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Scaffold-free in vitro organogenesis exploits the innate ability of cells to synthesise and deposit their own extracellular matrix to fabricate tissue-like assemblies. Unfortunately, cell-assembled tissue engineered concepts require prolonged ex vivo culture periods of very high cell numbers for the development of a borderline three-dimensional implantable device, which are associated with phenotypic drift and high manufacturing costs, thus, hindering their clinical translation and commercialisation. Herein, we report the accelerated (10 days) development of a truly three-dimensional (338.1 ± 42.9 µm) scaffold-free tissue equivalent that promotes fast wound healing and induces formation of neotissue composed of mature collagen fibres, using human adipose derived stem cells seeded at only 50,000 cells/cm2 on an poly (N-isopropylacrylamide-co-N-tert-butylacrylamide (PNIPAM86-NTBA14) temperature-responsive electrospun scaffold and grown under macromolecular crowding conditions (50 µg/ml carrageenan). Our data pave the path for a new era in scaffold-free regenerative medicine.

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BACKGROUND: Ditrosyl iron complexes (DNIC) are endogenous donors of nitric oxide. The possibility of their application to stimulate regeneration has been studied for more than 15 years. However, the most effective dose and form of delivery have not yet been determined. PURPOSE: The aim of this research was to develop a spray form of DNIC that accelerates wound healing. METHODS: We prepared a series of DNIC sprays with spray dosages of 10, 50 and 100 µg. We modelled full-thickness skin wounds in 24 Wistar rats and treated them with distilled water (n = 6), 10 (n = 6), 50 (n = 6) and 100 µg (n = 6) for three post-operative days. On the fourth day, the excised wound tissues were studied by morphological, immunohistochemical and morphometric methods. RESULTS: We demonstrated that 50 µg of DNIC spray had the most beneficial effect on wound healing: the thickness of the granulation tissue layer was 140% higher, vimentin positive fibroblasts predominated and the intensity of inflammation was significantly lower than in the control. There was a dose-dependent decrease in the functional activity of mast cells in the experimental groups compared to the control. CONCLUSION: DNIC spray is a potential effective dosage form for the treatment of large-area skin lesions.

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PURPOSE: To ascertain the role of autologous bone marrow-derived mesenchymal stem cells (BM-MSCs) in the tendon regeneration. METHODS: The study was conducted on 58 Achilles tendons from 29 laboratory Chinchilla adult rabbits. The central bundles of 48 tendons were partially removed and substituted with a tissue-engineered construct consisting of a collagen sponge either loaded with BM-MSCs (n = 24) or cell free (n = 24), placed inside a Vicryl mesh tube. The ends of the resected tendon were inserted in the construct to reach a direct contact with the sponge and sutured to the tube. The animals were sacrificed three and six months post-surgery. Ten intact tendons from five rabbits were used as an untreated control. The tissue samples (n = 30) were stained with haematoxylin and eosin, Picrosirius red, primary antibodies to collagen types I and III and studied by bright-field, phase-contrast, polarized light, and scanning electron microscopies followed by semi-quantitative morphometry. RESULTS: Six months results of cell-loaded scaffolds demonstrated parallel collagen fibres, spindle-shaped tenocytes, and neoangiogenesis. In the control cell-free group, the injured areas were filled with a nonspecific fibrotic tissue with minor foci of incomplete regeneration. The biomechanical tests of 28 tendons taken from 14 rabbits showed that the stiffness of the cell-based reconstructed tendons increased to 98% of the value for the intact samples. CONCLUSION: The obtained results support the hypothesis that the application of BM-MSCs in a tissue-engineered tendon construct leads to the restitution of the tendon tissue.

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Peri-implant fibrosis (PIF) increases the postsurgical risks after implantation and limits the efficacy of the implantable drug delivery systems (IDDS). Pirfenidone (PF) is an oral anti-fibrotic drug with a short (<3 h) circulation half-life and strong adverse side effects. In the current study, disk-shaped IDDS prototype combining polylactic acid (PLA) and PF, PLA@PF, with prolonged (~3 days) PF release (in vitro) was prepared. The effects of the PLA@PF implants on PIF were examined in the rabbit ear skin pocket model on postoperative days (POD) 30 and 60. Matching blank PLA implants (PLA0) and PLA0 with an equivalent single-dose PF injection performed on POD0 (PLA0+injPF) served as control. On POD30, the intergroup differences were observed in α-SMA, iNOS and arginase-1 expressions in PLA@PF and PLA0+injPF groups vs. PLA0. On POD60, PIF was significantly reduced in PLA@PF group. The peri-implant tissue thickness decreased (532 ± 98 µm vs. >1100 µm in control groups) approaching the intact derma thickness value (302 ± 15 µm). In PLA@PF group, the implant biodegradation developed faster, while arginase-1 expression was suppressed in comparison with other groups. This study proves the feasibility of the local control of fibrotic response on implants via modulation of foreign body reaction with slowly biodegradable PF-loaded IDDS.

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Mature hypertrophic scars (HSs) remain a challenging clinical problem, particularly due to the absence of biologically relevant experimental models as a standard rabbit ear HS model only reflects an early stage of scarring. The current study aims to adapt this animal model for simulation of mature HS by validating the time of the scar stabilization using qualitative and quantitative criteria. The full-thickness skin and perichondrium excision wounds were created on the ventral side of the rabbit ears. The tissue samples were studied on post-operation days (PODs) 30, 60, 90 and 120. The histopathological examination and morphometry were applied in parallel with biochemical analysis of protein and glycosaminoglycans (GAGs) content and amino acid composition. The supramolecular organization of collagen was explored by differential scanning calorimetry. Four stages of the rabbit ear HS maturation were delineated and attributed with the histolomorphometrical and physicochemical parameters of the tissue. The experimental scars formed in 30 days but stabilized structurally and biochemically only on POD 90-120. This evidence-based model can be used for the studies and testing of new treatments of the mature HSs.

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Laser-induced forward transfer is a versatile, non-contact, and nozzle-free printing technique which has demonstrated high potential for different printing applications with high resolution. In this article, three most widely used hydrogels in bioprinting (2% hyaluronic acid sodium salt, 1% methylcellulose, and 1% sodium alginate) were used to study laser printing processes. For this purpose, the authors applied a laser system based on a pulsed infrared laser (1064 nm wavelength, 8 ns pulse duration, 1 - 5 J/cm2 laser fluence, and 30 µm laser spot size). A high-speed shooting showed that the increase in fluence caused a sequential change in the transfer regimes: No transfer regime, optimal jetting regime with a single droplet transfer, high speed regime, turbulent regime, and plume regime. It was demonstrated that in the optimal jetting regime, which led to printing with single droplets, the size and volume of droplets transferred to the acceptor slide increased almost linearly with the increase of laser fluence. It was also shown that the maintenance of a stable temperature (±2°C) allowed for neglecting the temperature-induced viscosity change of hydrogels. It was determined that under room conditions (20°C, humidity 50%), the hydrogel layer, due to drying processes, decreased with a speed of about 8 µm/min, which could lead to a temporal variation of the transfer process parameters. The authors developed a practical algorithm that allowed quick configuration of the laser printing process on an applied experimental setup. The configuration is provided by the change of the easily tunable parameters: Laser pulse energy, laser spot size, the distance between the donor ribbon and acceptor plate, as well as the thickness of the hydrogel layer on the donor ribbon slide.

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Decellularized bovine pericardium (DBP)-based biomeshes are the gold standard in reconstructive surgery. In order to prolong their stability after the transplantation, various chemical cross-linking strategies are employed. However, structural and functional properties of the biomeshes differ in dependence on the cross-linker used. Here, we performed a bottom-up study of structural and functional alterations of DBP-based biomeshes following cross-linking with hexamethylene diisocyanate (HMDC), ethylene glycol diglycidyl ether (EGDE), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and genipin. The in vitro cytotoxicity tests supported their clinical applicability. Their structural differences (eg roughness, fibre thickness, pore morphology) were evaluated using the two-photon confocal laser scanning, atomic force, scanning electron and polarized light microscopies. HMDC and EDC samples appeared to be the roughest. Complex mechanical trials indicated the tendency to reduced Young's Modulus and mechanical anisotropy values of DBP upon cross-linking. The lowest mechanical anisotropy was found in EDC and genipin sample groups. In vitro collagenase susceptibility was the highest for EDC samples and the lowest for EGDE samples. The comparative analysis of the results allowed us to recognize the strengths and weaknesses of each cross-linker in relation to a particular clinical application.

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