Morphological and biochemical analysis of cells cultured on fibrous poly(ε-caprolactone) scaffolds with different degrees of fiber alignment produced by solution blow spinning.

BACKGROUND: Bioresorbable materials are compounds that decompose in physiological mediums both in vitro and in vivo and are used as an alternative to temporary implants in injured tissues. The aim of this study was to analyze the morphology and cytochemistry of cells grown on fibrous poly(ε-caprolactone) (PCL) scaffolds and to measure cell metabolism parameters by biochemical analysis of the conditioned culture medium from cells grown on the scaffolds. METHODS: Fibrous PCL scaffolds were used under the following conditions: unaligned fibers (NA), fibers aligned at 150 rpm (A150), and fibers aligned at 300 rpm (A300). Vero cells were cultured on these scaffolds for 24 h, 48 h, and 72 h. Samples were analyzed by SEM, MicroCT, cytochemistry, and culture medium biochemistry. RESULTS: The results of the cytochemical analysis showed cells were confluent and well spread on the culture plate, while cells grown on the polymeric scaffold, exhibited an elongated morphology. In the biochemical analyses, no significant differences were observed in the expression of alkaline phosphatase or in the levels of cholesterol or total protein in the culture medium. The different materials do not seem to promote changes in the expression or metabolism of these molecules. Only glucose was markedly reduced in the culture medium of cells grown on either aligned or unaligned scaffolds for 48 h or 72 h. This finding indicates the intense energy requirements of cells grown on these scaffolds. CONCLUSION: PCL fibers showed a great capacity to support cell growth. These data reinforce the interpretation that cells grow satisfactorily on PCL scaffolds.

The Impact of Biomaterial Surface Properties on Engineering Neural Tissue for Spinal Cord Regeneration.

Tissue engineering for spinal cord injury (SCI) remains a complex and challenging task. Biomaterial scaffolds have been suggested as a potential solution for supporting cell survival and differentiation at the injury site. However, different biomaterials display multiple properties that significantly impact neural tissue at a cellular level. Here, we evaluated the behavior of different cell lines seeded on chitosan (CHI), poly (ε-caprolactone) (PCL), and poly (L-lactic acid) (PLLA) scaffolds. We demonstrated that the surface properties of a material play a crucial role in cell morphology and differentiation. While the direct contact of a polymer with the cells did not cause cytotoxicity or inhibit the spread of neural progenitor cells derived from neurospheres (NPCdn), neonatal rat spinal cord cells (SCC) and NPCdn only attached and matured on PCL and PLLA surfaces. Scanning electron microscopy and computational analysis suggested that cells attached to the material's surface emerged into distinct morphological populations. Flow cytometry revealed a higher differentiation of neural progenitor cells derived from human induced pluripotent stem cells (hiPSC-NPC) into glial cells on all biomaterials. Immunofluorescence assays demonstrated that PCL and PLLA guided neuronal differentiation and network development in SCC. Our data emphasize the importance of selecting appropriate biomaterials for tissue engineering in SCI treatment.

Osteogenic differentiation of rat bone mesenchymal stem cells cultured on poly (hydroxybutyrate-co-hydroxyvalerate), poly (ε-caprolactone) scaffolds.

Bioresorbable biomaterials can fill bone defects and act as temporary scaffold to recruit MSCs to stimulate their differentiation. Among the different bioresorbable polymers studied, this work focuses on poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL). Were prepared blends of PHBV and PCL to obtain PHBV based biomaterials with good tenacity, important for bone tissue repair, associated with biocompatible properties of PCL. This study assesses the viability of Vero cells on scaffolds of PHBV, PCL, and their blends and the osteogenic differentiation of mesenchymal stem cells (MSCs). Materials were characterized in surface morphology, DSC and Impact Strength (IS). Vero cells and MSCs were assessed by MTT assay, cytochemical and SEM analysis. MSC osteogenic differentiation was evaluated through alizarin red staining and ALP activity. We found some roughness onto surface materials. DSC showed that the blends presented two distinct melting peaks, characteristic of immiscible blends. IS test confirmed that PHBV-PCL blends is an alternative for increase the tenacity of PHBV. MTT assay showed cells with high metabolic activities on extract toxicity test, but with low activity in the direct contact test. SEM analysis showed spreading cells with irregular and flattened morphology on different substrates. Cytochemical study revealed that MSCs maintained their morphology, although in smaller number for MSCs. The development of nodules of mineralized organic matrix in MSC cultures was identified by alizarin red staining and osteogenic differentiation was confirmed by the quantification of ALP activity. Thus, our scaffolds did not interfere on viability of Vero cells or the osteogenic differentiation of MSCs.

In Vitro Hydrodynamic Evaluation of a Scaffold for Heart Valve Tissue Engineering.

Although prosthetic heart valves have saved many lives, the search for a living substitute continues with the aid of tissue engineering. Much progress has been made so far, but the translation of this technology to clinical reality remains a challenge, especially due to the structural complexity of heart valves and the harsh environment they are in. In a joint effort, researchers from Federal University of ABC and Institute Dante Pazzanese of Cardiology have conceived a new bioresorbable scaffold for heart valve tissue engineering (HVTE), whose hydrodynamic performance was first assessed and described in this work. The scaffold was studied at the mitral position of a left heart simulator from Escola Politécnica of the University of São Paulo, under 60 bpm and with no cell seeding. In this condition, two-dimensional particle image velocimetry was performed to investigate the flow during diastolic and systolic phases. The results indicate that the scaffold can withstand the required intraventricular pressures for a simulated normal physiologic condition in a bioreactor. Furthermore, the averaged (N = 150) velocity vector maps showed a smooth and well-distributed flow during diastole and qualitatively demonstrated no-significant regurgitation at systole.

Comparative study of aligned and nonaligned poly(ε-caprolactone) fibrous scaffolds prepared by solution blow spinning.

Fibrous scaffolds have become popular in tissue engineering (TE) due to their morphological resemblance to extracellular matrix components. While electrospinning is the most common technique in the field, solution blow spinning is an emerging technique with great potential. One of its many advantages is that it can produce aligned fibers with a very simple experimental setup. This work aimed to fabricate poly(ε-caprolactone) mats with aligned fibers and compare them to nonaligned ones. For that, samples were produced using three rotational speeds of a cylindrical collector and characterized in terms of fiber alignment and diameter, mechanical properties, wettability, and biological response. Results showed that with a static collector, fibers were randomly deposited and nonaligned. As the speed was increased, the fibers began to align (as proven by image analysis), resulting in a change in mechanical behavior, but no differences in fiber diameter. Cells cultured on aligned samples were more elongated, and a higher alignment degree seemed to favor cellular growth. The results confirmed the potential of this up-and-coming technique to produce aligned fibers for TE. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1462-1470, 2019.

Mechanical behavior of bovine pericardium treated with hyaluronic acid derivative for bioprosthetic aortic valves.

We studied the mechanical behavior of bovine pericardium (BP) after anticalcification treatment using hyaluronic acid (HA) derivative. To simulate the physiological environment and stimulate the calcification process, the BP samples were immersed into simulated body fluid solution. We conducted scanning electron microscopy with energy dispersive X-ray spectrometry, and uniaxial mechanical tests of HA-treated and non-treated samples. Although our microstructural analyses indicated that the HA treatment actually prevents the formation of calcium phosphate deposits, the mechanical tests show significant increase of stiffness of the HA-treated samples. Using data from our mechanical tests as input parameters, we performed finite element (FE) computer simulations to estimate how this increase in the BP stiffness affects the stress distribution in the bioprosthetic leaflet. Although the maximum stress observed during the closing phase of the membrane in vivo is below the experimental yield stress in all cases we analyzed, our FE results indicate that increase of BP stiffness due to HA anticalcification treatment results in higher risk of disruption and failure of the leaflets in bioprosthetic heart valves. Since our FE results indicate that the commissure and the fixed edge are the regions that withstand the highest mechanical stresses during the closing phase, new designs of the valve might be efficient to enhance the endurance of the prosthesis. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2273-2280, 2019.

Mechanical and morphological evaluation of osteochondral implants in dogs.

The mechanical behavior of osteochondral defects was evaluated in this study with the intention of developing alternative procedures. Cylindrical pins (5.00 mm in diameter and in height) made of pHEMA hydrogel covered ultra-high molecular weight polyethylene (UHMWPE) or beta-tricalcium phosphate (beta-TCP) matrix were used. Ostoechondral defects were caused in the knees of adult dogs and the evaluation was carried out after a 9-month follow-up period. The mechanical behavior of the implants was evaluated by means of an indentation creep test that showed that the UHMWPE matrix maintained its viscoelastic behavior even after follow-up time, while the beta-TCP matrix osteochondral implants presented significant alterations. It is believed that the beta-TCP osteochondral implants were unable to withstand the load applied, causing an increase of complacency when compared to the UHMWPE osteochondral implants. Based on micro and macroscopic analysis, no significant wear was observed in either of the osteochondral implants when compared to the controls. However, morphological alterations, with fragmentation indices in the patella, were observed either due to friction with the hydrogel in the first postoperative months or due to forming of a dense conjunctive tissue. This wear mechanism caused on the counterface of the implant (patella) was observed, notwithstanding the osteochondral implant studied.

A chitosan-hyaluronic acid hydrogel scaffold for periodontal tissue engineering.

The current challenge in treating periodontitis is regenerating the periodontium. This motivates tissue-engineering researchers to develop scaffolds as artificial matrices that give mechanical support for osteoblasts, cementoblasts, gingival and periodontal ligament fibroblast cells. In this study, modified hyaluronic acid (HA) and chitosan (CS) were employed to create a hybrid CS-HA hydrogel scaffold for periodontal regeneration. CS, HA, and CS-HA scaffolds were obtained by freeze-drying technique, resulting in porous structures suitable for use in tissue engineering. Scaffolds were submitted to gamma and UV-sterilization without significant morphology changes. The ATR-FTIR spectra of CS-HA hydrogels showed peaks at 377 cm-1 , 1566 cm-1 , and 1614 cm-1 , representing secondary amide, primary amine, and carboxyl acid respectively, and it was also observed the emergence of peaks at 886 cm-1 , which probably represents the Schiff base formed in the case of hybrid CS-HA hydrogels. The scaffolds presented a high rate of PBS uptake, reaching values higher than 95%. Thermal degradation of HA scaffolds was around 225°C and CS was around 285°C. The ATR-FTIR spectra and swelling degree were slightly disturbed mainly after gamma sterilization, but degradation temperature did not change after sterilization. The performance of the CS-HA hydrogel scaffolds for in vitro cell culture was tested using NIH3T3 and MG63 cell lines. The Alamar Blue test showed a significant increase in cellular viability and high CD44 expression, suggesting that the cells migrated more when seeded onto the scaffolds. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1691-1702, 2016.

Comparative study of aligned and nonaligned poly(ε-caprolactone) fibrous scaffolds prepared by solution blow spinning

Fibrous scaffolds have become popular in tissue engineering (TE) due to their morphological resemblance to extracellular matrix components. While electrospinning is the most common technique in the field, solution blow spinning is an emerging technique with great potential. One of its many advantages is that it can produce aligned fibers with a very simple experimental setup. This work aimed to fabricate poly (ε-caprolactone) mats with aligned fibers and compare them to nonaligned ones. For that, samples were produced using three rotational speeds of a cylindrical collector and characterized in terms of fiber alignment and diameter, mechanical properties, wettability, and biological response. Results showed that with a static collector, fibers were randomly deposited and nonaligned. As the speed was increased, the fibers began to align (as proven by image analysis), resulting in a change in mechanical behavior, but no differences in fiber diameter. Cells cultured on aligned samples were more elongated, and a higher alignment degree seemed to favor cellular growth. The results confirmed the potential of this up-and-coming technique to produce aligned fibers for TE. (AU)

Biocompatibility study of polymeric biomaterials.

Polymeric hydrogels are used as wound dressing material since these materials show advantages such as pain relief, exudates absorption, barrier to microorganisms, permeability, and others. This article shows the results obtained in a study aiming to know the biological performance of different polymeric materials to be used in contact with skin: PVP hydrogels and acrylate adhesive. The biocompatibility was determined by in vitro assay of cytotoxicity and in vivo assay by using the contact test of irritability in rabbits. All the tested samples presented no toxicity and no dermal irritation.