Anna E. Guller

Laser-induced regeneration of cartilage

Laser radiation provides a means to control the fields of temperature and thermo mechanical stress, mass transfer, and modification of fine structure of the cartilage matrix. The aim of this outlook paper is to review physical and biological aspects of laser-induced regeneration of cartilage and to discuss the possibilities and prospects of its clinical applications. The problems and the pathways of tissue regeneration, the types and features of cartilage will be introduced first. Then we will review various actual and prospective approaches for cartilage repair; consider possible mechanisms of laser-induced regeneration. Finally, we present the results in laser regeneration of joints and spine disks cartilages and discuss some future applications of lasers in regenerative medicine.

Cytotoxicity and non-specific cellular uptake of bare and surface-modified upconversion nanoparticles in human skin cells

The cytotoxicity and non-specific cellular uptake of the most popular composition of upconversion nanoparticle (UCNP), NaYF4:Yb3+:Er3+, is reported using normal human skin cells, including dermal fibroblasts and immortalized human epidermal linear keratinocytes (HaCaT). A new hydrophilization reaction of as-synthesized UCNPs based on tetramethylammonium hydroxide (TMAH) enabled evaluation of the intrinsic cytotoxicity of bare UCNPs. The cytotoxicity effects of the UCNP surface-coating and polystyrene host were investigated over the concentration range 62.5–125 μg/mL with 24-h incubation, using a MTT test and optical microscopy. The fibroblast viability was not compromised by UCNPs, whereas the viability of keratinocytes varied from 52% ± 4% to 100% ± 10% than the control group, depending on the surface modification. Bare UCNPs reduced the keratinocyte viability to 76% ± 3%, while exhibiting profound non-specific cellular uptake. Hydrophilic poly(D,L-lactide)- and poly(maleic anhydride-alt-1-octadecene)-coated UCNPs were found to be least cytotoxic among the polymer-coated UCNPs, and were readily internalized by human skin cells. Polystyrene microbeads impregnated with UCNPs remained nontoxic. Surprisingly, no correlation was found between UCNP cytotoxicity and the internalization level in cells, although the latter ranged broadly from 0.03% to 59%, benchmarked against 100% uptake level of TMAH-UCNPs.

Specific Features of Early Stage of the Wound Healing Process Occurring Against the Background of Photodynamic Therapy Using Fotoditazin Photosensitizer–Amphiphilic Polymer Complexes

There is a growing demand on the studies of the wound healing potentials of photodynamic therapy. Here we analyze the effects of Fotoditazin, an e6 chlorine derivative, and its complexes with amphiphilic polymers, on the early stage of wound healing in a rat model. A skin excision wound model with prevented contraction was developed in male albino rats divided into eight groups according to the treatment mode. All animals received injections of one of the studied compositions into their wound beds and underwent low‐intensity laser irradiation or stayed un‐irradiated. The clinical monitoring and histological examination of the wounds were performed. It has been found that all the Fotoditazin formulations have significant effects on the early stage of wound healing. The superposition of the inflammation and regeneration was the main difference between groups. The aqueous solution of Fotoditazin alone induced a significant capillary hemorrhage, while its combinations with amphiphilic polymers did not. The best clinical and morphological results were obtained for the Fotoditazin–Pluronic F127 composition. Compositions of Fotoditazin and amphiphilic polymers, especially Pluronic F127, probably, have a great potential for therapy of wounds. Their effects can be attributed to the increased regeneration and suppressed reactions changes at the early stages of repair.

Regeneration of spine disc and joint cartilages under temporal and space modulated laser radiation

The effect of laser radiation on the generation of hyaline cartilage in spine disc and joints has been demonstrated. The paper considers physical processes and mechanisms of laser regeneration, presents results of investigations aimed to optimize laser settings and to develop feedback control system for laser reconstruction of spine discs. Possible mechanisms of laser-induced regeneration include: (1) Space and temporary modulated laser beam induces nonhomogeneous and pulse repetitive thermal expansion and stress in the irradiated zone of cartilage. Mechanical effect due to controllable thermal expansion of the tissue and micro and nano gas bubbles formation in the course of the moderate (up to 45-50 oC) heating of the NP activate biological cells (chondrocytes) and promote cartilage regeneration. (2) Nondestructive laser radiation leads to the formation of nano and micro-pores in cartilage matrix. That promotes water permeability and increases the feeding of biological cells. Results provide the scientific and engineering basis for the novel low-invasive laser procedures to be used in orthopedics for the treatment cartilages of spine and joints. The technology and equipment for laser reconstruction of spine discs have been tested first on animals, and then in a clinical trial. Since 2001 the laser reconstruction of intervertebral discs have been performed for 340 patients with chronic symptoms of low back or neck pain who failed to improve with non-operative care. Substantial relief of back pain was obtained in 90% of patients treated who returned to their daily activities. The experiments on reparation of the defects in articular cartilage of the porcine joints under temporal and spase modulated laser radiation have shown promising results.

Long Term Clinical Results in Laser Reconstruction of Spine Discs

Purpose: Determine the long-term clinical outcome of the laser reconstruction of intervertebral discs (LRD) in patients with chronic degenerative spine diseases. Methods: Ninety-seven patients with chronic back and neck pain caused by single and multi-level spinal discs degeneration were treated with non-ablative laser irradiation (1.56 μm Er:glass fiber laser) of the nucleus pulposus and the inner third of the annulus fibrosus through percutaneous needle puncture. The results were analyzed during five years after LRD by the means of clinical observation, radiological and biomechanical testing. Three surgical biopsies of the laser-irradiated disks were examined by the morphological methods. Subjective estimation of LRD influence of the patients’ life quality and back pain intensity has been performed with validated questionnaires of SF-36 and VAS. Results: Majority of the patients, who underwent LRD procedure, demonstrated an essential improvement in their health state, including decreasing of spine discs instability, pain relief and the general quality of their lives. There were no any complications related to the use of LRD. Five-year outcome have shown positive dynamics of MRI features of the treated discs in 77% of patients as well as an improvement in the SF-36 total score and VAS in 92 and 95.9% of patients respectively. The histological results have proved the growth of hyaline like cartilage in laser-treated zone. Conclusions: Five years outcome observations demonstrate stable positive structural changes in the intervertebral discs as well as the significant improvement in subjective feelings of the life quality and pain relief for the majority of the patients.

Dinitrosyl iron complexes with glutathione incorporated into a collagen matrix as a base for the design of drugs accelerating skin wound healing

Composites of a collagen matrix and dinitrosyl iron complexes with glutathione (DNIC-GS) (in a dose of 4.0 μmoles per item) in the form of spongy sheets (DNIC-Col) were prepared and then topically applied in rat excisional full-thickness skin wound model. The effects of DNIC-Col were studied in comparison with spontaneously healing wounds (SpWH) and wounds treated with collagen sponges (Col) without DNIC-GS. The composites induced statistically and clinically significant acceleration of complete wound closure (21±1 day versus 23±1 day and 26±1 day for DNIC-Col, Col and SpWH, respectively). Histological examination of wound tissues on days 4, 14, 18 and 21 after surgery demonstrated that this improvement was supported by enhanced growth, maturation and fibrous transformation of granulation tissue and earlier epithelization of the injured area in rats treated with DNIC-Col composites benchmarked against Col and SpWH. It is suggested that the positive effect of the new pharmaceutical material on wound healing is based on the release of NO from decomposing DNIC. This effect is believed to be potentiated by the synergy of DNIC and collagen.

Scar tissue classification using nonlinear optical microscopy and discriminant analysis

This paper addresses the scar tissue maturation process that occurs stepwise, and calls for reliable classification. The structure of collagen imaged by nonlinear optical microscopy (NLOM) in post-burn hypertrophic and mature scar, as well as in normal skin, appeared to distinguish these maturation steps. However, it was a discrimination analysis, demonstrated here, that automated and quantified the scar tissue maturation process. The achieved scar classification accuracy was as high as 96%. The combination of NLOM and discrimination analysis is believed to be instrumental in gaining insight into the scar formation, for express diagnosis of scar and surgery planning.

Морфологическое и морфометрическое исследование амиантоидной трансформации реберных хрящей в норме и при килевидной деформации грудной клетки у детей

Amianthoid transformation (AT) is the accumulations of abnormal collagen structures (amianthoid fibers) in the hyaline cartilages, tumors, and tendons. Neither functional value of costal cartilage matrix AT, nor its role in the pathogenesis of congenital chest deformities is known now. AIM to examine the morphological features of AT in the costal cartilage of children with the normal and keeled chest. SUBJECTS AND METHODS Costal cartilages were studied in 6 children with the normal chest (autopsy material) and in 5 ones with keeled chest (surgical material). Tissue fragments were fixed in 10% neutral formalin and embedded in compacted paraffin. The sections were stained with hematoxylin and eosin, picrofuchsin by van Gieson, with picrosirius, toluidine blue and by the Malaurie method modified by Gallego. The specimens were examined by light, phase-contrast, dark-field, fluorescence, and polarization microscopy. The frequency of AT sites and their area were morphometrically studied and the findings were then statistically processed. RESULTS Various types of AT in the costal cartilages were described as both the normal and keeled chest. According to their morphological features, classic, fine-fiber, twisted, and intralacunar types were identified. There were statistically significant increases in the incidence of all types (except the intralacunar one) and in the area of the fine-fiber AT type in keeled chest deformity as compared to health. There were positive correlations between the area of classic, intralacunar, and twisted types in both groups and between the area of a classic type and age in the controls. CONCLUSION A classification of AT areas varying in structures in health and disease has been given for the first time; their relation to each other and to the presence of keeled deformity shown, which, in our opinion, suggests that AT is implicated in the pathogenesis of the disease.

[Morphology of collagen matrices for tissue engineering (biocompatibility, biodegradation, tissue response)].

OBJECTIVE to perform a comparative morphological study of biocompatibility, biodegradation, and tissue response to implantation of collagen matrices (scaffolds) for tissue engineering in urology and other areas of medicine. MATERIAL AND METHODS Nine matrix types, such as porous materials reconstructed from collagen solution; a collagen sponge-vicryl mesh composite; decellularized and freeze-dried bovine, equine, and fish dermis; small intestinal submucosa, decellularized bovine dura mater; and decellularized human femoral artery, were implanted subcutaneously in 225 rats. The tissues at the implantation site were investigated for a period of 5 to 90 days. Classical histology and nonlinear optical microscopy (NLOM) were applied. RESULTS The investigations showed no rejection of all the collagen materials. The period of matrix bioresorption varied from 10 days for collagen sponges to 2 months for decellularized and freeze-dried vessels and vicryl meshes. Collagen was prone to macrophage resorption and enzymatic lysis, being replaced by granulation tissue and then fibrous tissue, followed by its involution. NLOM allowed the investigators to study the number, density, interposition, and spatial organization of collagen structures in the matrices and adjacent tissues, and their change over time during implantation. CONCLUSION The performed investigation could recommend three matrices: hybrid collagen/vicryl composite; decellularized bovine dermis; and decellularized porcine small intestinal submucosa, which are most adequate for tissue engineering in urology. These and other collagen matrices may be used in different areas of regenerative medicine.

AFM study of the extracellular connective tissue matrix in patients with pelvic organ prolapse

The method of atomic force microscopy (AFM) is used for the first time for morphological investigation of pathological changes in the extracellular matrix of skin connective tissue upon the prolapse of pelvic organs (common disorder among women). Skin samples of patients with clinically proven pelvic-organ prolapse and of patients that do not have any connective tissue related disease (control group) are investigated via AFM. The AFM study reveals that the extracellular matrix of the skin connective tissues from patients with pelvic-organ prolapse diverges from the normal in various organization levels including both micro- and nanotexture (packing of collagen fibers and fibrils, respectively). The results of AFM study of the normal and pathologically changed skin connective tissues are in good agreement with the data of clinical morphological analysis, which indicates the potential of AFM as an independent diagnostic tool.