Biomaterials (v.29, #19)

A simple chemical modification method using diazonium coupling chemistry was developed to tailor the structure and hydrophilicity of silk fibroin protein. The extent of modification using several aniline derivatives was characterized using UV–vis and 1H NMR spectroscopies, and the resulting protein structure was analyzed with ATR-FTIR spectroscopy. Introduction of hydrophobic functional groups facilitated rapid conversion of the protein from a random coil to a β-sheet structure, while addition of hydrophilic groups inhibited this process. hMSCs were grown on these modified silks to assess the biocompatibility of these materials. The hydrophilicity of the silk derivatives was found to affect the growth rate and morphology, but hMSCs were able to attach, proliferate and differentiate into an osteogenic lineage on all of the silk derivatives.
Keywords: Silk fibroin; Chemical modification; Diazonium coupling; Tyrosine; Biomaterials; Mesenchymal stem cells;

Physical properties and cellular responses to crosslinkable poly(propylene fumarate)/hydroxyapatite nanocomposites by Kee-Won Lee; Shanfeng Wang; Michael J. Yaszemski; Lichun Lu (2839-2848).
A series of crosslinkable nanocomposites has been developed using hydroxyapatite (HA) nanoparticles and poly(propylene fumarate) (PPF). PPF/HA nanocomposites with four different weight fractions of HA nanoparticles have been characterized in terms of thermal and mechanical properties. To assess surface chemistry of crosslinked PPF/HA nanocomposites, their hydrophilicity and capability of adsorbing proteins have been determined using static contact angle measurement and MicroBCA protein assay kit after incubation with 10% fetal bovine serum (FBS), respectively. In vitro cell studies have been performed using MC3T3-E1 mouse pre-osteoblast cells to investigate the ability of PPF/HA nanocomposites to support cell attachment, spreading, and proliferation after 1, 4, and 7 days. By adding HA nanoparticles to PPF, the mechanical properties of crosslinked PPF/HA nanocomposites have not been increased due to the initially high modulus of crosslinked PPF. However, hydrophilicity and serum protein adsorption on the surface of nanocomposites have been significantly increased, resulting in enhanced cell attachment, spreading, and proliferation after 4 days of cell seeding. These results indicate that crosslinkable PPF/HA nanocomposites are useful for hard tissue replacement because of excellent mechanical strength and osteoconductivity.
Keywords: Poly(propylene fumarate) (PPF); Hydroxyapatite (HA); Nanocomposite; Protein adsorption; Osteoblast response;

The effect of integrin-specific bioactive coatings on tissue healing and implant osseointegration by Timothy A. Petrie; Jenny E. Raynor; Catherine D. Reyes; Kellie L. Burns; David M. Collard; Andrés J. García (2849-2857).
Implant osseointegration, defined as bone apposition and functional fixation, is a requisite for clinical success in orthopaedic and dental applications, many of which are restricted by implant loosening. Modification of implants to present bioactive motifs such as the RGD cell-adhesive sequence from fibronectin (FN) represents a promising approach in regenerative medicine. However, these biomimetic strategies have yielded only marginal enhancements in tissue healing in vivo. In this study, clinical-grade titanium implants were grafted with a non-fouling oligo(ethylene glycol)-substituted polymer coating functionalized with controlled densities of ligands of varying specificity for target integrin receptors. Biomaterials presenting the α5β1-integrin-specific FN fragment FNIII7-10 enhanced osteoblastic differentiation in bone marrow stromal cells compared to unmodified titanium and RGD-presenting surfaces. Importantly, FNIII7-10-functionalized titanium significantly improved functional implant osseointegration compared to RGD-functionalized and unmodified titanium in vivo. This study demonstrates that bioactive coatings that promote integrin binding specificity regulate marrow-derived progenitor osteoblastic differentiation and enhance healing responses and functional integration of biomedical implants. This work identifies an innovative strategy for the rational design of biomaterials for regenerative medicine.
Keywords: Bone; Fibronectin; Cell adhesion; RGD; FAK; Integrins;

Articular cartilage repair using tissue engineering approach generally requires the use of an appropriate scaffold architecture that can support the formation of cartilage tissue. In this investigation, the potential of three-dimensional scaffolds made of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) was evaluated in rabbit articular cartilage defect model. Engineered PHBHHx cartilage constructs inoculated in vitro with rabbit chondrocytes for 30 days were examined. Subsequently the constructs inoculated with chondrocytes for 10 days were selected for transplantation into rabbits. After 16 weeks of in vivo implantation, both the engineered cartilage constructs and the bare scaffolds were found to be filled the defects with white cartilaginous tissue, with the engineered constructs showing histologically good subchondral bone connection and better surrounding cartilage infusion. Owing to pre-seeded chondrocytes in the PHBHHx scaffolds, better surface integrality and more accumulation of extracellular matrix (ECM) including type II collagen and sGAG were achieved in the engineered cartilage constructs. The repaired tissues possessed an average compressive modulus of 1.58 MPa. For comparison, the defects without repair treatments still showed defects with fibrous tissues. These results demonstrated that PHBHHx is a useful material for cartilage tissue engineering.
Keywords: Polyhydroxyalkanoates; PHB; PHBHHx; Chondrocytes; Transplantation; Cartilage tissue engineering;

Sustained release of sphingosine 1-phosphate for therapeutic arteriogenesis and bone tissue engineering by Lauren S. Sefcik; Caren E. Petrie Aronin; Kristen A. Wieghaus; Edward A. Botchwey (2869-2877).
Sphingosine 1-phosphate (S1P) is a bioactive phospholipid that impacts migration, proliferation, and survival in diverse cell types, including endothelial cells, smooth muscle cells, and osteoblast-like cells. In this study, we investigated the effects of sustained release of S1P on microvascular remodeling and associated bone defect healing in vivo. The murine dorsal skinfold window chamber model was used to evaluate the structural remodeling response of the microvasculature. Our results demonstrated that 1:400 (w/w) loading and subsequent sustained release of S1P from poly(lactic-co-glycolic acid) (PLAGA) significantly enhanced lumenal diameter expansion of arterioles and venules after 3 and 7 days. Incorporation of 5-bromo-2-deoxyuridine (BrdU) at day 7 revealed significant increases in mural cell proliferation in response to S1P delivery. Additionally, three-dimensional (3D) scaffolds loaded with S1P (1:400) were implanted into critical-size rat calvarial defects, and healing of bony defects was assessed by radiograph X-ray, microcomputed tomography (μCT), and histology. Sustained release of S1P significantly increased the formation of new bone after 2 and 6 weeks of healing and histological results suggest increased numbers of blood vessels in the defect site. Taken together, these experiments support the use of S1P delivery for promoting microvessel diameter expansion and improving the healing outcomes of tissue-engineered therapies.
Keywords: Tissue engineering; Controlled drug release; Phospholipids; Drug delivery; Bone healing; Growth factors;

Transplantation of islets of Langerhans (islets) is a promising method to treat insulin-dependent diabetes mellitus (type I diabetes). However, insulin independence is typically realized for only ∼30% of transplant recipients, even with sufficient numbers of islets from multiple donors. Innate immunological reactions triggered by blood coagulation play a key role in the loss of islets at the early stage. Here we propose a method to inhibit blood coagulation on the islet surface. A plasminogen activator, urokinase, was immobilized on the islet surface via a poly(vinyl alcohol) (PVA) derivative that carries alkyl chains and thiol groups. When the PVA derivative was added to an islet suspension, the alkyl side chains spontaneously anchored into the lipid bilayer membranes of islet cells. The surfaces of islets were covered with the PVA derivative. Urokinase modified with maleimide groups could be immobilized onto the islet surface by thiol/maleimide bonding with the layer of PVA derivatives. Urokinase-immobilized islets exhibited fibrinolytic properties, indicating that blood coagulation can be controlled on the islet surface. Urokinase immobilization on islets, which does not impair insulin release, represents a promising method to reduce early graft loss after intraportal islet transplantation.
Keywords: Islet; Poly(vinyl alcohol); Urokinase; Surface modification; Transplantation;

The effect of sustained delivery of vascular endothelial growth factor on angiogenesis in tissue-engineered intestine by Flavio G. Rocha; Cathryn A. Sundback; Nicholas J. Krebs; J. Kent Leach; David J. Mooney; Stanley W. Ashley; Joseph P. Vacanti; Edward E. Whang (2884-2890).
Our group has previously created a functional neointestine that is capable of restoring absorptive function. However, the endogenous level of vascular endothelial growth factor (VEGF) is markedly reduced in the construct compared to native bowel. Therefore, we wanted to locally deliver VEGF in a sustained fashion to upregulate angiogenesis in the neointestine. Rat recombinant VEGF was encapsulated in poly(lactide-co-glycolide) microspheres by a double emulsion method. Release kinetics and bioactivity were determined in vitro. Tissue-engineered intestine was generated by seeding donor neonatal rat intestinal organoid units onto a biodegradable polyglycolic acid scaffold along with VEGF-containing or empty microspheres, and wrapped in the omentum of recipient rats. After 4 weeks, the neointestinal cysts were analyzed for morphometry, VEGF levels, epithelial proliferation, and capillary density. Sustained release of biologically active VEGF was confirmed by in vitro studies. Intestinal constructs with VEGF microspheres were significantly larger than those containing empty microspheres. Tissue VEGF levels were significantly higher in neointestine loaded with encapsulated VEGF compared to those without growth factor. Epithelial cellular proliferation and capillary density were significantly increased in the VEGF-containing neointestinal constructs compared to empty constructs. Tissue-engineered intestine responds to sustained delivery of VEGF by upregulating microvasculature and epithelial proliferation.
Keywords: Small intestine; VEGF; Angiogenesis; Microspheres; PLGA;

Development of a composite vascular scaffolding system that withstands physiological vascular conditions by Sang Jin Lee; Jie Liu; Se Heang Oh; Shay Soker; Anthony Atala; James J. Yoo (2891-2898).
Numerous scaffolds that possess ideal characteristics for vascular grafts have been fabricated for clinical use. However, many of these scaffolds may not show consistent properties when they are exposed to physiologic vascular environments that include high pressure and flow, and they may eventually fail due to unexpected rapid degradation and low resistance to shear stress. There is a demand to develop a more durable scaffold that could withstand these conditions until vascular tissue matures in vivo. In this study, vascular scaffolds composed of poly(ɛ-caprolactone) (PCL) and collagen were fabricated by electrospinning. Morphological, biomechanical, and biological properties of these composite scaffolds were examined. The PCL/collagen composite scaffolds, with fiber diameters of approximately 520 nm, possessed appropriate tensile strength (4.0 ± 0.4 MPa) and adequate elasticity (2.7 ± 1.2 MPa). The burst pressure of the composite scaffolds was 4912 ± 155 mmHg, which is much greater than that of the PCL-only scaffolds (914 ± 130 mmHg) and native vessels. The composite scaffolds seeded with bovine endothelial cells (bECs) and smooth muscle cells (bSMCs) showed the formation of a confluent layer of bECs on the lumen and bSMCs on the outer surface of the scaffold. The PCL/collagen composite scaffolds are biocompatible, possess biomechanical properties that resist high degrees of pressurized flow over long term, and provide a favorable environment that supports the growth of vascular cells.
Keywords: Polycaprolactone; Collagen; Composite; Electrospinning; Mechanical properties; Physiological condition;

Current treatment options for restoring large skeletal muscle tissue defects due to trauma or tumor ablation are limited by the host muscle tissue availability and donor site morbidity of muscle flap implantation. Creation of implantable functional muscle tissue that could restore muscle defects may bea possible solution. To engineer functional muscle tissue for reconstruction, scaffolds that mimic native fibers need to be developed. In this study we examined the feasibility of using poly(ɛ-caprolactone) (PCL)/collagen based nanofibers using electrospinning as a scaffold system for implantable engineered muscle. We investigated whether electrospun nanofibers could guide morphogenesis of skeletal muscle cells and enhance cellular organization. Nanofibers with different fiber orientations were fabricated by electrospinning with a blend of PCL and collagen. Human skeletal muscle cells (hSkMCs) were seeded onto the electrospun PCL/collagen nanofiber meshes and analyzed for cell adhesion, proliferation and organization. Our results show that unidirectionally oriented nanofibers significantly induced muscle cell alignment and myotube formation as compared to randomly oriented nanofibers. The aligned composite nanofiber scaffolds seeded with skeletal muscle cells may provide implantable functional muscle tissues for patients with large muscle defects.
Keywords: Polycaprolactone; Collagen; Electrospinning; Cell alignment; Myotube formation; Tissue engineering;

Three-dimensional electrospun ECM-based hybrid scaffolds for cardiovascular tissue engineering by Sepideh Heydarkhan-Hagvall; Katja Schenke-Layland; Andrew P. Dhanasopon; Fady Rofail; Hunter Smith; Benjamin M. Wu; Richard Shemin; Ramin E. Beygui; William R. MacLellan (2907-2914).
Electrospinning using natural proteins or synthetic polymers is a promising technique for the fabrication of fibrous scaffolds for various tissue engineering applications. However, one limitation of scaffolds electrospun from natural proteins is the need to cross-link with glutaraldehyde for stability, which has been postulated to lead to many complications in vivo including graft failure. In this study, we determined the characteristics of hybrid scaffolds composed of natural proteins including collagen and elastin, as well as gelatin, and the synthetic polymer poly(ɛ-caprolactone) (PCL), so to avoid chemical cross-linking. Fiber size increased proportionally with increasing protein and polymer concentrations, whereas pore size decreased. Electrospun gelatin/PCL scaffolds showed a higher tensile strength when compared to collagen/elastin/PCL constructs. To determine the effects of pore size on cell attachment and migration, both hybrid scaffolds were seeded with adipose-derived stem cells. Scanning electron microscopy and nuclei staining of cell-seeded scaffolds demonstrated the complete cell attachment to the surfaces of both hybrid scaffolds, although cell migration into the scaffold was predominantly seen in the gelatin/PCL hybrid. The combination of natural proteins and synthetic polymers to create electrospun fibrous structures resulted in scaffolds with favorable mechanical and biological properties.
Keywords: Electrospinning; Gelatin; Collagen; Elastin; PCL; Tissue engineering;

The effect of pretreating morselized allograft bone with rhBMP-2 and/or pamidronate on the fixation of porous Ti and HA-coated implants by Jorgen Baas; Brian Elmengaard; Thomas B. Jensen; Thomas Jakobsen; Niels T. Andersen; Kjeld Soballe (2915-2922).
BMPs stimulate new bone formation, but may also accelerate bone resorption. We added rhBMP-2 and pamidronate to morselized bone allograft packed around uncemented HA-coated and non-coated porous Ti implants in sixteen dogs. Each dog received four implants where the allograft was added (1) nothing, (2) BMP, (3) BP, and (4) BMP + BP. After four weeks, the untreated control implants had better mechanical fixation than all other treatment groups. The rhBMP-2-treated group had abundant formation of new bone on and around the implant. However, almost all allografts were resorbed, rendering the implant mechanically unstable. In the pamidronate-treated group the allograft was preserved, but the implants were covered by fibrous tissue and there was almost no new bone formation. This was also the case for the combined BMP + BP group, although fibrous tissue was absent on these implants. The HA-coated implants had more than twice as good mechanical fixation and improved osseointegration compared to the corresponding Ti implants. RhBMP-2 raised the total metabolic turnover of bone within the allograft with a net negative result on implant fixation. Pamidronate virtually blocked bone metabolism, also when combined with rhBMP-2. The results warrant a conservative approach and emphasize the importance of identifying a therapeutic window for these substances prior to clinical use.
Keywords: BMP (bone morphogenetic protein); Bone graft; Bone ingrowth; Histomorphometry; Hydroxyapatite coating; Mechanical test;

Sutureless amniotic membrane transplantation for ocular surface reconstruction with a chemically defined bioadhesive by Maho Takaoka; Takahiro Nakamura; Hajime Sugai; Adam J. Bentley; Naoki Nakajima; Nigel J. Fullwood; Norihiko Yokoi; Suong-Hyu Hyon; Shigeru Kinoshita (2923-2931).
The purpose of this study was to evaluate the efficiency and safety of a sutureless amniotic membrane transplantation (AMT) for ocular surface reconstruction with a chemically defined bioadhesive (CDB). The CDB was synthesized from aldehyded polysaccharides and ɛ-poly(l-lysine), two kinds of medical and food additives, as starting materials. Biocompatibility assay indicated that the CDB showed excellent biocompatibility with in vitro and in vivo ocular surface tissues and most of the CDB was histologically degraded within 4 weeks. Sutureless AMT using the CDB was safely and successfully performed onto a rabbit scleral surface. Transplanted amniotic membrane (AM) evaluated by histological, electron microscopic- and immunohistochemical examination indicated that the CDB did not affect normal differentiation of the cells or the integrity of the surrounding tissue. Thus, this newly developed CDB was found to be very useful for sutureless AMT for ocular surface reconstruction, without considering the risk of infection. It has the ability to fix AM to the ocular surface for a long time-period without additional inflammation, scarring, or damage to the surrounding tissues.
Keywords: Ophthalmology; Transplantation; Sutureless; Ocular surface; Bioadhesive; Biocompatibility;

The inhibition of matrix metalloproteinase activity in chronic wounds by a polyacrylate superabsorber by Sabine Eming; Hans Smola; Berenike Hartmann; Gebhart Malchau; Ronny Wegner; Thomas Krieg; Sigrun Smola-Hess (2932-2940).
Excessive matrix metalloproteinase (MMP) levels have been observed in wound fluid of impaired healing wounds. This is thought to interfere with granulation tissue formation as newly formed extracellular matrix and cytokines are degraded and the wound becomes deadlocked, unable to progress to the next healing stages. In the cleansing phase, associated with high MMP activity levels, hydroactive wound dressings containing polyacrylate superabsorber particles are particularly effective. We tested whether these particles can block MMP activity in wound fluid obtained from chronic venous leg ulcers. Polyacrylate superabsorber particles inhibited MMP activity by more than 87% in a fluorogenic peptide substrate assay. Further analysis revealed two underlying molecular mechanisms. First, experiments showed direct binding of MMPs to the particles. Secondly, polyacrylate superabsorber particles can bind Ca2+ and Zn2+ ions competing with MMPs for divalent ions required for enzymatic activity. Furthermore, we provide the first evidence in vivo that MMPs bind effectively to polyacrylate superabsorber particles within the hostile environment of chronic wounds. We conclude that polyacrylate superabsorber particles can rescue the highly proteolytic microenvironment of non-healing wounds from MMP activity so that more conductive conditions allow healing to proceed.
Keywords: Absorption; Connective tissue; Matrix metalloproteinase; Polyacrylic acid; Wound dressing; Wound healing;