Anatoly B. Shekhter

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.

Laser Regeneration of Spine Discs Cartilage: Mechanism, In-Vivo Study and Clinical Applications

Laser Reconstruction of the spine discs (LRD) is a novel minimally invasive approach for the treatment of spine diseases. This approach belongs to the laser therapy, but it differs from low intensity laser therapy because LRD uses local and moderate heterogeneous laser heating that mostly does not effect directly on the cells; LRD procedure modifies the extra cellular matrix to provide better surroundings for the cells. Our main finding is that laser irradiation can activate the growth of hyaline cartilage. The predictability of the result, the locality and safety of laser effect allowed to use the technology for spine problems. LRD can be performed in an outpatient setting requiring only 30 min to complete without the need for general anesthesia. The new type of Erbium doped glass fiber laser (1.56 mm in wavelength) has been tested first on animals and then in a clinical trial. The mechanism of laser-induced tissue regeneration include: (1) formation of nanopores enchasing water permeability through end plates and annulus fibrosus of the disc that provide feeding for biological cells, and (2) activation of cell due to mechanical oscillations resulting from the periodically thermo expansion of nucleus pulposus under modulated laser irradiation. The clinical trials have shown positive results for 90% from 240 laser treated patients.

Medical Applications of Collagen and Collagen-Based Materials

Collagen and collagen-based materials have been successfully used in medicine for over 50 years. The number of scientific articles about the role of collagen in the construction of scaffolds for tissue engineering has risen precipitously in recent years. The review contains materials about historic and modern applications of collagen in medicine such as soluble collagen injections, solid constructs reconstructed from solution, and decellularized collagen matrices. The analysis of published data proves the efficacy of collagen material in the treatment of chronic wounds, burns, venous and diabetic ulcers, in plastic, reconstructive and general surgery, urology, proctology, gynecology, ophthalmology, otolaryngology, neurosurgery, dentistry, cardiovascular and bone and cartilage surgery, as well as in cosmetology. Further development of collagenoplasty requires addressing the problems of allergic complications, improvement of structure and maximizing therapeutic effects against pathological processes.

Collagenolytic enzymes and their applications in biomedicine

Nowadays, enzymatic therapy is a very promising line of treatment for many different diseases. There is a group of disorders and conditions, caused by fibrotic and scar processes and associated with the excessive accumulation of collagen that needs to be catabolized to normalize the connective tissue content. The human body normally synthesizes special extracellular enzymes, matrix metalloproteases (MMPs) by itself. These enzymes can cleave components of extracellular matrix (ECM) and different types of collagen and thus maintain the balance of the connective tissue components. MMPs are multifunctional enzymes and are involved in a variety of organism processes. However, under pathological conditions, the function of MMPs is not sufficient, and these enzymes fail to deal with disease. Thus, medical intervention is required. Enzymatic therapy is a very effective way of treating such collagen-associated conditions. It involves the application of exogenous collagenolytic enzymes that catabolize excessive collagen at the affected site and lead to the successful elimination of disease. Such collagenolytic enzymes are synthesized by many organisms: bacteria, animals (especially marine organisms), plants and fungi. The most studied and commercially available are collagenases from Clostridium histolyticum and from the pancreas of the crab Paralithodes camtschatica, due to their ability to effectively hydrolyse human collagen without affecting other tissues, and their wide pH ranges of collagenolytic activity. In the present review, we summarize not only the data concerning existing collagenase-based medications and their applications in different collagen-related diseases and conditions, but we also propose collagenases from different sources for their potential application in enzymatic therapy.