The effect of dextrin-rhEGF on the healing of full-thickness, excisional wounds in the (db/db) diabetic mouse

Chronic wounds, such as ulceration of the lower limb, represent a significant clinical challenge in today's ageing society. With the aim of identifying improved therapeutics, we have previously described a bioresponsive, dextrin-recombinant human epidermal growth factor conjugate (dextrin-rhEGF), that (i) protects rhEGF against proteolytic degradation by human chronic wound fluid; and (ii) mediates rhEGF release by α-amylase, capable of stimulating increased proliferation/migration in normal dermal and chronic wound fibroblasts; and keratinocytes, in vitro. The aim of this study was to extend these findings, by investigating the effects of dextrin-rhEGF on wound healing in the (db/db) diabetic mouse, a widely used in vivo model of delayed wound healing. Standardised, full-thickness excisional wounds, created in the dorsal flank skin, were treated topically with succinoylated dextrin (50 μg/mL), rhEGF (10 μg/mL) or dextrin-rhEGF (1 or 10 μg/mL). Treatments were applied immediately after injury and subsequently on post-wounding, days 3 and 8. Wound healing was assessed macroscopically, in terms of initiation of neo-dermal tissue deposition and wound closure (including wound contraction and re-epithelialisation), over a 16 day period. Wound healing was assessed histologically, in terms of granulation tissue formation/maturity; cranio-caudal wound contraction and wound angiogenesis (CD31 immuno-staining), using tissues harvested at day 16. Blood samples were also analysed for α-amylase and rhEGF concentrations. In this established impaired wound healing model, the topically-applied dextrin-rhEGF significantly accelerated wound closure and neo-dermal tissue formation at the macroscopic level; and significantly increased granulation tissue deposition and angiogenesis at the histological level (p < 0.05), relative to untreated, succinoylated dextrin and rhEGF alone controls. Overall, these findings support the further development of bioresponsive polymer conjugates, for tissue repair.     

Mucoadhesive polymers as platforms for peroral peptide delivery and absorption: synthesis and evaluation of different chitosan–EDTA conjugates

The purpose of the present study was to synthesize and evaluate mucoadhesive polymers, exhibiting a high capacity to bind bivalent cations which are essential co-factors for intestinal proteolytic enzymes. Under the formation of amide bonds, the complexing agent EDTA was covalently bound to the primary amino groups of chitosan. One gram of the resulting conjugate with the lowest amount of remaining free amino groups (0.1±0.03%; mean±SD, n=3) based on free chitosan as 1.0 was capable of binding 1.4±0.1 mM calcium, 2.0±0.1 mM zinc and 1.9±0.03 mM cobalt (mean±SD, n=3) under intestinal pH-conditions, respectively. Whereas proteolytic activity of the serine proteases trypsin (EC 3.4.21.4), -chymotrypsin (EC 3.4.21.1) and elastase (EC 3.4.21.36) could not be inhibited, proteolytic activity of the zinc proteases carboxypeptidase A (EC 3.4.17.1) and aminopeptidase N (EC 3.4.11.2) was strongly inhibited by the chitosan–EDTA conjugate. Moreover, it displays quick swelling properties in water and basic aqueous solutions. The adhesive force of the conjugate was even higher than of chitosan HCl. However, lowering the percentage of covalently attached EDTA on the polymer, leads to a significantly reduced adhesive force. According to these results, chitosan–EDTA conjugates exhibiting the lowest amount of remaining free amino groups, seem to be a useful tool in overcoming the enzymatic barrier for perorally administered therapeutic peptides.     

SIKVAV‐modified highly superporous PHEMA scaffolds with oriented pores for spinal cord injury repair

The architecture and mechanical properties of a scaffold for spinal cord injury treatment must provide tissue integration as well as effective axonal regeneration. Previous work has demonstrated the cell‐adhesive and growth‐promoting properties of the SIKVAV (Ser–Ile–Lys–Val–Ala–Val)‐modified highly superporous poly(2‐hydroxethyl methacrylate) (PHEMA) hydrogels. The aim of the current study was to optimize the porosity and mechanical properties of this type of hydrogel in order to develop a suitable scaffold for the repair of spinal cord tissue. Three types of highly superporous PHEMA hydrogels with oriented pores of ~60 µm diameter, porosities of 57–68% and equivalent stiffness characterized by elasticity moduli in the range 3–45 kPa were implanted into a spinal cord hemisection, and their integration into the host tissue, as well as the extent of axonal ingrowth into the scaffold pores, were histologically evaluated. The best tissue response was found with a SIKVAV‐modified PHEMA hydrogel with 68% porosity and a moderate modulus of elasticity (27 kPa in the direction along the pores and 3.6 kPa in the perpendicular direction). When implanted into a spinal cord transection, the hydrogel promoted tissue bridging as well as aligned axonal ingrowth. In conclusion, a prospective oriented scaffold architecture of SIKVAV‐modified PHEMA hydrogels has been developed for spinal cord injury repair; however, to develop an effective treatment for spinal cord injury, multiple therapeutic approaches are needed. Copyright © 2013 John Wiley & Sons, Ltd.     

Comparative facile methods for preparing graphene oxide–hydroxyapatite for bone tissue engineering

Motivated by the success of using graphene oxide (GO) as a nanofiller of composites, there is a drive to search for this new kind of carbon material as a bioactive component in ceramic materials. In the present study, biomineralized GO was prepared by two different approaches, represented by in situ sol–gel synthesis and biomimetic treatment. It was found that in the biocomposites obtained by the sol–gel approach, the spindle‐like hydroxyapatite nanoparticles, with a diameter of ca. 5 ± 0.37 nm and a length of ca. 70 ± 2.5 nm, were presented randomly and strongly on the surface. The oxygen‐containing functional groups, such as hydroxyl and carbonyl, present on the basal plane and edges of the GO sheets, play an important role in anchoring calcium ions, as demonstrated by FT–IR and TEM investigations. A different result was obtained for biocomposites after biomimetic treatment: an amorphous calcium phosphate on GO sheet was observed after 5 days of treatment. These different approaches resulted in a diverse effect on the proliferation and differentiation of osteogenic mesenchymal stem cells. In fact, in biocomposites prepared by the sol–gel approach the expression of an early marker of osteogenic differentiation, ALP, increases with the amount of GO in the first days of cell culture. Meanwhile, biomimetic materials sustain cell viability and proliferation, even if the expression of alkaline phosphatase activity in a basal medium is delayed. These findings may provide new prospects for utilizing GO‐based hydroxyapatite biocomposites in bone repair, bone augmentation and coating of biomedical implants and broaden the application of GO sheets in biological areas. Copyright © 2016 John Wiley & Sons, Ltd.     

Engineered skin graft with stromal vascular fraction cells encapsulated in fibrin–collagen hydrogel: A clinical study for diabetic wound healing

Despite the abundance of skin substitutes in the worldwide market, major hurdles in developing more complex tissues include the addition of skin appendages and vascular networks as the most important structure. The aim of this research was a clinical feasibility study of a novel prevascularized skin grafts containing the dermal and epidermal layer using the adipose stromal vascular fraction (SVF)‐derived endothelial cell population for vascular network regeneration. Herein, we characterized hydrogel with emphasis on biological compatibility and cell proliferation, migration, and vitality. The therapeutic potential of the prevascularized hydrogel transplanted on five human subjects as an intervention group with diabetic wounds was compared with nonvascularized skin grafts as the control on five patients. Wound planimetric and biometric analysis was performed using a Mann–Whitney nonparametric t‐test (p ≤ .05). The fibrin–collagen hydrogel was suitable for skin organotypic cell culture. There was a significant (p ≤ .05) increased in skin thickness and density in the vascular beds of the hypodermis measured with skin scanner compared with that in the control group. No significant macroscopic differences were observed between the intervention and control groups (p ≤ .05). In summary, we report for the first time the use of autologous dermal–epidermal skin grafts with intrinsic vascular plexus in a clinical feasibility study. The preliminary data showed that SVF‐based full‐thickness skin grafts are safe and accelerate the wound healing process. The next stage of the study is a full‐scale randomized clinical trial for the treatment of patients with chronic wounds.     

Repair of an osteochondral defect by sustained delivery of BMP‐2 or TGFβ1 from a bilayered alginate–PLGA scaffold

Regeneration of cartilage defects can be accelerated by localized delivery of appropriate growth factors (GFs) from scaffolds. In the present study we analysed the in vitro and in vivo release rates and delivery efficacies of transforming growth factor‐β1 (TGFβ1) and bone morphogenetic protein‐2 (BMP‐2) from a bilayered system, applied for osteochondral defect repair in a rabbit model. A bone‐orientated, porous PLGA cylinder was overlaid with GF containing PLGA microspheres, dispersed in an alginate matrix. Four microsphere formulations were incorporated: (a) blank ones; (b) microspheres containing 50 ng TGFβ1; (c) microspheres containing 2.5 µg BMP‐2; and (d) microspheres containing 5 µg BMP‐2. Release kinetics and tissue distributions were determined using iodinated (¹²⁵I) GFs. Bioactivity of in vitro released BMP‐2 and TGFβ1 was confirmed in cell‐based assays. In vivo release profiles indicated good GF release control. 20% of BMP‐2 and 15% of TGFβ1 were released during the first day. Virtually the total dose was delivered at the end of week 6. Significant histological differences were observed between untreated and GF‐treated specimens, there being especially relevant short‐term outcomes with 50 ng TGFβ1 and 5 µg BMP‐2. Although the evaluation scores for the newly formed cartilage did not differ significantly, 5 µg BMP‐2 gave rise to higher quality cartilage with improved surface regularity, tissue integration and increased collagen‐type II and aggrecan immunoreactivity 2 weeks post‐implantation. Hence, the bilayered system controlled GF release rates and led to preserved cartilage integrity from 12 weeks up to at least 24 weeks. Copyright © 2012 John Wiley & Sons, Ltd.     

A pedagogical framework for facilitating parents’ learning in nurse–parent partnership

Nursing work increasingly demands forms of expertise that complement specialist knowledge. In child and family nursing, this need arises when nurses work in partnership with parents of young children at risk. Partnership means working with parents in respectful, negotiated and empowering ways. Existing partnership literature emphasises communicative and relational skills, but this paper focuses on nurses’ capacities to facilitate parents’ learning. Referring to data from home visiting, day‐stay and specialist toddler clinic services in Sydney, a pedagogical framework is presented. Analysis shows how nurses notice aspects of children, parents and parent–child interactions as a catalyst for building on parents’ strengths, enhancing guided chance or challenging unhelpful constructs. Prior research shows the latter can be a sticking point in partnership, but this paper reveals diverse ways in which challenges are folded into learning process that position parents as agents of positive change. Noticing is dependent on embodied and communicative expertise, conceptualised in terms of sensory and reported channels. The framework offers a new view of partnership as mind‐expanding for the parent and specifies the nurse's role in facilitating this process.     

Multivalent interactions between biotin–polyrotaxane conjugates and streptavidin as a model of new targeting for transporters

Kinetic analysis of interactions between biotin–polyrotaxane or biotin–-cyclodextrin (biotin–-CD) conjugates and streptavidin was carried out as a model of new targeting to transporters using the surface plasmon resonance (SPR) technique. The biotin–polyrotaxane conjugates, in which biotin-introduced -CDs are threaded onto a poly(ethylene oxide) chain capped with bulky end-groups, are expected to increase the valency of biotin from monovalent to multivalent binding. The number of biotins conjugated with one polyrotaxane molecule varied from 11 to 78, and apparently increased the association equilibrium constant (K_a), assuming pseudo-first-order kinetics. A detailed dissociation kinetics was analyzed and the re-binding of the biotin–polyrotaxane conjugates was observed on the streptavidin-deposited SPR surface. The magnitude of the re-binding is likely to become larger with increasing the number of biotins, suggesting multivalent interaction on the SPR surface. To quantify the effect of valency, competitive inhibition assay was performed in terms of the supramolecular structure of the polyrotaxane. The inhibitory potency of the biotin–polyrotaxane conjugate was found to be 4–5 times greater than that of the biotin–-CD conjugate. Therefore, the biotin–polyrotaxane conjugates by supramolecular formation of the biotin–-CD conjugate significantly switches from monovalent to multivalent bindings to the model binding protein, streptavidin.     

Minimal contraction for tissue‐engineered skin substitutes when matured at the air–liquid interface

The structural stability of skin substitutes is critical to avoid aesthetic and functional problems after grafting, such as contractures and hypertrophic scars. The present study was designed to assess the production steps having an influence on the contractile behaviour of the tissue‐engineered skin made by the self‐assembly approach, where keratinocytes are cultured on tissue‐engineered dermis comprised of fibroblasts and the endogenous extracellular matrix they organized. Thus, different aspects were investigated, such as the assembly method of the engineered dermis (various sizes and anchoring designs) and the impact of epithelial cell differentiation (culture submerged in the medium or at the air–liquid interface). To evaluate the structural stability at the end of the production, the substitutes were detached from their anchorages and deposited on a soft substrate, and contraction was monitored over 1 week. Collected data were analysed using a mathematical model to characterize contraction. We observed that the presence of a differentiated epidermis significantly reduced the amount of contraction experienced by the engineered tissues, independently of the assembly method used for their production. When the epidermis was terminally differentiated, the average contraction was only 24 ± 4% and most of the contraction occurred within the first 12 h following deposition on the substrate. This is 2.2‐fold less compared to when the epidermis was cultured under the submerged condition, or when tissue‐engineered dermis was not overlaid with epithelial cells. This study highlights that the maturation at the air–liquid interface is a critical step in the reconstruction of a tissue‐engineered skin that possesses high structural stability. Copyright © 2012 John Wiley & Sons, Ltd.