Biomaterials (v.32, #18)

This paper constitutes the judgment on the four-paper debate in the same issue of this journal on the motion that ‘‘the use of short synthetic adhesion peptides, like RGD, is the best approach in the design of biomaterials that guide cell behavior for regenerative medicine and tissue engineering”. It is concluded that this motion is carried on the basis that the concept of installing multifunctionality and modularity in peptides is the more powerful concept, coupled with the potential attractiveness of retaining a chemically defined product, without any possibilities of disease transmission, and relative inexpensiveness and clearer regulatory pathways.
Keywords: Cell adhesion; Synthetic adhesion peptides; RGD; Extracellular matrix; Integrin binding;

Evolving the use of peptides as components of biomaterials by Joel H. Collier; Tatiana Segura (4198-4204).
This manuscript is part of a debate on the statement that “the use of short synthetic adhesion peptides, like RGD, is the best approach in the design of biomaterials that guide cell behavior for regenerative medicine and tissue engineering”. We take the position that although there are some acknowledged disadvantages of using short peptide ligands within biomaterials, it is not necessary to discard the notion of using peptides within biomaterials entirely, but rather to reinvent and evolve their use. Peptides possess advantageous chemical definition, access to non-native chemistries, amenability to de novo design, and applicability within parallel approaches. Biomaterials development programs that require such aspects may benefit from a peptide-based strategy.
Keywords: Peptide; Biomaterials; Extracellular matrix; Tissue engineering; Regenerative medicine;

Despite many years of in vitro research confirming the effectiveness of RGD in promoting cell attachment to a wide variety of biomaterials, animal studies evaluating tissue responses to implanted RGD-functionalized substrates have yielded more variable results. The goals of this report are to present some of the reasons why cell culture studies may not always reliably predict in vivo responses, and more importantly, to highlight potential applications that may benefit from the use of RGD peptides.
Keywords: RGD; Integrin; Extracellular matrix; Tissue regeneration; Cell adhesion;

The promise of biomaterials design for regenerative medicine tissue engineering is predicated on the fundamental ability to direct or guide specific and highly coordinated cellular behaviors that culminate in the creation of physiologically functional tissues and organs. To date, our efforts have focused primarily on the grafting and presentation of short synthetic peptides with just cause. Short peptides are capable of high levels of control, can be manufactured relatively easily in a highly reproducible manner under GMP guidelines and are readily modified to enable their integration with numerous current and emerging chemistries for biomaterials grafting. However, while extracellular matrix (ECM)-derived peptides have demonstrated their initial purpose of promoting cell adhesion, their general lack of specificity and significantly decreased receptor binding affinities have proven detrimental in attempts to regulate highly specific and integrated processes necessary for tissue regeneration. Unlike adhesion peptides, the natural ECM displays a complex interplay with cells by supporting environmentally sensitive and cell dependent integrin specificity and binding affinity. Furthermore, the adhesion ligands on ECM proteins display a finely tuned and evolutionarily directed spatial periodicity, of which is dynamically controlled through both mechanical and chemical modifications. These and other emerging concepts from matrix biology require our attention if biomaterials design is to fulfill its promise. Here, we are charged with debating the statement ‘The use of short synthetic adhesion peptides, like RGD, is the best approach in the design of biomaterials that guide cell behavior for regenerative medicine tissue engineering’. In this Leading Opinion Paper I will focus on aspects of natural ECM proteins and protein fragments that have proven difficult, if not impossible to date, to recapitulate in peptide-based systems. While this represents an argument against the use of peptides per se, it might also be viewed as outlining the challenges and opportunities for the biomaterials field.
Keywords: Cell adhesion; Synthetic adhesion peptides; RGD; Extracellular matrix; Integrin binding;

There are many ways to influence cell activities, and biomaterials with functional groups attached is an attractive method that clearly has the ability to modulate cell behavior. The evidence is clear that biomaterials, with or without growth factors and cells, have resulted in numerous products for the regenerative medicine field. In contrast the functionalized biomaterial products remain in the development phase.
Keywords: Cell adhesion; Adhesive peptides; RGD; Extracellular matrix; Integrin binding;

Recruitment of host’s progenitor cells to sites of human amniotic fluid stem cells implantation by Teodelinda Mirabella; Alessandro Poggi; Monica Scaranari; Massimo Mogni; Mario Lituania; Chiara Baldo; Ranieri Cancedda; Chiara Gentili (4218-4227).
The amniotic fluid is a new source of multipotent stem cells with a therapeutic potential for human diseases. Cultured at low cell density, human amniotic fluid stem cells (hAFSCs) were still able to generate colony-forming unit-fibroblast (CFU-F) after 60 doublings, thus confirming their staminal nature. Moreover, after extensive in vitro cell expansion hAFSCs maintained a stable karyotype. The expression of genes, such as SSEA-4, SOX2 and OCT3/4 was confirmed at early and later culture stage. Also, hAFSCs showed bright expression of mesenchymal lineage markers and immunoregulatory properties. hAFSCs, seeded onto hydroxyapatite scaffolds and subcutaneously implanted in nude mice, played a pivotal role in mounting a response resulting in the recruitment of host’s progenitor cells forming tissues of mesodermal origin such as fat, muscle, fibrous tissue and immature bone. Implanted hAFSCs migrated from the scaffold to the skin overlying implant site but not to other organs. Given their in vivo: (i) recruitment of host progenitor cells, (ii) homing towards injured sites and (iii) multipotentiality in tissue repair, hAFSCs are a very appealing reserve of stem cells potentially useful for clinical application in regenerative medicine.
Keywords: Mesenchymal stem cells; Tissue engineering; Angiogenesis; Inflammation; In vivo test; In vitro test;

The modulation of myogenic cells differentiation using a semiconductor-muscle junction by Marco Quarta; Michele Scorzeto; Marta Canato; Marco Dal Maschio; Davide Conte; Bert Blaauw; Stefano Vassanelli; Carlo Reggiani (4228-4237).
The present study is aimed to design a prototype of hybrid silicon-muscle cell junction, analog to an artificial neuromuscular junction prototype and relevant to the development of advanced neuro-prostheses and bionic systems. The device achieves focal Electric Capacitive Stimulation (ECS) by coupling of single cells and semiconductors, without electrochemical reaction with the substrate. A voltage change applied to a stimulation spot beneath an electrogenic cell leads to a capacitive current (charge accumulation) that opens voltage-gated ion channels in the membrane and generates an action potential. The myo-electronic junction was employed to chronically stimulate muscle cells via ECS and to induce cytosolic calcium transients in myotubes, fibers isolated from mouse FDB (fast [Ca2+]i transients) and surprisingly also in undifferentiated myoblasts (slow [Ca2+]i waves). The hybrid junction elicited, via chronic ECS, a differential reprogramming of single muscle cells by inducing early muscle contraction maturation and plasticity effects, such as NFAT-C3 nuclear translocation. In addition, in the presence of agrin, chronic ECS induced a modulation of AchR clustering which simulates in vitro synaptogenesis. This methodology can coordinate the myogenic differentiation, thus offering direct but non-invasive single cell/wiring, providing a platform for regenerative medicine strategies.
Keywords: Calcium; Cell culture; Electrical Stimulation; Muscle; Plasticity; Silicon; Titanium;

The acceleration of wound healing in primates by the local administration of immunostimulatory CpG oligonucleotides by Masaki Yamamoto; Takashi Sato; Joel Beren; Daniela Verthelyi; Dennis M. Klinman (4238-4242).
The process of wound healing involves complex interactions between circulating immune cells and local epithelial and endothelial cells. Studies in murine models indicate that cells of the innate immune system activated via their Toll-like receptors (TLR) can accelerate wound healing. This work examines whether immunostimulatory CpG oligodeoxynucleotides (ODN) designed to trigger human immune cells via TLR9 can promote the healing of excisional skin biopsies in rhesus macaques. Results indicate that ‘K’ type CpG ODN significantly accelerate wound closure in non-human primates (p < 0.05). Contributing to this outcome was a CpG-dependent increase in both the production of basic fibroblast growth factor and in keratinocyte migration. Of interest, IL-1α and TGFα normally present at sites of skin injury facilitated these effects. Current findings support the conclusion that the local administration of CpG ODN may provide an effective strategy for accelerating wound healing in humans.
Keywords: CpG oligonucleotide; Primate; TLR9; Wound healing;

Promotion of skin regeneration in diabetic rats by electrospun core-sheath fibers loaded with basic fibroblast growth factor by Ye Yang; Tian Xia; Wei Zhi; Li Wei; Jie Weng; Cong Zhang; Xiaohong Li (4243-4254).
Diabetic skin ulcer is difficult to heal due to the lack of cellular and molecular signals required for normal wound repair. Emulsion electrospinning was adopted to imbed basic fibroblast growth factor (bFGF) into ultrafine fibers with a core-sheath structure to promote the wound healing process. An initially burst release as low as 14.0 ± 2.2% was achieved, followed by gradual release for around 4 wk. In vitro investigations on mouse embryo fibroblasts indicated that bFGF-loaded fibrous mats enhanced cell adhesion, proliferation, and secretion of extracellular matrix (ECM). Skin wounds were created in the dorsal area of diabetic rats for in vivo evaluation of skin regeneration after covered with bFGF-loaded fibrous mats. Compared with fibrous mats infiltrated with free bFGF, bFGF-loaded scaffolds revealed significantly higher wound recovery rate with complete re-epithelialization and regeneration of skin appendages. Higher density and mature capillary vessels were generated during 2 wk after treatment with bFGF-loaded fibers, and there was no fiber fragment observed in the histological sections at week 4 after operation. The gradual release of bFGF from fibrous mats enhanced collagen deposition and ECM remodeling, and the arrangement and component of collagen fibers were similar to normal tissues. The above results demonstrate the potential use of bFGF-loaded electrospun fibrous mats to rapidly restore the structural and functional properties of wounded skin for patients with diabetic mellitus.
Keywords: Emulsion electrospinning; Core-sheath structure; Growth factor-loaded fibrous mats; Diabetic ulcer; Skin regeneration;

Antimicrobial functionalized genetically engineered spider silk by Sílvia C. Gomes; Isabel B. Leonor; João F. Mano; Rui L. Reis; David L. Kaplan (4255-4266).
Genetically engineered fusion proteins offer potential as multifunctional biomaterials for medical use. Fusion or chimeric proteins can be formed using recombinant DNA technology by combining nucleotide sequences encoding different peptides or proteins that are otherwise not found together in nature. In the present study, three new fusion proteins were designed, cloned and expressed and assessed for function, by combining the consensus sequence of dragline spider silk with three different antimicrobial peptides. The human antimicrobial peptides human neutrophil defensin 2 (HNP-2), human neutrophil defensins 4 (HNP-4) and hepcidin were fused to spider silk through bioengineering. The spider silk domain maintained its self-assembly features, a key aspect of these new polymeric protein biomaterials, allowing the formation of β-sheets to lock in structures via physical interactions without the need for chemical cross-linking. These new functional silk proteins were assessed for antimicrobial activity against Gram –Escherichia coli and Gram + Staphylococcus aureus and microbicidal activity was demonstrated. Dynamic light scattering was used to assess protein aggregation to clarify the antimicrobial patterns observed. Attenuated-total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and circular dichroism (CD) were used to assess the secondary structure of the new recombinant proteins. In vitro cell studies with a human osteosarcoma cell line (SaOs-2) demonstrated the compatibility of these new proteins with mammalian cells.
Keywords: Spider silk; Antimicrobial activity; Recombinant proteins; Self-assembly; Cell viability; Bone tissue engineering;

Patterned cardiomyocytes on microelectrode arrays as a functional, high information content drug screening platform by Anupama Natarajan; Maria Stancescu; Vipra Dhir; Christopher Armstrong; Frank Sommerhage; James J. Hickman; Peter Molnar (4267-4274).
Cardiac side effects are one of the major causes of drug candidate failures in preclinical drug development or in clinical trials and are responsible for the retraction of several already marketed therapeutics. Thus, the development of a relatively high-throughput, high information content tool to screen drugs and toxins would be important in the field of cardiac research and drug development. In this study, recordings from commercial multielectrode arrays were combined with surface patterning of cardiac myocyte monolayers to enhance the information content of the method; specifically, to enable the measurement of conduction velocity, refractory period after action potentials and to create a functional re-entry model. Two drugs, 1-Heptanol, a gap junction blocker, and Sparfloxacin, a fluoroquinone antibiotic, were tested in this system. 1-Heptanol administration resulted in a marked reduction in conduction velocity, whereas Sparfloxacin caused rapid, irregular and unsynchronized activity, indicating fibrillation. As shown in these experiments, patterning of cardiac myocyte monolayers solved several inherent problems of multielectrode recordings, increased the temporal resolution of conduction velocity measurements, and made the synchronization of external stimulation with action potential propagation possible for refractory period measurements. This method could be further developed as a cardiac side effect screening platform after combination with human cardiomyocytes.
Keywords: Biosensor; Cardiac tissue engineering; Cardiomyocyte; Electrode; In vitro test; Micropatterning;

Immunostimulatory activity of polysaccharide–poly(I:C) nanoparticles by Gizem Tincer; Seda Yerlikaya; Fuat C. Yagci; Tamer Kahraman; Osman M. Atanur; Oktay Erbatur; Ihsan Gursel (4275-4282).
Immunostimulatory properties of mushroom derived polysaccharides (PS) as stand-alone agents were tested. Next, PS were nanocomplexed with polyI:C (pIC) to yield stable nanoparticles around 200 nm in size evidenced by atomic force microscopy and dynamic light scattering analyses. PSs were selectively engaged by cells expressing TLR2 and initiated NFκB dependent signaling cascade leading to a Th1-biased cytokine/chemokine secretion in addition to bactericidal nitric oxide (NO) production from macrophages. Moreover, cells treated with nanoparticles led to synergistic IL6, production and upregulation of TNFα, MIP3α, IFNγ and IP10 transcript expression. In mice, PS-Ovalbumin-pIC formulation surpassed anti-OVA IgG responses when compared to either PS-OVA or pIC-OVA mediated immunity. Our results revealed that signal transduction initiated both by TLR2 and TLR3 via co-delivery of pIC by PS in nanoparticle depot delivery system is an effective immunization strategy. The present work implicate that the PS and nucleic acid based nanoparticle approach along with protein antigens can be harnessed to prevent infectious diseases.
Keywords: Polysaccharides; Poly(I:C); Nanoparticles; Targeted delivery system; TLR; Immunogenicity;

Ternary complexes of amphiphilic polycaprolactone-graft-poly (N,N-dimethylaminoethyl methacrylate), DNA and polyglutamic acid-graft-poly(ethylene glycol) for gene delivery by Shutao Guo; Yuanyu Huang; Wendi Zhang; Weiwei Wang; Tuo Wei; Daoshu Lin; Jinfeng Xing; Liandong Deng; Quan Du; Zicai Liang; Xing-Jie Liang; Anjie Dong (4283-4292).
Binary complexes of cationic polymers and DNA were used commonly for DNA delivery, whereas, the excess cationic charge of the binary complexes mainly leads to high toxicity and unstability in vivo. In this paper, ternary complexes by coating polyglutamic acid-graft-poly(ethylene glycol)(PGA-g-mPEG) onto binary complexes of polycaprolactone-graft-poly(N,N-dimethylaminoethyl methacrylate) (PCL-g-PDMAEMA) nanoparticles (NPs)/DNA were firstly developed for effective and targeted gene delivery. The coating of PGA-g-mPEG was able to decrease the zeta potential of the nano-sized DNA complexes nearly to electroneutrality without interferring with DNA condensation ability. As a result, the stability, the escape ability from endosomes and the transfection efficiency of the complexes were enhanced. The ternary complexes of PCL-g-PDMAEMA NPs/DNA/PGA-g-mPEG demonstrated lower cytotoxicity in CCK-8 measurements and higher gene transfection efficiency than the binary complexes in vitro. In addition, Lactate dehydrogenase (LDH) assay was performed to quantify the membrane-damaging effects of the complexes, which is consistent with the conclusion of CCK-8 measurement for cytotoxicity assay. The in vivo imaging measurement and histochemical analysis of tumor sessions confirmed that the intravenous administration of the ternary complexes with red fluorescent protein (RFP) as payload led to protein expression in tumor, which was further enhanced by the targeted coating of PGA-g-PEG-folate.
Keywords: Gene delivery; PCL-g-PDMAEMA; Amphiphilic copolymers; Ternary complexes; Tumor target;

Angiopep-conjugated poly(ethylene glycol)-co-poly(ε-caprolactone) nanoparticles as dual-targeting drug delivery system for brain glioma by Hongliang Xin; Xinyi Jiang; Jijin Gu; Xianyi Sha; Liangcen Chen; Kitki Law; Yanzuo Chen; Xiao Wang; Ye Jiang; Xiaoling Fang (4293-4305).
Dual-targeting nanoparticle drug delivery system was developed by conjugating Angiopep with PEG–PCL nanoparticles (ANG-NP) through bifunctional PEG to overcome the limitations of low transport of chemotherapeutics across the Blood–brain barrier (BBB) and poor penetration into tumor tissue. ANG-NP can target the low-density lipoprotein receptor-related protein (LRP) which is over-expressed on the BBB and glioma cells. Compared with non-targeting nanoparticles, a significantly higher amount of rhodamine isothiocyanate-labeled dual-targeting nanoparticles were endocytosed by U87 MG cells. The antiproliferative and cell apoptosis assay of paclitaxel-loaded ANG-NP (ANG-NP-PTX) demonstrated that ANG-NP-PTX resulted in enhanced inhibitory effects to U87 MG glioma cells. The transport ratios across the BBB model in vitro were significantly increased and the cell viability of U87 MG glioma cells after crossing the BBB was obviously decreased by ANG-NP-PTX. Enhanced accumulation of ANG-NP in the glioma bed and infiltrating margin of intracranial U87 MG glioma tumor-bearing in vivo model were observed by real time fluorescence image. In conclusion, Angiopep-conjugated PEG–PCL nanoparticles were prospective in dual-targeting drug delivery system for targeting therapy of brain glioma.
Keywords: Dual targeting; Angiopep; LRP; Blood–brain barrier; Brain glioma; PTX;

Systemic delivery of siRNA to tumors using a lipid nanoparticle containing a tumor-specific cleavable PEG-lipid by Hiroto Hatakeyama; Hidetaka Akita; Erika Ito; Yasuhiro Hayashi; Motoi Oishi; Yukio Nagasaki; Radostin Danev; Kuniaki Nagayama; Noritada Kaji; Hiroshi Kikuchi; Yoshinobu Baba; Hideyoshi Harashima (4306-4316).
Previously, we developed a multifunctional envelope-type nano device (MEND) for efficient delivery of nucleic acids. For tumor delivery of a MEND, PEGylation is a useful method, which confers a longer systemic circulation and tumor accumulation via the enhanced permeability and retention (EPR) effect. However, PEGylation inhibits cellular uptake and subsequent endosomal escape. To overcome this, we developed a PEG-peptide-DOPE (PPD) that is cleaved in a matrix metalloproteinase (MMP)-rich environment. In this study, we report on the systemic delivery of siRNA to tumors by employing a MEND that is modified with PPD (PPD-MEND). An in vitro study revealed that PPD modification accelerated both cellular uptake and endosomal escape, compared to a conventional PEG modified MEND. To balance both systemic stability and efficient activity, PPD-MEND was further co-modified with PEG-DSPE. As a result, the systemic administration of the optimized PPD-MEND resulted in an approximately 70% silencing activity in tumors, compared to non-treatment. Finally, a safety evaluation showed that the PPD-MEND showed no hepatotoxicity and innate immune stimulation. Furthermore, in a DNA microarray analysis in liver and spleen tissue, less gene alternation was found for the PPD-MEND compared to that for the PEG-unmodified MEND due to less accumulation in liver and spleen.
Keywords: Multifunctional envelope-type nano device (MEND); Systemic siRNA delivery; Cleavable PEG; Matrix metalloproteinase; PEG dilemma; EPR effect;