Study of Antimicrobial Peptide Dendrimers on Wound healing in SKin Tissue Engineering

Infections are one of the most common complications in burn injuries. In severe burns, the wound surface is optimal for microbial proliferation as the exudate is a protein rich environment. Autograft and cultured grafts are the main treatments used nowadays. However, they require a two-step surgical procedure, a collecting step and a grafting step, which slows down the treatment. In order to circumvent this two-step procedure, the CHUV has developed biological bandages made of collagen sheets seeded with fetal skin fibroblasts. The specific gene regulation and growth factor production of fetal cells allows a better healing. The goal of this project was to study the combination of antimicrobial peptide dendrimers with the existing biological bandages, for an improved tissue engineering skin construct having antimicrobial properties. To begin, the cytotoxic effect of two antimicrobial peptides, G3KL and G3RL, was tested on fetal and adult skin fibroblasts. The results showed that fetal cells were more sensitive to the peptides than adult cells. A high peptide concentration induced a higher death rate but in some cases also a higher amount of live cells. In parallel a scratch assay was performed and showed that peptides did not influence cell migration. Then, the efficacy of the antimicrobial peptides was tested against P.aeruginosa by a time kill and zone of inhibition assay. The results showed that both peptides were able to slow down the bacterial growth and that G3KL could even reduce the amount of bacteria. Finally, the biocompatibility of the peptides was tested with a chorioallantoic membrane assay (CAM). The results showed no adverse effect on the membrane or its vascularization and suggested a potential angiogenic effect of G3KL. The gene expression of fetal cells cultured with peptides was measured with a quantitative PCR and showed a small up-regulation of VEGFC, suggesting an explanation for the results obtained with the CAM.

Tissue engineering for prenatal applications

Anna-Sofia Johanna Kiveliö

Fetal therapies have become available for a restricted number of life-threatening clinical conditions. Harnessing tissue engineering for prenatal applications has not been widely pursued even though isolating cells from fetal and extraembryonic tissues has been routinely done for years. The objectives of this thesis were twofold: i) the development of materials based and tissue engineering based strategies to prevent iatrogenic preterm prelabour rupture of fetal membranes (iPPROM) after diagnostic or therapeutic interventions into the amniotic cavity and ii) the generation of tissue substitutes from patient derived fetal cells, which can be employed for prenatal or perinanal transplantation to restore or replace defective tissues. For the prevention of iPPROM, mussel-mimetic tissue adhesive (mussel glue) a biomaterial which was recently described to not compromise cell viability and to have good tissue sealing capability was compared to fibrin glue. In this in vivo study we assessed whether in a mid-gestational rabbit model punctured fetal membranes could be efficiently sealed with mussel glue. Mussel glue showed comparable in vivo performance to fibrin glue in sealing fetal membranes though no apparent healing of the membranes could be observed in any of the samples. The limited ability of naturally derived scaffolds to promote fetal membrane healing inspired the engineering of synthetic plugging material with specifically tailored biological and mechanical properties which could activate the cells in the amnion and induce a healing response. In this thesis the modularly designed biomimetic poly(ethylene glycol) (PEG)-based hydrogel platform (called TG-PEG from here on) was used together with fetal cells to demonstrate that upon presentation of appropriate biological cues in 3D tissue mimicking environment, mesenchymal progenitor cells from amnion can be mobilized, induced to proliferate and supported in maintaining their native extracellular matrix production, thus creating a suitable environment for healing to take place. These data provide the basis for future engineering of materials with defined mechanical and biochemical properties and the ability to present migration and proliferation inducing factors, namely PDGF, bFGF, or EGF which could be key in resolving the clinical problem of iPPROM and allowing the field of fetal surgery to move forward. Cleft palate, where the bones of the palatal halves fail to fuse properly, is one of the most common birth defects. Tissue engineering has been envisioned as a treatment option but current approaches have been limited by the lack of i) appropriate autologous cell sources and ii) structural organization and vascularization. Tissue engineering of cleft palates using of autologous amniocentesis-derived and thus ethically unproblematic fetal amniotic fluid cells (AFCs) could be done parallel to the ongoing pregnancy with living reconstruction material being ready when the child is born. We describe using TG-PEG hydrogels to first evaluate 3D osteogenic differentiation of AFCs and creation of vascular structures from AFC derived endothelial cells (enAFC) and undifferentiated AFC either in random or organized channel cocultures in vitro and in vivo. Next, these approaches were combined in an osteogenic matrix with a channel perfused with enAFC and finally the integration and functional properties of these fetal bone constructs was tested in ectopic mouse model.

EPFL2015

Peptide ligands for targeting and retention of nanoparticles and protein therapeutics in articular cartilage

Dominique André Rothenfluh

Osteoarthritis (OA) is one of the most common causes of pain and disability. Currently, non-surgical treatment is limited to analgesic and anti-inflammatory drugs, which are not capable of either modifying the disease process or regenerate damaged parts in articular cartilage. From a clinical point of view, small focal lesions in articular cartilage are frequent incidental findings mainly in the hip and knee but also in other joints during open and arthroscopic joint preserving surgical procedures. There is therefore an obvious demand for (bio)-pharmaceutical treatment options to complement surgical procedures in order to regenerate damaged parts in articular cartilage and generally attenuate disease progression. However, pharmaceutical approaches are generally associated with low bioavailabilities of the therapeutic molecules in the cartilage matrix, the primary site of the disease process in OA. The low bioavailability is due to the fact that the joint assumes a remote location being connected to the systemic circulation via the synovial fluid, which is an ultrafiltrate of blood secreted into the joint space. Articular cartilage, i.e. the lining of the joint surfaces providing nearly frictionless motion, is avascular and therefore not connected to the systemic circulation at all and thus depends on nutrients from the synovial fluid being transported to the cells, i.e. chondrocytes, by convective transport, which is inherently impaired due to its dense extracellular matrix. In addition, small molecules which may end up in the joint space via the synovial fluid systemically or after local administration by intra-articular injection are rapidly cleared out of the joint through lymphatic uptake. In order for (bio)-pharmaceutical treatment options to be efficient and effective, the therapeutic needs to reach the site of the disease process, here articular cartilage, and stay there long enough to exert its desired function. At first, we sought to engineer a nanoparticle-based drug delivery system which is capable of targeting the cartilage matrix and is subsequently immobilized there to release its cargo over time. Towards this end, we used a technique called peptide-on-phage display to carry out rounds of affinity selection against bovine cartilage in order to discover potential 6-mer peptides as bioaffinity ligands to a cartilage extracellular matrix component. Phage display relies on the genotype-phenotype linkage of inserts in the gene coding for minor coat protein of the phage virion, which is then displayed as a fusion protein on its surface. The sequence displayed by recovered binding phages can therefore be determined by sequencing of the phage DNA. After five rounds of affinity selection, i.e. recovering the binding phages, amplifying and re-exposing them to bovine articular cartilage, the peptide sequence WYRGRL could be discovered, which appeared in 94 out of 96 sequenced phage clones. WYRGRL was shown to bind specifically to articular cartilage and not to the synovial membrane. In addition, adding synovial fluid does not impair its capability of binding to articular cartilage. In a competetive binding assay of phages displaying WYRGRL and its synthesized free peptide an IC50 of binding to articular cartilage of 140 nM could be calculated. The free peptide was subsequently immobilized in a thiopropyl sepharose column for affinity purification in order to identify the binding partner of WYRGRL in the cartilage extracellular matrix. Affinity purification yielded one single protein band in SDS-PAGE which was identified to be collagen II alpha1 by in-gel trypsin digestion and analysis of protein fragments by mass-spectrometry. The peptide WYRGRL therefore provides means of immobilizing macromolecules in the collagen II network of articular cartilage. As a proof of principle, the peptide was conjugated to the surface of poly(propylene sulfide) nanoparticles, which were in average between 31 to 38 nm in size. It was covalently bound to the emulsifier for nanoparticle synthesis which remains displayed on the surface after polymerization. To visualize the nanoparticles in vivo by confocal laser scanning microscopy, they were labelled with a fluorophore for visualization. The nanoparticles were injected into the knee joints of mice, which were sacrificed after 24, 48 and 96 h. Compared to control nanoparticles displaying the scrambled peptide sequence, a 44.8-fold higher accumulation of nanoparticles in the extracellular matrix after 24 h could be observed, which was 71.7-fold higher after 48 h and 27.6-fold higher after 96 hours. Conjugation of WYRGRL to nanoparticles therefore efficiently immobilized them in the extracellular matrix of articular cartilage, an effect that was not present in nanoparticles with a scrambled peptide sequence attached to their surface. Nanoparticles could also be observed in chondrocytes in vivo, although to a lesser extent than in the extracellular matrix. In the light of potential adverse effects on cells, internalization of nanoparticles in immature murine articular chondrocytes (IMAC) and their effect on cell viability as well as phenotype specific gene-expression was studied. In accordance with the observations in vivo, conjugated and non-conjugated nanoparticles were efficiently internalized by cultured IMACs ranging from 74.8% to 77.5% after 24 h. Nanoparticle internalization was shown to be associated with elevetad rates of cell death and a decrease in the phenotypic gene expression, especially collagen II and aggrecan, but also collagen I, whereas cathepsin B as a de-differentiation marker and lysosomal enzyme was not downregulated. After 96 h, gene expression levels seemed to recover and approach baseline values. At the concentrations used, internalized nanoparticles seemed to be toxic to IMACs and interfere with phenotypic gene expression and potentially alter the cell phenotype, which needs to be investigated further for potential clinical applications. As it was shown that the peptide WYRGRL effectively immobilized poly(propylene sulfide) nanoparticles in the extracellular matrix of articular cartilage, we sought to extend this concept to protein-based therapeutics. Of interest here is FGF-18 which had been shown to be an important trophic factor on chondrocytes, inducing synthesis of matrix molecules. It had also been shown that FGF-18 was capable of repairing focal cartilage lesions in a model of rapidly progressing OA in rats, which required bi-weekly intra-articular injections, however. A fusion protein of human FGF-18 and the collagen II-binding peptide WYRGRL was expressed in E. coli and purified by heparin-affinity chromatography. It was demonstrated in vitro by surface plasmon resonance, that the fusion of FGF-18 and WYRGRL exhibits slower release after binding to immobilized collagen II than wild-type FGF-18. In a model of surgically induced knee joint instability in mice, injection of the fusion protein of FGF-18 at the time of surgery and after 2 weeks prevented the onset of OA in these animals over 5 weeks and demonstrated significantly improved histological scores compared to the wild-type protein and control animals. Enhanced retention of FGF-18 in the cartilage matrix resulted therefore to shift from bi-weekly to less than bi-monthly intra-articular administration of the therapeutic. In conclusion, the present thesis introduces an enhanced retention technology for articular cartilage which has been demonstrated to immobilize nanoparticles and protein-based therapeutics in the cartilage matrix by binding to the collagen II network. The potential reduction of intra-articular injections as a result of immobilizing either the carrier or the therapeutic is of significant clinical relevance and may prove to be pivotal for future (bio)-phamaceutical approaches for cartilage regeneration.

EPFL2009

Hydrogels for Urinary Tract Tissue Engineering

Lionel Ambroise Micol

Several urinary tract disorders, such as hypospadias or bladder cancer, require reconstructive surgery to preserve the patient's quality of life and at times even save their life. The current available surgical treatments, involving grafts from other tissue origin, can, however, lead to long-term complications in up to 50% of these patients. New alternatives are therefore considered among which tissue-engineered grafts are thought to be the most promising approach. Tissue-engineered grafts have so far been shown to require the presence of cells, which involves different steps prior to implantation including cell isolation and expansion, seeding on a matrix, and further culture. These steps are time-consuming, costly, and raise safety concerns regarding the in vitro cultured cells. In this thesis, we sought to further improve some tissue-engineered grafts with approaches that would either reduce or replace the cell culture steps. One of these approaches relied on the plastic compression of collagen gels. Ready cell-seeded plastic compressed collagen gels can be rapidly formed within minutes and have much better mechanical properties than regular collagen gels that are known to be fragile. In order to have a stronger gel we adapted the plastic compression concept and developed high-density collagen gels tubes (hdCGTs) for urinary tract tissue-engineering. We demonstrated that they were suitable for smooth muscle cell (SMC) proliferation and that their mechanical response to dilation and extension were compatible with ureter and urethra physiologic values. Hence, these ready-seeded hdCGTS represent promising tissue-engineered constructs that can shorten the production time of cell-seeded grafts for urinary tract regeneration. We further tested our hdCGTs in a rabbit model to investigate their regenerative capacity in vivo. Autologous rabbit SMCs were isolated from a bladder biopsy to form cellular constructs, while acellular constructs were used as control. Both types of constructs were sealed with fibrin glue at the site of a newly created urethral defect. Rabbits were evaluated by retrograde urethrography after 1 or 3 months. Despite a high rate of fistula and a relative narrowing of graft calibers, all rabbits survived and showed micturition within 1-3 days following surgery. SMC-seeded grafts showed, however, a better outcome at 3 months in accordance to literature. Since plastic compressed collagen gels are still too weak to be sutured, we decided to strengthen them by combining them with a polymer mesh. We investigated flat collagen-poly(lactic acid-co-ε-caprolactone) (PLAC) hybrid scaffolds seeded with human bladder cells for their behaviour in the subcutaneous space of nude mice. Host cell infiltration, scaffold degradation, and the presence of the seeded bladder cells were analyzed at different time points on a total period of 6 months. The hybrids showed a lower inflammatory reaction in vivo than PLAC meshes alone, and first signs of polymer degradation were visible at six months. The collagen-PLAC hybrids have potential for urinary tract tissue regeneration as they show efficient cell seeding and proliferation as well as proper mechanical properties for surgical handling. Another approach towards improving tissue-engineered grafts was to use off-the-shelf smart biomatrices to replace the cell culture steps. This involved the development of cell-controlled release of insulin-like growth factor-1 (IGF1) in an acellular collagen matrix. IGF1 induces SMC migration, proliferation, and differentiation, and can hence promote the invasion and population of the acellular matrix by cells in the surrounding tissue once implanted in vivo. In this approach, we designed and produced a recombinant collagen-binding IGF1. The IGF1 domain would then be released after cleavage from an affinity binding domain to collagen under the action of cell-secreted matrix metallo-proteases (MMP). Although our recombinant IGF1 showed the expected bioactivity on SMCs and sensitivity to MMP-2, its affinity for type I collagen (Col-I) could not be demonstrated. We further investigated covalent binding alternatives involving transglutaminase (TG) crosslinking or IGF binding protein (IGFBP)-mediated binding to collagen. TG crosslinking did not allow for sufficient amount of a TG-IGF, a recombinant form of IGF1 presenting a substrate sequence for plasma TG, to be bound to Col-I. The entrapment of IGFBP5 within plastic compressed collagen gels however suggests that collagen compression and collagen-binding means could be combined in order to allow for a more controlled-release profile of IGF1 from collagen gels. Lastly, we sought to develop a new bioengineered solution for bladder sphincter disorders, such as urinary incontinence or vesico-ureteral reflux. These disorders are treated with open surgeries susceptible of complications and extended hospital stay. Endoscopic treatment by injection of bulking agents is thus usually preferred, although they often necessitate additional interventions for a sustained effect. With the idea to suppress this need for additional intervention and thus to limit discomfort for patients as well as costs, we sought for a permanent bulking agent. We therefore developed an injectable poly(acrylonitrile) hydrogel paste injected into the submucosal space of pig bladders. The implants were harvested at days 7, 14, 21, 28, 84, and 168 and analyzed morphologically and by histology. The persistence of the implants was demonstrated, indicating that this injectable bulking agent could potentially be used as biomaterial for the treatment of urinary incontinence and vesico-ureteral reflux.

EPFL2011