Sensory perturbations using suture and sutureless repair of transected median nerve in rats

The effects of changes to cold, mechanical, and heat thresholds following median nerve transection with repair by sutures (Su) or Rose Bengal adhesion (RA) were compared to sham-operated animals. Both nerve-injured groups showed a transient, ipsilateral hyposensitivity to mechanical and heat stimuli followed by a robust and long-lasting hypersensitivity (6–7 weeks) with gradual recovery towards pre-injury levels by 90 days post-repair. Both tactile and thermal hypersensitivity were seen in the contralateral limb that was similar in onset but differed in magnitude and resolved more rapidly compared to the injured limb. Prior to injury, no animals showed any signs of aversion to cold plate temperatures of 4–16?°C. After injury, animals showed cold allodynia, lasting for 7 weeks in RA-repaired rats before recovering towards pre-injury levels, but were still present at 12 weeks in Su-repaired rats. Additionally, sensory recovery in the RA group was faster compared to the Su group in all behavioural tests. Surprisingly, sham-operated rats showed similar bilateral behavioural changes to all sensory stimuli that were comparable in onset and magnitude to the nerve-injured groups but resolved more quickly compared to nerve-injured rats. These results suggest that nerve repair using a sutureless approach produces an accelerated recovery with reduced sensorimotor disturbances compared to direct suturing. They also describe, for the first time, that unilateral forelimb nerve injury produces mirror-image-like sensory perturbations in the contralateral limb, suggesting that the contralateral side is not a true control for sensory testing. The potential mechanisms involved in this altered behaviour are discussed.     

Investigation of the differentiation of ex vivo nerve and fat tissues using laser‐induced breakdown spectroscopy (LIBS): Prospects for tissue‐specific laser surgery

In the present study, the elemental compositions of fat and nerve tissue during their plasma mediated laser ablation are studied in the context of tissue differentiation for laser surgery applications by using Laser‐Induced Breakdown Spectroscopy (LIBS). Tissue samples of porcine fat and nerve were prepared as ex vivo experimental objects. Plasma mediated laser ablation is performed using an Nd : YAG laser in open air and under normal stray light conditions. The performed measurements suggest that the two tissue types show a high similarity in terms of qualitative elemental composition while at the same time revealing a distinct difference in the concentration of the constituent elements. Different analysis approaches are evaluated and discussed to optimize the tissue‐differentiation performance of the LIBS approach.

Plasma mediated laser tissue ablation.     

Interplay of temperature,thermal‐stresses and strains in laser‐assisted modification of collagenous tissues: Speckle‐contrast and OCT‐based studies

Moderate heating of collagenous tissues such as cartilage and cornea by infrared laser irradiation can produce biologically nondestructive structural rearrangements and relaxation of internal stresses resulting in the tissue reshaping. The reshaping results and eventual changes in optical and biological properties of the tissue strongly depend on the laser‐irradiation regime. Here, a speckle‐contrast technique based on monochromatic illumination of the tissue in combination with strain mapping by means of optical coherence elastography (OCE) is applied to reveal the interplay between the temperature and thermal stress fields producing tissue modifications. The speckle‐based technique ensured en face visualization of cross correlation and contrast of speckle images, with evolving proportions between contributions of temperature increase and thermal‐stresses determined by temperature gradients. The speckle‐technique findings are corroborated by quantitative OCE‐based depth‐resolved imaging of irradiation‐induced strain‐evolution. The revealed relationships can be used for real‐time control of the reshaping procedures (e.g., for laser shaping of cartilaginous implants in otolaryngology and maxillofacial surgery) and optimization of the laser‐irradiation regimes to ensure the desired reshaping using lower and biologically safer temperatures. The figure of waterfall OCE‐image demonstrates how the strain‐rate maximum arising in the heating‐beam center gradually splits and drifts towards the zones of maximal thermal stresses located at the temperature‐profile slopes.     

Advances of tooth‐derived stem cells in neural diseases treatments and nerve tissue regeneration

Nerous system diseases, both central and peripheral, bring an incredible burden onto patients and enormously reduce their quality of life. Currently, there are still no effective treatments to repair nerve lesions that do not have side effects. Stem cell–based therapies, especially those using dental stem cells, bring new hope to neural diseases. Dental stem cells, derived from the neural crest, have many characteristics that are similar to neural cells, indicating that they can be an ideal source of cells for neural regeneration and repair. This review summarizes the neural traits of all the dental cell types, including DPSCs, PDLCs, DFCs, APSCs and their potential applications in nervous system diseases. We have summed up the advantages of dental stem cells in neural repair, such as their neurotrophic and neuroprotective traits, easy harvest and low rejective reaction rate, among others. Taken together, dental stem cells are an ideal cell source for neural tissue regeneration and repair.     

The importance of the neuro‐immuno‐cutaneous system on human skin equivalent design

The skin is a highly complex organ, responsible for sensation, protection against the environment (pollutants, foreign proteins, infection) and thereby linked to the immune and sensory systems in the neuro‐immuno‐cutaneous (NIC) system. Cutaneous innervation is a key part of the peripheral nervous system; therefore, the skin should be considered a sensory organ and an important part of the central nervous system, an ‘active interface’ and the first connection of the body to the outside world. Peripheral nerves are a complex class of neurons within these systems, subsets of functions are conducted, including mechanoreception, nociception and thermoception. Epidermal and dermal cells produce signalling factors (such as cytokines or growth factors), neurites influence skin cells (such as via neuropeptides), and peripheral nerves have a role in both early and late stages of the inflammatory response. One way this is achieved, specifically in the cutaneous system, is through neuropeptide release and signalling, especially via substance P (SP), neuropeptide Y (NPY) and nerve growth factor (NGF). Cutaneous, neuronal and immune cells play a central role in many conditions, including psoriasis, atopic dermatitis, vitiligo, UV‐induced immunosuppression, herpes and lymphomas. Therefore, it is critical to understand the connections and interplay between the peripheral nervous system and the skin and immune systems, the NIC system. Relevant in vitro tissue models based on human skin equivalents can be used to gain insight and to address impact across research and clinical needs.     

Identification of peptide‐binding sites within BSA using rapid,laser‐induced covalent cross‐linking combined with high‐performance mass spectrometry

We are developing a rapid, time‐resolved method using laser‐activated cross‐linking to capture protein‐peptide interactions as a means to interrogate the interaction of serum proteins as delivery systems for peptides and other molecules. A model system was established to investigate the interactions between bovine serum albumin (BSA) and 2 peptides, the tridecapeptide budding‐yeast mating pheromone (α‐factor) and the decapeptide human gonadotropin‐releasing hormone (GnRH). Cross‐linking of α‐factor, using a biotinylated, photoactivatable p‐benzoyl‐L‐phenylalanine (Bpa)–modified analog, was energy‐dependent and achieved within seconds of laser irradiation. Protein blotting with an avidin probe was used to detect biotinylated species in the BSA‐peptide complex. The cross‐linked complex was trypsinized and then interrogated with nano‐LC–MS/MS to identify the peptide cross‐links. Cross‐linking was greatly facilitated by Bpa in the peptide, but some cross‐linking occurred at higher laser powers and high concentrations of a non‐Bpa–modified α‐factor. This was supported by experiments using GnRH, a peptide with sequence homology to α‐factor, which was likewise found to be cross‐linked to BSA by laser irradiation. Analysis of peptides in the mass spectra showed that the binding site for both α‐factor and GnRH was in the BSA pocket defined previously as the site for fatty acid binding. This model system validates the use of laser‐activation to facilitate cross‐linking of Bpa‐containing molecules to proteins. The rapid cross‐linking procedure and high performance of MS/MS to identify cross‐links provides a method to interrogate protein‐peptide interactions in a living cell in a time‐resolved manner.     

Direct transplantation of uncultured hair‐follicle pluripotent stem (hfPS) cells promotes the recovery of peripheral nerve injury

We previously showed that the stem cell marker nestin is expressed in hair follicle stem cells which suggested their pluripotency. We subsequently showed that the nestin‐expressing hair‐follicle pluripotent stem (hfPS) cells can differentiate in culture to neurons, glial cells, keratinocytes, and other cell types and can promote regeneration of peripheral nerve and spinal cord injuries upon injection to the injured nerve or spinal cord. The location of the hfPS cells has been termed the hfPS cell area (hfPSCA). Previously, hfPS cells were cultured for 1–2 months before transplantation to the injured nerve or spinal cord which would not be optimal for clinical application of these cells for nerve or spinal cord repair, since the patient should be treated soon after injury. In the present study, we addressed this issue by directly using the upper part of the hair follicle containing the hfPSCA, without culture, for injection into the severed sciatic nerve in mice. After injection of hfPSCA, the implanted hfPS cells grew and promoted joining of the severed nerve. The transplanted hfPS cells differentiated mostly to glial cells forming myelin sheaths, which promoted axonal growth and functional recovery of the severed nerve. These results suggest that the direct transplantation of the uncultured upper part of the hair follicle containing the hfPSA is an important method to promote the recovery of peripheral nerve injuries and has significant clinical potential. J. Cell. Biochem. 110: 272–277, 2010. © 2010 Wiley‐Liss, Inc.     

Real‐time monitoring of incision profile during laser surgery using shock wave detection

Lack of sensory feedback during laser surgery prevents surgeons from discerning the exact location of the incision, which increases duration and complexity of the treatment. In this study we demonstrate a new method for monitoring of laser ablation procedures. Real‐time tracking of the exact three dimensional (3D) lesion profile is accomplished by detection of shock waves emanating from the ablation spot and subsequent reconstruction of the incision location using time‐of‐flight data obtained from multiple acoustic detectors. Here, incisions of up to 9 mm in depth, created by pulsed laser ablation of fresh bovine tissue samples, were successfully monitored in real time. It was further observed that, by utilizing as little as 12 detection elements, the incision profile can be characterized with accuracy below 0.5 mm in all three dimensions and in good agreement with histological examinations. The proposed method holds therefore promise for delivering high precision real‐time feedback during laser surgeries. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Laser tissue welding analyzed using fluorescence,Stokes shift spectroscopy,and Huang‐Rhys parameter

Near infrared (NIR) continuous wave laser radiation at the 1,450 nm wavelength was used to weld porcine aorta and skin samples via the absorption of combitional vibrational modes of native water in the tissues. The fluorescence spectra were measured from the key native molecules of welded and non‐welded tissues at specific excitation and emission wavelengths from collagen, elastin, and tryptophan. The changes in the fluorescence intensities and differences in Stokes shift (Δν_ss) of key native fluorophores were measured to differentiate the Huang‐Rhys parameter values (S) of the chromophores. The strength of coupling depends on the local electron‐vibration intra‐tissue molecular environment and the amount of polar solvent water surrounding the net charges on collagen, elastin, and tryptophan. The S values for both non‐welded and welded tissues were almost the same and less than 3, suggesting minimal changes in the local molecular environment as a result of welding. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Bioassembly of three‐dimensional embryonic stem cell‐scaffold complexes using compressed gases

Tissues are composed of multiple cell types in a well‐organized three‐dimensional (3D) microenvironment. To faithfully mimic the tissue in vivo, tissue‐engineered constructs should have well‐defined 3D chemical and spatial control over cell behavior to recapitulate developmental processes in tissue‐ and organ‐specific differentiation and morphogenesis. It is a challenge to build a 3D complex from two‐dimensional (2D) patterned structures with the presence of cells. In this study, embryonic stem (ES) cells grown on polymeric scaffolds with well‐defined microstructure were constructed into a multilayer cell‐scaffold complex using low pressure carbon dioxide (CO₂) and nitrogen (N₂). The mouse ES cells in the assembled constructs were viable, retained the ES cell‐specific gene expression of Oct‐4, and maintained the formation of embryoid bodies (EBs). In particular, cell viability was increased from 80% to 90% when CO₂ was replaced with N₂. The compressed gas‐assisted bioassembly of stem cell‐polymer constructs opens up a new avenue for tissue engineering and cell therapy. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Photobiomodulation of a flowable matrix in a human skin ex vivo model demonstrates energy‐based enhancement of engraftment integration and remodeling

The use of dermal substitutes to treat skin defects such as ulcers has shown promising results, suggesting a potential role for skin substitutes for treating acute and chronic wounds. One of the main drawbacks with the use of dermal substitutes is the length of time from engraftment to graft take, plus the risk of contamination and failure due to this prolonged integration. Therefore, the use of adjuvant energy‐based therapeutic modalities to augment and accelerate the rate of biointegration by dermal substitute engraftments is a desirable outcome. The photobiomodulation (PBM) therapy modulates the repair process, by stimulating cellular proliferation and angiogenesis. Here, we evaluated the effect of PBM on a collagen‐glycosaminoglycan flowable wound matrix (FWM) in an ex vivo human skin wound model. PBM resulted in accelerated rate of re‐epithelialization and organization of matrix as seen by structural arrangement of collagen fibers, and a subsequent increased expression of alpha‐smooth muscle actin (α‐SMA) and vascular endothelial growth factor A (VEGF‐A) leading to an overall improved healing process. The use of PBM promoted a beneficial effect on the rate of integration and healing of FWM. We therefore propose that the adjuvant use of PBM may have utility in enhancing engraftment and tissue repair and be of value in clinical practice.