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Background: Age-driven immune signals cause a state of chronic low-grade inflammation and in consequence affect bone healing and cause challenges for clinicians when repairing critical-sized bone defects in elderly patients., Methods: Poly(L-lactide-co-ɛ-caprolactone) (PLCA) scaffolds are functionalized with plant-derived nanoparticles from potato, rhamnogalacturonan-I (RG-I), to investigate their ability to modulate inflammation in vitro in neutrophils and macrophages at gene and protein levels. The scaffolds' early and late host response at gene, protein and histological levels is tested in vivo in a subcutaneous rat model and their potential to promote bone regeneration in an aged rodent was tested in a critical-sized calvaria bone defect. Significant differences were tested using one-way ANOVA, followed by a multiple-comparison Tukey's test with a p value ≤ 0.05 considered significant., Results: Gene expressions revealed PLCA scaffold functionalized with plant-derived RG-I with a relatively higher amount of galactose than arabinose (potato dearabinated (PA)) to reduce the inflammatory state stimulated by bacterial LPS in neutrophils and macrophages in vitro. LPS-stimulated neutrophils show a significantly decreased intracellular accumulation of galectin-3 in the presence of PA functionalization compared to Control (unmodified PLCA scaffolds). The in vivo gene and protein expressions revealed comparable results to in vitro. The host response is modulated towards anti-inflammatory/ healing at early and late time points at gene and protein levels. A reduced foreign body reaction and fibrous capsule formation is observed when PLCA scaffolds functionalized with PA were implanted in vivo subcutaneously. PLCA scaffolds functionalized with PA modulated the cytokine and chemokine expressions in vivo during early and late inflammatory phases. PLCA scaffolds functionalized with PA implanted in calvaria defects of aged rats downregulating pro-inflammatory gene markers while promoting osteogenic markers after 2 weeks in vivo., Conclusion: We have shown that PLCA scaffolds functionalized with plant-derived RG-I with a relatively higher amount of galactose play a role in the modulation of inflammatory responses both in vitro and in vivo subcutaneously and promote the initiation of bone formation in a critical-sized bone defect of an aged rodent. Our study addresses the increasing demand in bone tissue engineering for immunomodulatory 3D scaffolds that promote osteogenesis and modulate immune responses., (© 2022. The Author(s).)

Clinical results obtained when degradable polymer-based medical devices are used in breast reconstruction following mastectomy are promising. However, it remains challenging to develop a large scaffold structure capable of providing both sufficient external mechanical support and an internal cell-like environment to support breast tissue regeneration. We propose an internal-bra-like prototype to solve both challenges. The design combines a 3D-printed scaffold with knitted meshes and electrospun nanofibers and has properties suitable for both breast tissue regeneration and support of a silicone implant. Finite element analysis (FEA) was used to predict the macroscopic and microscopic stiffnesses of the proposed structure. The simulations show that introduction of the mesh leads to a macroscopic scaffold stiffness similar to the stiffness of breast tissue, and mechanical testing confirms that the introduction of more layers of mesh in the modular design results in a lower elastic modulus. The compressive modulus of the scaffold can be tailored within a range from hundreds of kPa to tens of kPa. Biaxial tensile testing reveals stiffening with increasing strain and indicates that rapid strain-induced softening occurs only within the first loading cycle. In addition, the microscopic local stiffness obtained from FEA simulations indicates that cells experience significant heterogeneous mechanical stimuli at different places in the scaffold and that the local mechanical stimulus generated by the strand surface is controlled by the elastic modulus of the polymer, rather than by the scaffold architecture. From in vitro experiments, it was observed that the addition of knitted mesh and an electrospun nanofiber layer to the scaffold significantly increased cell seeding efficiency, cell attachment, and proliferation compared to the 3D-printed scaffold alone. In summary, our results suggest that the proposed design strategy is promising for soft tissue engineering of scaffolds to assist breast reconstruction and regeneration., Competing Interests: The authors declare the following competing financial interest(s): Dr. Hailong Liu, Dr. Astrid Ahlinder, Dr. Tiziana Fuoco and Prof. Anna Finne-Wistrand declare that they are involved in Akira Science AB which sells one of the polymers and the degradable adhesive used in the research. There has been no significant financial support for this work that could have influenced its outcome., (© 2021 The Authors. Published by American Chemical Society.)

We have developed an innovative methodology to overcome the lack of techniques for real-time assessment of degradable biomedical polymers at physiological conditions. The methodology was established by combining polymer characterization techniques with electrochemical sensors. The in vitro hydrolytic degradation of a series of aliphatic polyesters was evaluated by following the molar mass decrease and the mass loss at different incubation times while tracing pH and l-lactate released into the incubation media with customized miniaturized electrochemical sensors. The combination of different analytical approaches provided new insights into the mechanistic and kinetics aspects of the degradation of these biomedical materials. Although molar mass had to reach threshold values for soluble oligomers to be formed and specimens' resorption to occur, the pH variation and l-lactate concentration were direct evidence of the resorption of the polymers and indicative of the extent of chain scission. Linear models were found for pH and released l-lactate as a function of mass loss for the l-lactide-based copolymers. The methodology should enable the sequential screening of degradable polymers at physiological conditions and has potential to be used for preclinical material's evaluation aiming at reducing animal tests.

Various 3D printing techniques currently use degradable polymers such as aliphatic polyesters to create well-defined scaffolds. Even though degradable polymers are influenced by the printing process, and this subsequently affects the mechanical properties and degradation profile, degradation of the polymer during the process is not often considered. Degradable scaffolds are today printed and cell-material interactions evaluated without considering the fact that the polymer change while printing the scaffold. Our methodology herein was to vary the printing parameters such as temperature, pressure, and speed to define the relationship between printability, polymer microstructure, composition, degradation profile during the process, and rheological behavior. We used high molecular weight medical-grade (co)polymers, poly(l-lactide- co -ε-caprolactone) (PCLA), poly(l-lactide- co -glycolide) (PLGA), and poly(d,l-lactide- co -glycolide) (PDLGA), with l-lactide content ranging from 25 to 100 mol %, for printing in an extrusion-based printer (3D Bioplotter). Optical microscopy confirmed that the polymers were printable at high resolution and good speed, until a certain degree of degradation. The results show also that printability can not be claimed just by optimizing printing parameters and highlight the importance of a careful analysis of how the polymer's structure and properties vary during printing. The polymers thermally decomposed from the first processing minute and caused a decrease in the average block length of the lactide blocks in the copolymers and generated lower crystallinity. Poly(l-lactide) (PLLA) and PCLA are printable at a higher molecular weight, less degradation before printing was possible, compared to PLGA and PDLGA, a result explained by the higher complex viscosity and more elastic polymeric melt of the copolymer containing glycolide (GA) and lactide (LA). In more detail, copolymers comprised of LA and ε-caprolactone (CL) formed lower molecular weight compounds over the course of printing, while the PLGA copolymer was more susceptible to intermolecular transesterification reactions, which do not affect the overall molecular weight, but cause changes in the copolymer microstructure. This results in a longer printing time for PLGA than PLLA and PCLA.

The advancement of 3D printing technologies in the fabrication of degradable scaffolds for tissue engineering includes, from the standpoint of the polymer chemists, an urgent need to develop new materials that can be used as ink and are suitable for medical applications. Here, we demonstrate that a copolymer of ε-caprolactone (CL) with low amounts of p -dioxanone (DX) (15 mol %) is a degradable and printable material that suits the requirements of melt extrusion 3D printing technologies, including negligible degradation during thermal processing. It is therefore a potential candidate for soft tissue regeneration. The semicrystalline CL/DX copolymer is processed at a lower temperature than a commercial polycaprolactone (PCL), shaped as a filament for melt extrusion 3D printing and as porous and pliable scaffolds with a gradient design. Scaffolds have Young's modulus in the range of 60-80 MPa, values suitable for provision of structural support for damaged soft tissue such as breast tissue. SEM and confocal microscope indicate that the CL/DX copolymer scaffolds support adipose stem cell attachment, spreading, and proliferation.

We have developed a straightforward strategy to obtain semicrystalline and random copolymers of ε-caprolactone (CL) and p -dioxanone (DX) with thermal stabilities similar to poly(ε-caprolactone), PCL, but with a faster hydrolytic degradation rate. CL/DX copolymers are promising inks when printing scaffolds aimed for tissue engineering. Such copolymers behave similar to PCL and resorb faster. The copolymers were synthesized by bulk ring-opening copolymerization, achieving a high yield; a molecular weight, M n , of 57-176 kg mol -1 . The copolymer microstructure consisted of long CL blocks that are separated by isolated DX units. The block length and the melting point were a linear function of the DX content. The copolymers crystallize as an orthorhombic lattice that is typical for PCL, and they formed more elastic, softer, and less hydrophobic films with faster degradation rates than PCL. Relatively high thermal degradation temperatures (above 250 °C), similar to PCL, were estimated by thermogravimetric analysis, and copolymer filaments for three-dimensional printing and scaffolds were produced without thermal degradation.-1 . The copolymer microstructure consisted of long CL blocks that are separated by isolated DX units. The block length and the melting point were a linear function of the DX content. The copolymers crystallize as an orthorhombic lattice that is typical for PCL, and they formed more elastic, softer, and less hydrophobic films with faster degradation rates than PCL. Relatively high thermal degradation temperatures (above 250 °C), similar to PCL, were estimated by thermogravimetric analysis, and copolymer filaments for three-dimensional printing and scaffolds were produced without thermal degradation.

A challenge in the extrusion-based bioprinting is to find a bioink with optimal biological and physicochemical properties. The aim of this study was to evaluate the influence of wood-based cellulose nanofibrils (CNF) and bioactive glass (BaG) on the rheological properties of gelatin-alginate bioinks and the initial responses of bone cells embedded in these inks. CNF modulated the flow behavior of the hydrogels, thus improving their printability. Chemical characterization by SEM-EDX and ion release analysis confirmed the reactivity of the BaG in the hydrogels. The cytocompatibility of the hydrogels was shown to be good, as evidenced by the viability of human osteoblast-like cells (Saos-2) in cast hydrogels. For bioprinting, 4-layer structures were printed from cell-containing gels and crosslinked with CaCl 2 . Viability, proliferation and alkaline phosphatase activity (ALP) were monitored over 14 d. In the BaG-free gels, Saos-2 cells remained viable, but in the presence of BaG the viability and proliferation decreased in correlation with the increased viscosity. Still, there was a constant increase in the ALP activity in all the hydrogels. Further bioprinting experiments were conducted using human bone marrow-derived mesenchymal stem cells (hBMSCs), a clinically relevant cell type. Interestingly, hBMSCs tolerated the printing process better than Saos-2 cells and the ALP indicated BaG-stimulated early osteogenic commitment. The addition of CNF and BaG to gelatin-alginate bioinks holds great potential for bone tissue engineering applications.

The history of resorbable polymers containing glycolide, lactide, ε-caprolactone and trimethylene carbonate, with a special emphasis being placed on the time frame of the 1960s-1990s is described. Reviewing the history is valuable when looking into the future perspectives regarding how and where these monomers should be used. This story includes scientific evaluations indicating that these polymers are safe to use in medical devices, while the design of the medical device is not considered in this report. In particular, we present the data regarding the tissue response to implanted polymers, as well as the toxicity and pharmacokinetics of their degradation products. In the translation of these polymers from "the bench to the bedside," various challenges have been faced by surgeons, medical doctors, biologists, material engineers and polymer chemists. This Perspective highlights the visionary role played by the pioneers, addressing the problems that occurred on a case by case basis in translational medicine.

l-Lactide/trimethylene carbonate copolymers have been produced as multifilament fibers by high-speed melt-spinning. The relationship existing between the composition, processing parameters and physical properties of the fibers has been disclosed by analyzing how the industrial process induced changes at the macromolecular level, i.e., the chain microstructure and crystallinity development. A poly(l-lactide) and three copolymers having trimethylene carbonate contents of 5, 10 and 18 mol % were synthesized with high molecular weight ( M n ) up to 377 kDa and narrow dispersity. Their microstructure, crystallinity and thermal properties were dictated by the composition. The spinnability was then assessed for all the as-polymerized materials: four melt-spun multifilament fibers with increasing linear density were collected for each (co)polymer at a fixed take-up speed of 1800 m min -1 varying the mass throughput during the extrusion. A linear correlation resulted between the as-spun fiber properties and the linear density. The as-spun fibers could be further oriented, developing more crystallinity and improving their tensile properties by a second stage of hot-drawing. This ability was dependent on the composition and crystallinity achieved during the melt-spinning and the parameters selected for the hot-drawing, such as temperature, draw ratio and input speed. The crystalline structure evolved to a more stable form, and the degree of crystallinity increased from 0-52% to 25-66%. Values of tensile strength and Young's modulus up to 0.32-0.61 GPa and 4.9-8.4 GPa were respectively achieved.

A low dose of 1μg rhBMP-2 was immobilised by four different functionalising techniques on recently developed poly(l-lactide)-co-(ε-caprolactone) [(poly(LLA-co-CL)] scaffolds. It was either (i) physisorbed on unmodified scaffolds [PHY], (ii) physisorbed onto scaffolds modified with nanodiamond particles [nDP-PHY], (iii) covalently linked onto nDPs that were used to modify the scaffolds [nDP-COV] or (iv) encapsulated in microspheres distributed on the scaffolds [MICS]. Release kinetics of BMP-2 from the different scaffolds was quantified using targeted mass spectrometry for up to 70days. PHY scaffolds had an initial burst of release while MICS showed a gradual and sustained increase in release. In contrast, NDP-PHY and nDP-COV scaffolds showed no significant release, although nDP-PHY scaffolds maintained bioactivity of BMP-2. Human mesenchymal stem cells cultured in vitro showed upregulated BMP-2 and osteocalcin gene expression at both week 1 and week 3 in the MICS and nDP-PHY scaffold groups. These groups also demonstrated the highest BMP-2 extracellular protein levels as assessed by ELISA, and mineralization confirmed by Alizarin red. Cells grown on the PHY scaffolds in vitro expressed collagen type 1 alpha 2 early but the scaffold could not sustain rhBMP-2 release to express mineralization. After 4weeks post-implantation using a rat mandible critical-sized defect model, micro-CT and Masson trichrome results showed accelerated bone regeneration in the PHY, nDP-PHY and MICS groups. The results demonstrate that PHY scaffolds may not be desirable for clinical use, since similar osteogenic potential was not seen under both in vitro and in vivo conditions, in contrast to nDP-PHY and MICS groups, where continuous low doses of BMP-2 induced satisfactory bone regeneration in both conditions. The nDP-PHY scaffolds used here in critical-sized bone defects for the first time appear to have promise compared to growth factors adsorbed onto a polymer alone and the short distance effect prevents adverse systemic side effects., (Copyright © 2014. Published by Elsevier B.V.)

Our recent in vitro study demonstrated that endothelial cells (ECs) might influence the differentiation of bone marrow stromal cells (BMSCs). Therefore, the aim of this study was to describe this effect in vivo, using a rat calvarial bone defect model. BMSCs were isolated from femurs of two-donor Lewis rats and expanded in α-minimum essential medium containing 10% fetal bovine serum. One fifth of BMSCs were induced and differentiated into ECs in an Endothelial Cell Growth Medium-2 and then characterized by a flow cytometry. The remaining BMSCs were cultured in freshly prepared osteogenic stimulatory medium, containing dexamethasone, ascorbic acid and β-glycerophosphate. Either BMSCs alone (BMSC-group) or co-cultured ECs/BMSCs (CO-group) were seeded into poly(L-lactide-co-1,5-dioxepan-2-one) [poly(LLA-co-DXO)] scaffolds, cultured in spinner flasks, and then implanted into symmetrical calvarial defects prepared in recipient rats. The animals were sacrificed after 2 months. The formation of new bone was evaluated by radiography and histology and by the expression of osteogenic markers using reverse transcriptase-polymerized chain reaction (RT-PCR). To investigate vessel formation, histological staining was performed with EC's markers. The radiographical and histological results showed more rapid bone formation in the CO- than in the BMSC-group. However, the expression of EC's marker was similar on both groups by histological analysis after 2 months postoperatively. Furthermore, the CO-group exhibited greater expression of osteogenic markers as demonstrated by RT-PCR. The results are consistent with the previous in vitro findings that poly(LLA-co-DXO) scaffold might be suitable candidate for bone tissue engineering. In vivo, bone regeneration was enhanced by a construct of the polymer scaffold loaded with co-cultured cells., (2010 Wiley Periodicals, Inc.)

A simple and effective method of fabricating scaffolds with open pore structures was successfully used on several copolymers. The method, which is straightforward and fast, was developed to overcome problems such as low pore interconnectivity and to achieve thick three-dimensional scaffolds. Copolymers are of particular interest because it is possible to tune their mechanical and degradable properties, and in this work, copolymers of L-lactide (LLA) and trimethylene carbonate (TMC) were synthesized through ring-opening polymerization. The copolymers formed had molecular weights ranging from close to 60000 g/mol to over 300000 g/mol and they were composed of 12-55 molar percentages of TMC and 88-45 molar percentages of LLA. The synthesized copolymers were evaluated as scaffold materials using a combined phase separation and particulate leaching technique, in which sugar templates were used as the leachable porosifiers. Differences in molecular weights, molar compositions, and degrees of crystallinity were all factors that influenced the properties of the prepared scaffolds. The copolymers with high LLA contents and high degrees of crystallinity were best suited for the scaffold fabrication technique used and gave degradable scaffolds with interconnected pores.

By using less catalyst in the ring-opening polymerization of epsilon-caprolactone, a residual tin content of 5 ppm was reached without the need for additional purification. The initial amount of tin (II) 2-ethylhexanoate [Sn(Oct)(2)] was varied using catalyst:monomer ratios of 1:1000, 1:10,000, and 1:20,000. The impact on the final conversion, reaction control, average molecular weight, and polydispersity was studied. The amount of Sn(Oct)(2) could be significantly reduced without influencing the reaction results. The residual amount of tin was reduced from 176 ppm with a catalyst:monomer ratio of 1:1000 in the polymer, to 5 ppm with the ratio 1:10,000. It was thus concluded that a catalyst:monomer ratio of 1:10,000 or lower is required to achieve a polymer with tin content suitable for biomedical applications. The materials were also tested in a proliferation study with mesenchymal stem cells from mouse. Porous scaffolds were fabricated from the polymers, using a salt leaching technique, and the cell growth on the porous scaffolds as well as on homogeneous films was determined by light absorbance measurements. In this study, the cell proliferation results showed that cells could grow on all polymers with an efficiency equal to or better than that on normal tissue culture plastic., (2008 Wiley Periodicals, Inc.)

The influences of the stereochemical structure, the molecular weight, and the number of molecular branches for poly(lactide) (PLA) on enzymatic hydrolysis rates of PLA monolayers were studied by atomic force microscopy (AFM) and the Langmuir-Blodgett (LB) technique. Monolayers of six kinds of PLA with different molecular weights, stereochemical structure, and numbers of molecular branches were prepared by LB techniques and then characterized by AFM in air. The PLA molecules covered homogeneously with a silicon substrate and did not form lamellar crystals in the monolayer. We determined the initial hydrolysis rate of PLA monolayers in presence of proteinase K by volumetric analysis from the continuous AFM height images. The presence of D-lactyl unit reduced the hydrolysis rate of the monolayer. The hydrolysis rate for the linear PLLA samples increased with a decrease in the molecular weight. In contrast, the rates of erosion for branched PLLA monolayers were independent of the molecular weight of samples. The erosion rate of branched PLLA monolayers was found to be dependent on the average molecular weight of PLLA segment in branched molecules, not on the overall molecular weight of samples. From these results, furthermore, the hydrolysis mode of PLAs by proteinase K is discussed.