Polylactic acid as a biocompatible polymer for three-dimensional printing of interim prosthesis: Mechanical characterization.

Mechanical properties of polylactic acid (PLA), which is a biopolymer obtained via 3D-printing, were compared with conventional resins for the realization of interim prosthesis. A PLA built by fused deposition modeling and traditional interim resins (Unifast®, Integrity®, Temporary CB®) were divided into 4 groups (n=10). Each group was investigated for Young modulus, flexural strength, microhardness and analysis of the fractured surface. Data were analyzed by Kruskal-Wallis and ANOVA (α=0.05). The porosity of the PLA was calculated from the crystallinity degree and density. PLA-group showed an elastic modulus and flexural strength in the same range than Integrity®-group, better than Unifast®-group and inferior to Temporary CB®-group (p<0.05). PLA-group microhardness was equivalent to Unifast®-group and inferior to Integrity® and Temporary CB® groups (p<0.05). Due to mechanical properties similar to conventional resins and the low porosity rate, this biocompatible 3D-printed polymer may be an interesting alternative to conventional polymer to build temporary prosthesis.

Selective Laser Melted Titanium Alloy for Transgingival Components: Influence of Surface Condition on Fibroblast Cell Behavior.

PURPOSE: To mechanically characterize and assess the biological properties of Ti6Al4V surfaces obtained by Selective Laser Melting in order to determine whether this process is conceivable for production of implant-supported prostheses and particularly trans-gingival components. As-built and polished surfaces were studied in comparison with components obtained by computer numerical control machining technology in order to consider whether the properties are in the same range as the conventional method currently used. MATERIALS AND METHODS: Cylindrical specimens of Ti6Al4V (n = 6) were built with Selective Laser Melting for the characterization of mechanical properties according to ISO 22674 and discs (n = 12) were fabricated in the same conditions for cytotoxicity evaluation. Discs (n = 12) of Ti6Al4V were also obtained by computer numerical control machining as control. Half of the number of discs (n = 6) from each process were polished, to simulate the laboratory protocol for polishing of transmucosal components and half of the discs remained unaltered (as-built). Surface roughness measurements of disc specimens (as-built and polished) were compared with computer numerical control milling specimens (as-built and polished). Proliferation of human gingival fibroblasts on Ti6Al4V surfaces was also assessed for each condition. Viability and cell morphology were then evaluated qualitatively. Ra and Sa data were compared using Student's t-test (α = 0.05) and metabolic activity data were compared using Kruskal-Wallis statistical test (α = 0.05). RESULTS: Selective Laser Melting specimens showed elongation at break greater than 2% and 0.2% yield strength better than 500MPa which complied with ISO 22674 standards. Although Selective Laser Melting samples displayed significantly increased roughness on as-built surfaces compared to computer numerically controlled milling samples (p < 0.05), no statistically significant difference was observed after mechanical polishing (p = 0.279). Regarding metabolic activity, no statistical difference was observed between groups at day 3 (p > 0.05) and fibroblasts showed a viability higher than 97% on all discs. Cell shapes on polished samples suggested moderate adhesion compared to unpolished samples. CONCLUSION: With the manufacturing parameters selected in this study, Selective Laser Melting of Ti6Al4V appeared to be compatible with a prosthetic application type 4 according to ISO 22674. Surfaces obtained, followed by recommended postprocessing provided components with equivalent biological properties compared to computer numerical control machining technology.

In vitro and in vivo proves of concept for the use of a chemically cross-linked poly(ester-urethane-urea) scaffold as an easy handling elastomeric biomaterial for bone regeneration.

Bone loss can occur as a result of various pathologies, traumas and injuries and poor bone healing leads to functionally debilitating condition, loss of self-sufficiency and deterioration in life quality. Given the increasing incidence of facial trauma and the emergence of new procedural techniques, advanced scaffolds are currently developed as substitutes for bone tissue engineering. In this study, we investigated the capability of a chemically cross-linked ε-caprolactone-based poly(ester-urethane-urea) (PCLU) scaffold to support bone regeneration. In vitro assays demonstrated that PCLU scaffolds could be colonized by cells through direct cell seeding and cell migration from outside to scaffold inside. Moreover, PCLU scaffolds could provide a suitable environment for stem cells proliferation in a 3D spatial arrangement, and allowed osteogenic differentiation under appropriate induction. In vivo results revealed the osteogenic properties of PCLU scaffolds through a drilled-hole femoral bone defect repair improvement in rats. Using histology and microtomography analysis, we showed that PCLU scaffolds fit well the bone cavity and were eventually entrapped between the newly formed trabeculae. Finally, no sign of inflammation or rejection was noticed. We envision that PCLU scaffolds can provide the clinicians with a substitute having appropriate characteristics for the treatment of bone defects.

The Use of Platelet-Rich Plasma to Promote Cell Recruitment into Low-Molecular-Weight Fucoidan-Functionalized Poly(Ester-Urea-Urethane) Scaffolds for Soft-Tissue Engineering.

Due to their elastomeric behavior, polyurethane-based scaffolds can find various applications in soft-tissue engineering. However, their relatively inert surface has to be modified in order to improve cell colonization and control cell fate. The present study focuses on porous biodegradable scaffolds based on poly(ester-urea-urethane), functionalized concomitantly to the scaffold elaboration with low-molecular-weight (LMW) fucoidan; and their bio-activation with platelet rich plasma (PRP) formulations with the aim to promote cell response. The LMW fucoidan-functionalization was obtained in a very homogeneous way, and was stable after the scaffold sterilization and incubation in phosphate-buffered saline. Biomolecules from PRP readily penetrated into the functionalized scaffold, leading to a biological frame on the pore walls. Preliminary in vitro assays were assessed to demonstrate the improvement of scaffold behavior towards cell response. The scaffold bio-activation drastically improved cell migration. Moreover, cells interacted with all pore sides into the bio-activated scaffold forming cell bridges across pores. Our work brought out an easy and versatile way of developing functionalized and bio-activated elastomeric poly(ester-urea-urethane) scaffolds with a better cell response.

Characterization of elastomeric scaffolds developed for tissue engineering applications by compression and nanoindentation tests, µ-Raman and µ-Brillouin spectroscopies.

In tissue engineering, porous biodegradable scaffolds are developed with morphological, chemical and mechanical properties to promote cell response. Therefore, the scaffold characterization at a (sub)micrometer and (bio)molecular level is paramount since cells are sensitive to the chemical signals, the rigidity, and the spatial structuring of their microenvironment. In addition to the analysis at room temperature by conventional quasi-static (0.1-45 Hz) mechanical tests, the ultrasonic (10 MHz) and µ-Brillouin inelastic light scattering (13 GHz) were used in this study to assess the dynamical viscoelastic parameters at different frequencies of elastomeric scaffolds. Time-temperature superposition principle was used to increase the high frequency interval (100 MHz-100 THz) of Brillouin experiments providing a mean to analyse the viscoelastic behavior with the fractional derivative viscoelastic model. Moreover, the µ-Raman analysis carried out simultaneously during the µ-Brillouin experiment, gave the local chemical composition.

Preliminary In Vitro Assessment of Stem Cell Compatibility with Cross-Linked Poly(ε-caprolactone urethane) Scaffolds Designed through High Internal Phase Emulsions.

By using a high internal phase emulsion process, elastomeric poly(ε-caprolactone urethane) (PCLU) scaffolds were designed with pores size ranging from below 150 µm to 1800 µm and a porosity of 86% making them suitable for bone tissue engineering applications. Moreover, the pores appeared to be excellently interconnected, promoting cellularization and future bone ingrowth. This study evaluated the in vitro cytotoxicity of the PCLU scaffolds towards human mesenchymal stem cells (hMSCs) through the evaluation of cell viability and metabolic activity during extract test and indirect contact test at the beginning of the scaffold lifetime. Both tests demonstrated that PCLU scaffolds did not induce any cytotoxic response. Finally, direct interaction of hMSCs and PCLU scaffolds showed that PCLU scaffolds were suitable for supporting the hMSCs adhesion and that the cells were well spread over the pore walls. We conclude that PCLU scaffolds may be a good candidate for bone tissue regeneration applications using hMSCs.

The grafting of a thin layer of poly(sodium styrene sulfonate) onto poly(ε-caprolactone) surface can enhance fibroblast behavior.

Poly(sodium styrene sulfonate) (pNaSS) was grafted onto poly(ε-caprolatone) (PCL) surfaces via ozonation and graft polymerization. The effect of ozonation and polymerization time, as well as the Mohr's salt concentration in the grafting solution, on the degree of grafting was investigated. The degree of grafting was determined through toluidine blue staining. The surface chemical change was characterized by attenuated total reflection Fourier transform infrared spectroscopy, energy-dispersive X-ray spectroscopy and X-ray photoelectron spectroscopy. The result demonstrated that the grafting did not induce any degradation of PCL, and that pNaSS was grafted onto PCL as a thin and covalently stable layer. Furthermore, the modified PCL surface reveals a significant increase in the metabolic activity of fibroblastic cells, as well as a better cell spreading with higher adhesion strength. Consequently, bioactivity of PCL is greatly enhanced by immobilizing a thin layer of pNaSS onto its surface. The grafting of pNaSS is a promising approach to increase the bioactivity of PCL-based materials used in tissue engineering applications, such as ligament reconstruction.

Increasing the bioactivity of elastomeric poly(ε-caprolactone) scaffolds for use in tissue engineering.

BACKGROUND: Biodegradable polymers used in tissue engineering applications, such as poly(ε-caprolactone) (PCL), are hydrophobic leading to a lack of favorable cell signalization and finally to a poor cell adhesion, proliferation and differentiation. To overcome this problem, scaffolds undergo generally a surface modification. OBJECTIVE: Our laboratory has demonstrated that the grafting of poly(sodium styrene sulfonate) (pNaSS) onto titanium or poly(ethylene terephthalate) surfaces, leads to a more specific protein adsorption and a better control of cell proliferation. The objective of this work is to develop, through a straightforward way, bioactive elastomeric PCL scaffolds by grafting pNaSS. METHODS: Porous elastomeric PCL scaffolds were developed using a particulate-leaching process. pNaSS was grafted into the scaffold by a "grafting from" technique. In vitro tests were carried out to assess cell adhesion and protein expression. RESULTS: pNaSS was grafted homogeneously onto PCL scaffolds without degrading the biodegradable polymer or the porous structure. The in vitro studies have shown that pNaSS grafted onto PCL improves the cell response with a better expression of collagen, fibronectin and integrin α1. CONCLUSIONS: The grafting of pNaSS onto biomaterial surfaces is a versatile method that can provide a new generation of biodegradable scaffolds which could be "biointegrable".

Cellular integration and vascularisation promoted by a resorbable, particulate-leached, cross-linked poly(ε-caprolactone) scaffold.

Flexible, strong scaffolds were created by crosslinking PCL with 1,6-hexamethylenediisocyanate, using paraffin beads as a porogen. Particulate leaching generated homogeneous scaffolds with interconnected spherical pores of 5-200 µm. Subcutaneous implantation in rats for 3 months resulted in minimal scaffold resorption and a non-inflammatory regenerative host response, with complete infiltration by alternatively-activated CD68(+) macrophages. In addition, scaffolds were populated extensively along microfractures by a stromal matrix, which was highly vascularised and contained a subset of stromal cells that expressed the anti-inflammatory CD163 antigen. Such microfractures may be an important physical feature for directing stromal integration and vascularisation events.

The relationship between the mechanical properties and cell behaviour on PLGA and PCL scaffolds for bladder tissue engineering.

Previous work on 2D synthetic films showed growth of human bladder stromal cells was enhanced on materials with lower moduli that mimic the elastic properties of native tissue. This study developed 3D synthetic foam scaffolds for soft tissue engineering by emulsion freeze-drying. Foams of poly(lactide-co-glycolide) (PLGA) and poly(epsilon-caprolactone) (PCL) were extensively characterised using scanning electron microscopy, mercury porosimetry, dynamic mechanical analysis, degradation analysis, size exclusion chromatography and differential scanning calorimetry. Foams of 85-88% porosity and 35 microm pore diameter were selected for further study; the storage modulus of PCL foams was around half that of PLGA (2 MPa vs 4 MPa) and closer to the reported value for native bladder tissue. Urinary tract stromal cells showed a 4.4 and 2.4-fold higher attachment and rate of growth, respectively, on PCL scaffolds, as assessed by a modified 3-[4,5-dimethyl(thiazol-2yl)-3,5-diphery] tetrazolium bromide assay. A greater contractile force was exerted by cells seeded in PLGA than on PCL scaffolds, raising the possibility that the reduced rate of proliferation of cells on PLGA scaffolds may reflect differentiation into a contractile phenotype. This study has generated PCL foam scaffolds with properties that may be pertinent to the tissue engineering of the bladder and other soft tissues.

Influence of the physical properties of two-dimensional polyester substrates on the growth of normal human urothelial and urinary smooth muscle cells in vitro.

Although synthetic biomaterials have a wide range of promising applications in regenerative medicine and tissue engineering, there is limited insight into the basic materials properties that influence cellularisation events. The aim of this study was to investigate the influence of the physical properties of polyester films on the adherence and growth of normal human urothelial and urinary smooth muscle (SM) cells, as part of a programme for the development of potential biomaterials for bladder tissue engineering. Films of different thickness were produced by spin coating from solution. Cell attachment and proliferation were analysed and revealed a reproducible and significant growth advantage over the initial 7 days for both cell types on poly(lactide-co-glycolide) (PLGA) versus poly(epsilon-caprolactone) (PCL), and on thick versus thin films. In order to understand the basis of the variation in cell growth, the surface morphology, degradation behaviour and mechanical properties of the films were investigated. The pattern of cell attachment and growth was found to be unrelated to surface topography and no distinction in film degradation behaviour was found to account for differences in cell growth, except at late time points (14 days), where degradation of thin PLGA films became significant. By contrast, the flexural loss and storage moduli were found to be reduced in films composed of PLGA versus PCL, and also as film thickness increased, indicating that mechanical properties of biomaterials can influence cell growth. We conclude that elastic modulus is relevant to biology at the cellular scale and may also be influential at the tissue/organ level, and is a critical parameter to be considered during development of synthetic biomaterials for tissue engineering.