth in epithelial wounds shown in a porcine skin organ culture model. These findings suggest that hydrogels containing HA and sHA can be engineered for smart and effective wound dressings. STATEMENT OF SIGNIFICANCE: Immobilization and sustained release of recombinant proteins from functional biomaterials might overcome the limited success of direct application of non-protected solute growth factors during the treatment of impaired wound healing. We developed HA/collagen-based hydrogels supplemented with acrylated sulfated HA for binding and release of HB-EGF. We analyzed the molecular basis of HB-EGF interaction with HA and its chemical derivatives by in silico modeling and surface plasmon resonance. These hydrogels bind HB-EGF reversibly. Using different in vitro assays and organ culture we demonstrate that the introduction of sulfated HA into the hydrogels significantly increases the effectivity of HB-EGF action on target cells. Therefore, sulfated HA-containing hydrogels are promising functional biomaterials for the development of mediator releasing wound dressings.

Mono(2-ethylhexyl) phthalate (MEHP) and mono(2-ethyl-5-oxohexyl) phthalate (MEOHP) but not di(2-ethylhexyl) phthalate (DEHP) bind productively to the peroxisome proliferator-activated receptor γ.

RATIONALE: The most frequently occurring phthalate, di(2-ethylhexyl) phthalate (DEHP), causes adverse effects on glucose homeostasis and insulin sensitivity in several cell models and epidemiological studies. However, thus far, there is no information available on the molecular interaction of phthalates and one of the key regulators of the metabolism, the peroxisome proliferator-activated receptor gamma (PPARγ). Since the endogenous ligand of PPARγ, 15-deoxy-delta-12,14-prostaglandin J2 (15Δ-PGJ2 ), features structural similarity to DEHP and its main metabolites produced in human hepatic metabolism, mono(2-ethylhexyl) phthalate (MEHP) and mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), we tested the hypothesis of direct interactions between PPARγ and DEHP or its transformation products. METHODS: Hydrogen/deuterium exchange mass spectrometry (HDX-MS) and docking were conducted to obtain structural insights into the interactions and surface plasmon resonance (SPR) analysis to reveal information about binding levels. To confirm the activation of PPARγ upon ligand binding on the cellular level, the GeneBLAzer® bioassay was performed. RESULTS: HDX-MS and SPR analyses demonstrated that the metabolites MEHP and MEOHP, but not DEHP itself, bind to the ligand binding pocket of PPARγ. This binding leads to typical activation-associated conformational changes, as observed with its endogenous ligand 15Δ-PGJ2 . Furthermore, the reporter gene assay confirmed productive interaction. DEHP was inactive up to a concentration of 14 µM, while the metabolites MEHP and MEOHP were active at low micromolar concentrations. CONCLUSIONS: In summary, this study gives structural insights into the direct interaction of PPARγ with MEHP and MEOHP and shows that the DEHP transformation products may modulate the lipid metabolism through PPARγ pathways.

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.

One step 3D printing of surface functionalized composite scaffolds for tissue engineering applications.

their surface properties. A main challenge in this area is the development of processing routes enabling for a simple but efficient surface design of complex shaped geometries. Against this background, this work aimed at the implementation of self-assembly principles for surface functionalization of 3D-printed poly(lactic-co-glycolic acid) (PLGA)-based constructs with macro- and microporous geometries via precision extruding deposition. METHODS: Three-component melts from PLGA, CaCO3 and amphiphilic polymers (poly(2-oxazoline) block copolymer) were printed and their bulk and surface properties were studied. RESULTS: Melts with up to 30 mass % of CaCO3 could be successfully printed with homogeneously distributed mineral particles. PLGA degradation during the printing process was temperature and time dependent: the molecular weight reached 10 to 15% of the initial values after ca. 120 min of heat exposure. Filament surfaces from melts containing CaCO3 show an increasing microroughness along with increasing CaCO3 content. Surface roughness and amphiphilic polymer content improve scaffold wettability with both factors showing synergistic effects. The CaCO3 content of the melts affected the inner filament structure during in vitro degradation in PBS, resulting in a homogeneous mineral particle-associated microporosity for mineral contents of 20 mass % and above. CONCLUSIONS: These results provide novel insights into the behavior of three-component melts from PLGA, CaCO3 and amphiphilic polymers during precision extruding deposition and show for the first time that self-assembly processes can be used to tailor scaffolds surface properties under such processing conditions.

Sulfation degree not origin of chondroitin sulfate derivatives modulates keratinocyte response.

Chondroitin sulfate (CS) sulfation-dependently binds transforming growth factor-ß1 (TGF-ß1) and chronic wounds often accompany with epidermal hyperproliferation due to downregulated TGF-ß signaling. However, the impact of CS on keratinocytes is unknown. Especially biotechnological-chemical strategies are promising to replace animal-derived CS. Thus, this study aims to evaluate the effects of CS derivatives on the interaction with vascular endothelial growth factor-A (VEGF-A) and on keratinocyte response. Over-sulfated CS (sCS3) interacts stronger with VEGF-A than CS. Furthermore, collagen coatings with CS variants are prepared by in vitro fibrillogenesis. Stability analyses demonstrate that collagen is firmly integrated, while the fibril diameters decrease with increasing sulfation degree. CS variants sulfation-dependently decelerate keratinocyte (HaCaT) migration and proliferation in a scratch assay. HaCaT cultured on sCS3-containing coatings produced increased amounts of solute active TGF-ß1 which could be translated into biomaterials able to decrease epidermal hyperproliferation in chronic wounds. Overall, semi-synthetic and natural CS yield to comparable responses.

Utilizing DNA for functionalization of biomaterial surfaces.

DNA sequences are widely used for gene transfer into cells including a number of substrate surface-based supporting systems, but due to its singular structure property profile, DNA also offers multiple options for noncanonical applications. The special case of using DNA and oligodeoxyribonucleotide (ODN) structures for surface functionalization of biomedical implants is summarized here with the major focus on (a) immobilization or anchoring of nucleic acid structures on substrate surfaces, (b) incorporation of biologically active molecules (BAM) into such systems, and (c) biological characteristics of the resulting surfaces in vitro and in vivo. Sterilizations issues, important for potential clinical applications, are also considered.