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BackgroundMesenchymal stromal cells attract much interest in tissue regeneration because of their capacity to differentiate into mesodermal origin cells, their paracrine properties and their possible use in autologous transplantations. The aim of this study was to investigate the safety and reparative potential of implanted human mesenchymal stromal cells (hMSCs), prepared under Good Manufacturing Practice (GMP) conditions utilizing human mixed platelet lysate as a culture supplement, in a collagenase Achilles tendon injury model in rats.MethodsEighty-one rats with collagenase-induced injury were divided into two groups. The first group received human mesenchymal stromal cells injected into the site of injury 3 days after lesion induction, while the second group received saline. Biomechanical testing, morphometry and semiquantitative immunohistochemistry of collagens I, II and III, versican and aggrecan, neovascularization, and hMSC survival were performed 2, 4, and 6 weeks after injury.ResultsHuman mesenchymal stromal cell-treated rats had a significantly better extracellular matrix structure and a larger amount of collagen I and collagen III. Neovascularization was also increased in hMSC-treated rats 2 and 4 weeks after tendon injury. MTCO2 (Cytochrome c oxidase subunit II) positivity confirmed the presence of hMSCs 2, 4 and 6 weeks after transplantation. Collagen II deposits and alizarin red staining for bone were found in 6 hMSC- and 2 saline-treated tendons 6 weeks after injury. The intensity of anti-versican and anti-aggrecan staining did not differ between the groups.ConclusionshMSCs can support tendon healing through better vascularization as well as through larger deposits and better organization of the extracellular matrix. The treatment procedure was found to be safe; however, cartilage and bone formation at the implantation site should be taken into account when planning subsequent in vivo and clinical trials on tendinopathy as an expected adverse event.

Many connective tissue diseases and defects are associated with poor synthesis or excessive degradation of collagen. The modern tissue engineering approach is to replace the defective site via the implantation of a biocompatible scaffold which serves as a carrier for cell incorporation, proliferation, and growth. Collagen is widely used in the field of clinical medicine in connection with both hard and soft tissue applications. However, certain collagen properties such as poor dimensional stability, poor in vivo mechanical strength, low degree of elasticity, variable nature in terms of enzymatic degradation, crosslinking density, fiber size, trace impurities, and side effects frequently limit both its analysis and application. This review focuses particularly on the processing and modification of collagen type I with respect to its biological and mechanical properties. The processing of collagen into scaffolds is crucial to mimic successfully the extracellular matrices. Moreover, the review suggests several ways in which the most common problems related to the isolation, handling, electrospinning, and crosslinking of collagen can be overcome while maintaining its native character as much as possible. Further, the review provides a summary of the analytical methods available for the physicochemical characterization of collagen with respect to both its molecular and submolecular structure.

Collagen composite scaffolds have been used for a number of studies in tissue engineering. The hydration of such highly porous and hydrophilic structures may influence mechanical behaviour and porosity due to swelling. The differences in physical properties following hydration would represent a significant limiting factor for the seeding, growth and differentiation of cells in vitro and the overall applicability of such hydrophilic materials in vivo. Scaffolds based on collagen matrix, poly(DL-lactide) nanofibers, calcium phosphate particles and sodium hyaluronate with 8 different material compositions were characterised in the dry and hydrated states using X-ray microcomputed tomography, compression tests, hydraulic permeability measurement, degradation tests and infrared spectrometry. Hydration, simulating the conditions of cell seeding and cultivation up to 48 h and 576 h, was found to exert a minor effect on the morphological parameters and permeability. Conversely, hydration had a major statistically significant effect on the mechanical behaviour of all the tested scaffolds. The elastic modulus and compressive strength of all the scaffolds decreased by ~95%. The quantitative results provided confirm the importance of analysing scaffolds in the hydrated rather than the dry state since the former more precisely simulates the real environment for which such materials are designed.

BackgroundCollagen-based scaffolds provide a promising option for the treatment of bone defects. One of the key parameters of such scaffolds consists of porosity, including pore size. However, to date, no agreement has been found with respect to the methodology for pore size evaluation. Since the determination of the exact pore size value is not possible, the comparison of the various methods applied is complicated. Hence, this study focuses on the comparison of two widely-used methods for the characterization of porosity—scanning electron microscopy (SEM) and micro-computed tomography (micro-CT).Methods7 types of collagen-based composite scaffold models were prepared by means of lyophilization and collagen cross-linking. Micro-CT analysis was performed in 3D and in 2D (pore size parameters were: major diameter, mean thickness, biggest inner circle diameter and area-equivalent circle diameter). Afterwards, pore sizes were analyzed in the same specimens by an image analysis of SEM microphotographs. The results were statistically evaluated. The comparison of the various approaches to the evaluation of pore size was based on coefficients of variance and the semi-quantitative assessment of selected qualities (e.g. the potential for direct 3D analysis, whole specimen analysis, non-destructivity).ResultsThe pore size values differed significantly with respect to the parameters applied. Median values of pore size values were ranging from 20 to 490 µm. The SEM values were approximately 3 times higher than micro-CT 3D values for each specimen. The Mean thickness was the most advantageous micro-CT 2D approach. Coefficient of variance revealed no differences among pore size parameters (except major diameter). The semi-quantitative comparison approach presented pore size parameters in descending order with regard to the advantages thereof as follows: (1) micro-CT 3D, (2) mean thickness and SEM, (3) biggest inner circle diameter, major diameter and area equivalent circle diameter.ConclusionThe results indicated that micro-CT 3D evaluation provides the most beneficial overall approach. Micro-CT 2D analysis (mean thickness) is advantageous in terms of its time efficacy. SEM is still considered as gold standard for its widespread use and high resolution. However, exact comparison of pore size analysis in scaffold materials remains a challenge.Electronic supplementary materialThe online version of this article (10.1186/s12938-018-0543-z) contains supplementary material, which is available to authorized users.

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Various types of nanofibers are increasingly used in tissue engineering, mainly for their ability to mimic the architecture of tissue at the nanoscale. We evaluated the adhesion, growth, viability, and differentiation of human osteoblast-like MG 63 cells on polylactide (PLA) nanofibers prepared by needle-less electrospinning and loaded with 5 or 15 wt % of hydroxyapatite (HA) nanoparticles. On day 7 after seeding, the cell number was the highest on samples with 15 wt % of HA. This result was confirmed by the XTT test, especially after dynamic cultivation, when the number of metabolically active cells on these samples was even higher than on control polystyrene. Staining with a live/dead kit showed that the viability of cells on all nanofibrous scaffolds was very high and comparable to that on control polystyrene dishes. An enzyme-linked immunosorbent assay revealed that the concentration of osteocalcin was also higher in cells on samples with 15 wt % of HA. There was no immune activation of cells (measured by production of TNF-alpha), associated with the incorporation of HA. Moreover, the addition of HA suppressed the creep behavior of the scaffolds in their dry state. Thus, nanofibrous PLA scaffolds have potential for bone tissue engineering, particularly those with 15 wt % of HA.