Biomaterials (v.27, #32)
Synthesis and characterization of hyperbranched polyglycerol hydrogels
by Marion H.M. Oudshoorn; Robert Rissmann; Joke A. Bouwstra; Wim E. Hennink (pp. 5471-5479).
Hyperbranched polyglycerol (HyPG; Mn 2000g/mol) was derivatized with glycidyl methacrylate (GMA) in dimethyl sulfoxide using 4-( N,N-dimethylamino)pyridine as a catalyst to obtain methacrylated HyPG (HyPG-MA). The degree of substitution (DS, the percentage of derivatized hydroxyl groups), established by NMR and RP-HPLC, was fully controlled in the range of 0.7–70 by varying the molar ratio of GMA to HyPG in the reaction mixture. This indicates that for e.g. a DS of 28, 9 out of the 32 hydroxyl groups of a HyPG molecule were esterified with methacryloyl groups. Under the selected conditions, the reaction reached an equilibrium within 4h. Furthermore, it was demonstrated that under the applied conditions the reaction was reversible. Hydrogels were obtained by crosslinking HyPG-MA in aqueous solutions using potassium peroxodisulfate (KPS) and N,N,N′,N′-tetramethylethylenediamine (TEMED) as initiator and catalyst, respectively. Within 10min, 99% of the methacryloyl groups were polymerized. Rheological analysis showed that the storage modulus of these gels could be tailored by varying the concentration of HyPG-MA in the aqueous solution as well as by the DS. Moreover, the obtained hydrogels have a limited swelling capacity indicating that rather dimensionally stable networks were obtained. As an alternative for radical polymerization with KPS and TEMED, the HyPG-MA could also be crosslinked by photopolymerization using Irgacure 2959 as photoinitiator. A methacrylate conversion of 99% was obtained within 3min of illumination. As for the gels prepared with KPS and TEMED, networks formed by photopolymerization also had a high shear storage modulus and showed limited swelling. Hydrogels based on HyPG have great potential as drug delivery matrices and for tissue engineering purposes.
Keywords: Hyperbranched polyglycerol; Hydrogels; Methacrylation; Polymerization; Viscoelasticity
Freeze casting of hydroxyapatite scaffolds for bone tissue engineering
by Sylvain Deville; Eduardo Saiz; Antoni P. Tomsia (pp. 5480-5489).
Although extensive efforts have been put into the development of porous scaffolds for bone regeneration, with encouraging results, all porous materials have a common limitation: the inherent lack of strength associated with porosity. Hence, the development of porous hydroxyapatite scaffolds has been hindered to non-load bearing applications. We report here how freeze casting can be applied to synthesize porous scaffolds exhibiting unusually high compressive strength, e.g. up to 145MPa for 47% porosity and 65MPa for 56% porosity. The materials are characterized by well-defined pore connectivity along with directional and completely open porosity. Various parameters affecting the porosity and compressive strength have been investigated, including initial slurry concentration, freezing rate, and sintering conditions. The implications and potential application as bone substitute are discussed. These results might open the way for hydroxyapatite-based materials designed for load-bearing applications. The biological response of these materials is yet to be tested.
Keywords: Hydroxyapatite; Bone tissue engineering; Ceramic structure; Porosity; Freeze casting
Chemical and biological properties of supramolecular polymer systems based on oligocaprolactones
by Patricia Y.W. Dankers; Ellen N.M. van Leeuwen; Gaby M.L. van Gemert; A.J.H. Spiering; Martin C. Harmsen; Linda A. Brouwer; Henk M. Janssen; Anton W. Bosman; Marja J.A. van Luyn; E.W. Meijer (pp. 5490-5501).
We show that materials with a diverse range of mechanical and biological properties can be obtained using a modular approach by simply mixing different ratios of oligocaprolactones that are either end-functionalized or chain-extended with quadruple hydrogen bonding ureido-pyrimidinone (UPy) moieties. The use of two UPy-synthons allows for easy synthesis of UPy-modified polymers resulting in high yields. Comparison of end-functionalized UPy-polymers with chain-extended UPy-polymers shows that these polymers behave distinctively different regarding their material and biological properties. The end-modified UPy-polymer is rather stiff and brittle due to its high crystallinity. Disks made of this material fractures after subcutaneous implantation. The material shows a low inflammatory response which is accompanied by the formation of a fibrous capsule, reflecting the inertness of the material. The chain-extended UPy-material on the contrary is practically free of crystalline domains and shows clear flexible properties. This material deforms after in-vivo implantation, accompanied with cellular infiltration. By mixing both polymers, materials with intermediate properties concerning their mechanical and biological behaviour can be obtained. Surprisingly, a 20:80 mixture of both polymers with the chain-extended UPy-polymer in excess shows flexible properties without visible deformation upon implantation for 42 days. This mixture, a blend formed by intimate mixing through UPy–UPy interaction, also shows a mild tissue response accompanied with the formation of a thin capsule. The material does not become more crystalline upon implantation. Hence, this mixture might be an ideal scaffold material for soft tissue engineering due to its flexibility and diminished fibrous tissue formation, and illustrates the strength of the modular approach.
Keywords: Foreign body response; Polycaprolactone; In vivo test; Scaffold; Self-assembly
Activation of phospholipase D1 by surface roughness of titanium in MG63 osteoblast-like cell
by Myung-Joo Kim; Myung-Un Choi; C.-W. Chang-Whe Kim (pp. 5502-5511).
Although it is recognized that the surface roughness of titanium (Ti) promotes the osteogenic differentiation, the related mechanisms and factors remain elusive. The purpose of this study was to explore the potential correlation among phospholipase D (PLD) activity, Ti surface roughness and subsequent osteoblast differentiation. The machined Ti disks were sandblasted with aluminum oxide particles to produce surfaces of varying roughness (n=160). Normal or transfected MG63cells with PLD genes were cultured on roughened Ti specimens and assayed for PLD, alkaline phosphatase (ALP) and osteocalcin. The statistical significance was evaluated by analysis of variance. The activity, mRNA and protein levels of PLD significantly increased in MG63 cells with a roughness-dependent pattern (P<0.05). The ALP activity and osteocalcin production, promoted by Ti surface roughness, were enhanced by the PLD activator and inhibited by the PLD blocker. It was also found that the PLD1 isoform responds to Ti surface roughness and regulates selectively the ALP activity. These observations strongly suggest that PLD1 mediates the cellular signaling of and modulates osteoblast differentiation induced by Ti surface roughness in MG63 osteoblast-like cell.
Keywords: Ti surface roughness; Osteoblast differentiation; Alkaline phosphatase activity; Osteocalcin production; Phospholipase DAbbreviations; ALP; Alkaline phosphatase; DAG; Diacylglycerol; 2*3-DPG; 2*3-diphosphoglycerate; PA; Phosphatidic acid; PBt; Phosphatidylbutanol; PC; Phosphatidylcholine; PIP2; Phosphatidylinositol 4*5-bisphosphate; PKC; Protein kinase C; PLA2; Phospholipase A; 2; PLC; Phospholipase C; PLD; Phospholipase D
In vitro anti-bacterial and biological properties of magnetron co-sputtered silver-containing hydroxyapatite coating
by W. Chen; Y. Liu; H.S Courtney; M. Bettenga; C.M. Agrawal; J.D. Bumgardner; J.L. Ong (pp. 5512-5517).
Bacterial infection after implant placement is a significant rising complication. In order to reduce the incidence of implant-associated infections, several biomaterial surface treatments have been proposed. In this study, the effect of in vitro antibacterial activity and in vitro cytotoxicity of co-sputtered silver (Ag)-containing hydroxyapatite (HA) coating was evaluated. Deposition was achieved by a concurrent supply of 10W to the Ag target and 300W to the HA target. Heat treatment at 400°C for 4h was performed after 3h deposition. X-ray diffraction, contact angles measurements, and surface roughness were used to characterize the coating surfaces. The RP12 strain of Staphylococcus epidermidis (ATCC 35984) and the Cowan I strain of Staphylococcus aureus were used to evaluate the antibacterial activity of the Ag-HA coatings, whereas human embryonic palatal mesenchyme cells, an osteoblast precursor cell line, were used to evaluate the in vitro cytotoxicity of the coatings. X-ray diffraction analysis performed in this study indicated peaks corresponding to Ag and HA on the co-sputtered Ag-HA surfaces. The contact angles for HA and Ag-HA surfaces were observed to be significantly lower when compared to Ti surfaces, whereas no significant difference in surface roughness was observed for all groups. In vitro bacterial adhesion study indicated a significantly reduced number of S. epidermidis and S. aureus on Ag-HA surface when compared to titanium (Ti) and HA surfaces. In addition, no significant difference in the in vitro cytotoxicty was observed between HA and Ag-HA surfaces. Overall, it was concluded that the creation of a multifunctional surface can be achieved by co-sputtering the osteoconductive HA with antibacterial Ag.
Keywords: Hydroxyapatite coating; Cytotoxicity; Bacterial adhesion; Silver
The effect of micropores in the surface of temperature-responsive culture inserts on the fabrication of transplantable canine oral mucosal epithelial cell sheets
by Daisuke Murakami; Masayuki Yamato; Kohji Nishida; Takeshi Ohki; Ryo Takagi; Joseph Yang; Hideo Namiki; Teruo Okano (pp. 5518-5523).
Primary canine oral mucosal epithelial cells were cultured on temperature-responsive dishes and cell culture inserts to fabricate transplantable epithelial cell sheets. When 3T3 feeder layers and fetal bovine serum were eliminated from dish culture, the harvested cell sheets became significantly more fragile. In contrast, when epithelial cells were cultured on inserts having submicron-scale pores, cell sheet fragility was eliminated. Keratin expression profiles showed no differences among the harvested cell sheets, but the expression of p63, a putative stem/progenitor marker, was strongly dependent on the presence of 3T3 feeder layers and serum. These results suggest that the maintenance of stem/progenitor cells is influenced by the apical/basal supply of nutrients as well as culture supplements.
Keywords: Cell culture; Co-culture; Epithelial cell; Stem cell; Thermally responsive material
Modulation of anisotropy in electrospun tissue-engineering scaffolds: Analysis of fiber alignment by the fast Fourier transform
by Chantal Ayres; Gary L. Bowlin; Scott C. Henderson; Leander Taylor; Jackie Shultz; John Alexander; Todd A. Telemeco; David G. Simpson (pp. 5524-5534).
We describe the use of the fast Fourier transform (FFT) in the measurement of anisotropy in electrospun scaffolds of gelatin as a function of the starting conditions. In electrospinning, fiber alignment and overall scaffold anisotropy can be manipulated by controlling the motion of the collecting mandrel with respect to the source electrospinning solution. By using FFT to assign relative alignment values to an electrospun matrix it is possible to systematically evaluate how different processing variables impact the structure and material properties of a scaffold. Gelatin was suspended at varying concentrations (80, 100, 130, 150mg/ml) and electrospun from 2,2,2 trifluoroethanol onto rotating mandrels (200–7000 RPM). At each starting concentration, fiber diameter remained constant over a wide range of mandrel RPM. Scaffold anisotropy developed as a function of fiber diameter and mandrel RPM. The induction of varying degrees of anisotropy imparted distinctive material properties to the electrospun scaffolds. The FFT is a rapid method for evaluating fiber alignment in tissue-engineering materials.
Keywords: Electrospinning; Anisotropy; Gelatin; ECM; Fast Fourier transform; Material properties
Mineralization capacity of Runx2/Cbfa1-genetically engineered fibroblasts is scaffold dependent
by Jennifer E. Phillips; Dietmar W. Hutmacher; Robert E. Guldberg; A.J. Garcia Andrés J. García (pp. 5535-5545).
Development of tissue-engineered constructs for skeletal regeneration of large critical-sized defects requires the identification of a sustained mineralizing cell source and careful optimization of scaffold architecture and surface properties. We have recently reported that Runx2-genetically engineered primary dermal fibroblasts express a mineralizing phenotype in monolayer culture, highlighting their potential as an autologous osteoblastic cell source which can be easily obtained in large quantities. The objective of the present study was to evaluate the osteogenic potential of Runx2-expressing fibroblasts when cultured in vitro on three commercially available scaffolds with divergent properties: fused deposition-modeled polycaprolactone (PCL), gas-foamed polylactide- co-glycolide (PLGA), and fibrous collagen disks. We demonstrate that the mineralization capacity of Runx2-engineered fibroblasts is scaffold dependent, with collagen foams exhibiting ten-fold higher mineral volume compared to PCL and PLGA matrices. Constructs were differentially colonized by genetically modified fibroblasts, but scaffold-directed changes in DNA content did not correlate with trends in mineral deposition. Sustained expression of Runx2 upregulated osteoblastic gene expression relative to unmodified control cells, and the magnitude of this expression was modulated by scaffold properties. Histological analyses revealed that matrix mineralization co-localized with cellular distribution, which was confined to the periphery of fibrous collagen and PLGA sponges and around the circumference of PCL microfilaments. Finally, FTIR spectroscopy verified that mineral deposits within all Runx2-engineered scaffolds displayed the chemical signature characteristic of carbonate-containing, poorly crystalline hydroxyapatite. These results highlight the important effect of scaffold properties on the capacity of Runx2-expressing primary dermal fibroblasts to differentiate into a mineralizing osteoblastic phenotype for bone tissue engineering applications.
Keywords: Runx2/Cbfa1; Osteoblast; Fibroblast; Osteogenic differentiation; Scaffold; Bone tissue engineering
The evaluation of a small-diameter polysaccharide-based arterial graft in rats
by Marc Chaouat; Catherine Le Visage; Aude Autissier; Frederic Chaubet; Didier Letourneur (pp. 5546-5553).
Cardiovascular diseases may require surgery such as arterial bypasses that are usually performed with synthetic PTFE and Dacron grafts with diameter above 6mm. For smaller diameter replacement, healthy vascular tissue from the patient is not always available to carry out this type of graft. The purpose of this study was to evaluate the effectiveness of a small-diameter polysaccharide-based scaffold as an alternative arterial replacement. Tubular polysaccharide-based grafts of 2mm internal diameter were prepared and moulded by a cross-linking technique. Fifteen Wistar adult rats underwent infrarenal aortic bypass with these grafts using microsurgical techniques. Grafts withstood aortic blood pressure and exhibited physiological blood flow, as evaluated with ultrasound techniques and angiographies at 4 and 8 weeks post-surgery. Harvested grafts were morphologically evaluated by light microscopy and immunohistochemistry. Neointima formation at 8 weeks was evidenced through collagen deposition and smooth muscle-like cells circumferential growth on the luminal surface without intimal hyperplasia or aneurysm formation. In conclusion, we described a 2mm polysaccharide-based arterial material being investigated in vivo and demonstrating patency for up to 8 weeks post-surgery with neointima formation and absence of intimal hyperplasia.
Keywords: Hydrogel; Arterial tissue engineering; Polysaccharide; Vascular grafts; In vivo test
Development of an accommodating intra-ocular lens—In vitro prevention of re-growth of pig and rabbit lens capsule epithelial cells
by T.G. Theo G. van Kooten; Steven Koopmans; Thom Terwee; Sverker Norrby; J.M.M. Hooymans; Henk J. Busscher (pp. 5554-5560).
Cataract surgery is routinely performed to replace the clouded lens by a rigid polymeric intra-ocular lens unable to accommodate. By implanting a silicone gel into an intact capsular bag the accommodating properties of the natural lens can be maintained or enhanced. The implantation success of accommodating lenses is hampered by the occurrence of capsular opacification (PCO) due to lens epithelial cell (LEC) growth. In order to prevent LEC proliferation, a treatment regime using actinomycin D, cycloheximide and water was developed. The effectiveness of treatment was analyzed using an in vitro, MTT-based cell culture system and an ex vivo pig eye model in which the implanted lens-in-the-bag is cultured as a whole. LEC were exposed to treatment solutions for 5min, then the cells were allowed to recover and to re-colonize the substratum. MTT conversion by cells was transiently inhibited by cycloheximide dissolved in water and by water alone. Exposure to actinomycin D resulted in a lasting inhibition of MTT conversion and consequently cell proliferation. These in vitro data could not be fully reproduced in the ex vivo pig eye model due to essential differences between both models. Treatment with actinomycin D containing solutions, however, resulted in a nearly complete absence of cells on the capsular wall. The pig eye model is a promising approach to further evaluate the effects of peri-surgical treatment during the accommodating intra-ocular lens implantation.
Keywords: Intra-ocular lens; Lens epithelial cell; Confocal microscopy; Silicone gel; MTT assay; Capsular opacification
Coating of titanium implants with collagen, RGD peptide and chondroitin sulfate
by Stefan Rammelt; Till Illert; Susanne Bierbaum; Dieter Scharnweber; Hans Zwipp; Wolfgang Schneiders (pp. 5561-5571).
Coating of orthopaedic implants with extracellular bone matrix components was performed to enhance bone healing. Titanium pins of 0.8mm diameter were coated with type I collagen (Ti/Coll), RGD peptide (Ti/RGD) or type I collagen and chondroitin sulfate (Ti/Coll/CS). Uncoated pins (Ti) served as control. The pins were inserted as intramedullary nails into the tibia of male adult Wistar rats. Six specimens of each group were retrieved at 4, 7, 14 and 28 days. All implants healed uneventfully without adverse reactions. ED 1-positive macrophages appeared in higher numbers around Ti/RGD at day 4 and around Ti at day 14 after implantation (p<0.05). TRAP-positive osteoclasts and precursors were abundant around Ti/Coll/CS at day 7 (p<0.05). A significant increase in osteopontin-positive osteoblasts was seen around Ti/Coll/CS implants at days 7 and 14, and around Ti/RGD at day 14 (p<0.05). At day 28, 62% of Ti, 76% of Ti/Coll, 85%* of Ti/RGD and 89%* of Ti/CoIl/CS (*p<0.05) implants were covered with newly formed lamellar bone. The addition of extracellular matrix components significantly enhances bone remodelling in the early stages of bone healing around Ti implants, eventually leading to increased new bone formation at the implant surface after 4 weeks.
Keywords: Bone remodelling; Titanium; Collagen; Chondroitin sulfate; RGD peptide
Enhanced bone ingrowth into hydroxyapatite with interconnected pores by Electrical Polarization
by Soichiro Itoh; Satoshi Nakamura; Miho Nakamura; Kenichi Shinomiya; Kimihiro Yamashita (pp. 5572-5579).
Hydroxyapatite (HA) ceramics are used as implants to repair damaged/removed bone, and negative or positive electrical polarization enhances osteoblast and decreases osteoclast activity, respectively, in vivo. We compared the ability of electrically polarized and non-polarized HA with interconnected pores (IPHA) implants to promote bone growth. Polarized or non-treated IPHAs were implanted into the right or left femoral condyle of rabbits ( N=10 in each group), and we performed histological examination, including enzymatic staining for osteoblasts and osteoclasts, 3 and 6 weeks after implantation. We observed improved bone ingrowth and increased osteoblast activity in polarized implants with complete bone penetration into polarized implants occurring as early as 3 weeks after surgery. In contrast, non-polarized implants were not fully ossified at 6 weeks after surgery. Furthermore, positively charged implant regions had decreased osteoclast activity compared to negatively charged or uncharged regions. We propose two different models to explain these observations.
Keywords: Bone ingrowth; Ceramic structure; Electrical stimulation; Hydroxyapatite; Osteoblast; Osteoclast
The effect of α-tocopherol on the oxidation and free radical decay in irradiated UHMWPE
by Ebru Oral; Shannon L. Rowell; Orhun K. Muratoglu (pp. 5580-5587).
We developed a radiation cross-linked ultra-high molecular weight polyethylene (UHMWPE) stabilized with α-tocopherol (Vitamin E) as a bearing material in total joint replacements. The stabilizing effect of α-tocopherol on free radical reactions in UHMWPE is not well understood. We investigated the effect of α-tocopherol on the oxidation and transformation of residual free radicals during real-time aging of α-tocopherol-doped, irradiated UHMWPE ( αTPE) and irradiated UHMWPE (control). Samples were aged at 22°C (room temperature) in air, at 40°C in air and at 40°C in water for 7 months. During the first month, αTPE showed some oxidation at the surface, which stayed constant thereafter. Control exhibited substantial oxidation in the subsurface region, which increased with time. The alkyl/allyl free radicals transformed to oxygen centered ones in both materials; this transformation occurred faster in α-TPE. In summary, the real-time oxidation behavior of α-TPE was consistent with that observed using accelerated aging methods. This new UHMWPE is oxidation resistant and is expected to maintain its properties in the long term.
Keywords: Total hip arthroplasty; Highly cross-linked polyethylene; Oxidation resistance; Free radicals; Biomaterials
Controlled release of sirolimus from a multilayered PLGA stent matrix
by Xintong Wang; Subbu S. Venkatraman; Freddy Y.C. Boey; Joachim S.C. Loo; Lay Poh Tan (pp. 5588-5595).
The release of sirolimus from a bi-layer biodegradable polymeric film is reported in this study. Approved drug-eluting metal stents use a thin polymer coating to control drug release, but the degree of control is limited. In a fully polymeric stent, the use of multilayers allows a range of release kinetics. A bi-layer system, with PLLA as the supporting layer and PLGA as the drug-eluting layer, was used in this study to simulate release of sirolimus from a stent. The results show that the release of sirolimus is diffusion and degradation-controlled, and that the amount of sirolimus loading does not affect its release kinetics. The release of sirolimus is, however, accelerated by the addition of a plasticizer, such as PEG, as water uptake is increased. An increased water uptake increases polymer degradation, and changes the dominant mode of release to degradation-control. The release of sirolimus can, on the other hand, be retarded by using a coating of a biodegradable polyester with a lauryl ester end group. Therefore, multilayered systems offer many options for controlling sirolimus release over months.
Keywords: Sirolimus; Controlled release; PLGA; Multilayered; Degradation control; Diffusion control
Ultra-low sized cross-linked polyvinylpyrrolidone nanoparticles as non-viral vectors for in vivo gene delivery
by Amit Saxena; Subho Mozumdar; Atul Kumar Johri (pp. 5596-5602).
In principle, the technique of gene delivery involves taking complete or parts of genes that can code specific message and delivering them to selected cells in the body. Such a transfer of plasmid DNA into mammalian cells has posed major challenges for gene therapy. This study shows the encapsulation of a plasmid DNA in cross-linked polyvinylpyrrolidone (PVP) nanoparticles of size less than 100nm. This kind of encapsulation provides complete protection to the plasmid DNA from external DNase environment and generates the hope that the resulting formulation can be developed into a potential vector for effective gene delivery. In order to check this potentially, the reporter gene pSV β-gal was encapsulated and in vitro transfection efficiency of this system was found to be nearly 80% compared to the commercially available transfection reagent PolyfectR. Further, in vivo biodistribution studies indicated that this system could be used safely for effective gene delivery.
Keywords: PVP nanoparticles; Non-viral vectors; Transfection; Gene delivery
Alginate-based microcapsules for immunoisolation of pancreatic islets
by Paul de Vos; Marijke M. Faas; Berit Strand; Ricardo Calafiore (pp. 5603-5617).
Transplantation of microencapsulated cells is proposed as a therapy for the treatment of a wide variety of diseases since it allows for transplantation of endocrine cells in the absence of undesired immunosuppression. The technology is based on the principle that foreign cells are protected from the host immune system by an artificial membrane. In spite of the simplicity of the concept, progress in the field of immunoisolation has been hampered for many years due to biocompatibility issues. During the last years important advances have been made in the knowledge of the characteristics and requirements capsules have to meet in order to provide optimal biocompatibility and survival of the enveloped tissue. Novel insight shows that not only the capsules material but also the enveloped cells should be hold responsible for loss of a significant portion of the immunoisolated cells and, thus, failure of the grafts on the long term. Microcapsules without cells can be produced as such that they remain free of any significant foreign body response for prolonged periods of time in both experimental animals and humans. New approaches in which newly discovered inflammatory responses are silenced bring the technology of transplantation of immunoisolated cells close to clinical application.
Keywords: Microencapsulation; Macroencapsulation; Islets; Alginate; Immunoisolation; Vascularization
Three-dimensional cell colonization in a sulfate rich environment
by Jeremy Tillman; Annett Ullm; Sundararajan V. Madihally (pp. 5618-5626).
Glycosaminoglycans (GAGs) have been explored for regenerating various tissues due to their involvement in diverse bioregulatory activity. However, understanding their influence on cell colonization in three-dimension (3-D) has been difficult due to variation in their molecular weight, degree of sulfation, and lack of in vitro models. This research focused on developing an in vitro model and evaluating the influence of MW (5, 10, and 500kDa) of negatively charged dextran sulfate (DS), a semisynthetic GAG analog, on cell colonization. DS was combined with chitosan, a positively charged polymer in solution and porous 3-D matrices were formed inside 24-well plates using controlled rate freezing and lyophilization technique by two schemes: (i) chitosan structures were formed and then allowed to react with DS; (ii) DS was reacted with chitosan in solution and then matrices were formed. Scanning electron microscopy analysis showed that forming matrices after reacting DS with chitosan was more suitable for tissue regeneration. Analysis for the quantity and stability of DS by toluidine blue assay indicated significant presence of DS in the 3-D matrices even after seven days of incubation in phosphate buffered saline solution. Matrices formed by reacting 4% 5kDa, 2% 10kDa and 1% 500kDa DS solution with chitosan had optimum porosity and mechanical stability. Next, 25,000 fibroblasts per matrix were seeded onto 3-D matrices and analyzed for proliferation by MTT-formazan assay, cytoskeletal organization by actin staining, and histological analysis by H/E staining. These results showed that cell growth was better on low MW containing 2-D membranes but high MW DS containing 3-D matrix supported cell growth similar to chitosan. Also, cells showed peripheral actin distribution in 3-D matrices. Analysis of fibronectin binding by ELISA showed negligible binding to all the DS-containing matrices, unlike chitosan. In summary, results show cell colonization on negatively charged matrices, similar to chitosan.
Keywords: Dextran sulfate; Fibronectin; Porous structure; 3-D; Chitosan; Fibroblast