Porous Polycaprolactone Scaffolds Research Articles

4026 Dissecting the role of microenvironment heterogeneity on metastatic tumor cell phenotype at an engineered metastatic niche

OBJECTIVES/GOALS: Breast cancer metastases are stochastic and difficult to detect. Therapy is often ineffective due to phenotypic changes of tumor cells at these sites. We engineered a synthetic metastatic niche to study the role of phenotypic transitions in the microenvironment on tumor cell phenotype. METHODS/STUDY POPULATION: The engineered metastatic niche is composed of a porous polycaprolactone scaffold implanted subcutaneously in Balb/c mice. The mice received an orthotopic inoculation of 4T1 cells (murine triple negative breast cancer) in the fourth right mammary fat pad and the disease was allowed to progress for 7-21 days (pre-metastatic to overt metastatic disease). The scaffolds and lungs (native metastatic site) were explanted and analyzed by single cell RNA-seq via Drop-seq. Cell phenotypes were identified and tracked over time with the Seurat and Monocle3 pipelines. Assessment of the impact of these cell populations on tumor cell phenotype was conducted through Transwell co-cultures. RESULTS/ANTICIPATED RESULTS: Healthy scaffolds are primarily composed of macrophages, dendritic cells, and fibroblasts – consistent with a foreign body response. Despite differences in the lung and scaffold prior to tumor inoculation, both tissues were marked by >5-fold increase in neutrophils/MDSCs. Additionally, 79% of genes at the scaffold that significantly changed over time were also identified in the lung, indicating key similarities in niche maturation. However, many immune cells at the scaffold had distinct phenotypes, with pro-inflammatory/cytotoxic characteristics. These changes clearly impacted tumor cell phenotype, as cells from the scaffold increased tumor cell migration and apoptosis in vitro. DISCUSSION/SIGNIFICANCE OF IMPACT: Early phenotypic changes at the engineered metastatic niche can identify signs of metastasis prior to colonization of tumor cells. Furthermore, dynamics of immune and stromal cells change throughout niche maturation, influencing tumor cell phenotype and may suggest targeted therapies. CONFLICT OF INTEREST DESCRIPTION: Lonnie Shea, Jacqueline Jeruss, and Grace Bushnell are named inventors on patents or patent applications.

An Interleukin 4 Loaded Bi-phasic 3D Printed Scaffold Promotes Osteochondral Defect Regeneration

Multilayer scaffolds fabricated by 3D printing or other techniques have been applied to repair and regenerate osteochondral defects. However, it still remains a challenge of repairing and regenerating the articular cartilage and subchondral bone simultaneously. In this study, to enhance the repair efficacy of osteochondral defect, an interleukin-4 (IL4) incorporated bi-phasic scaffold was designed and fabricated by using digital light processing (DLP) and fused deposition modeling (FDM) printing techniques. The upper layer was the IL4 loaded radially-oriented gelatin methacrylate (GelMA) scaffold (GelMA-IL4) which was produced by DLP printing; its physical and biological properties were suitable for cartilage repair, and the IL4 reduced the negative effect of IL1β and M1 macrophages on chondrocytes in the inflammatory environment in vitro. The lower layer was the porous polycaprolactone and hydroxyapatite (PCL-HA) scaffold that was fabricated by FDM printing. It promoted osteogenic differentiation of cells. In the rabbit model with osteochondral defect, the IL4 loaded bi-phasic scaffold (bi-scaffold+IL4) showed an excellent performance in promoting the repair and regeneration of bone and significantly enhanced cartilage regeneration with more and healthier neo-cartilage tissue formation. Thus, the IL4 loaded bi-phasic scaffold has shown great potential to enhance regeneration of subchondral bone and cartilage and provides a promising strategy for repair of osteochondral defects.

Comparative study of different nitrogen-containing plasma modifications applied on 3D porous PCL scaffolds and 2D PCL films

Additive manufacturing of 3D-scaffolds and surface modification of 2D-substrates using nitrogen-containing plasmas have separately advanced the tissue engineering field for their cyto-responsive topographical and chemical features. However, their synergistic implementation remains largely unsuccessful because of the non-homogeneous surface treatment throughout porous structures. Therefore, this study focuses on the 3D-printing of polycaprolactone (PCL) scaffolds and the optimization of their surface chemistry via 3 plasma modifications: N2 activation, NH3:He (1:9) activation and allylamine polymerization. For an adequate optimization, a precise surface chemical analysis is performed throughout scaffold cross-sections by local measurements using an X-ray photoelectron spectroscopy (XPS) machine connected to a secondary electron image camera. Overall higher nitrogen incorporation and amino-selectivity are detected after NH3 (5.0%) compared to N2 plasma activation (2.8%). A remarkably high average nitrogen content (12.4%) primarily incorporated as C-N bonds is perceived on plasma polymerized allylamine coatings that exhibit an excellent uniformity throughout the scaffolds. In fact, high energy densities lead to a deposition of coatings with a symmetrical shallow nitrogen gradient towards the scaffold inner struts that still present a much higher nitrogen content (7%) when comparing with the state-of-the-art. Overall, this study constitutes a perfect-picture reference for plasma treatment of porous scaffolds.

Boosting the Osteogenic and Angiogenic Performance of Multiscale Porous Polycaprolactone Scaffolds by In Vitro Generated Extracellular Matrix Decoration.

Tissue engineering (TE)-based bone grafts are favorable alternatives to autografts and allografts. Both biochemical properties and the architectural features of TE scaffolds are crucial in their design process. Synthetic polymers are attractive biomaterials to be used in the manufacturing of TE scaffolds, due to various advantages, such as being relatively inexpensive, enabling precise reproducibility, possessing tunable mechanical/chemical properties, and ease of processing. However, such scaffolds need modifications to improve their limited interaction with biological tissues. Structurally, multiscale porosity is advantageous over single-scale porosity; therefore, in this study, we have considered two key points in the design of a bone repair material; (i) manufacture of multiscale porous scaffolds made of photocurable polycaprolactone (PCL) by a combination of emulsion templating and three-dimensional (3D) printing and (ii) decoration of these scaffolds with the in vitro generated bone-like extracellular matrix (ECM) to create biohybrid scaffolds that have improved biological performance compared to PCL-only scaffolds. Multiscale porous scaffolds were fabricated, bone cells were cultured on them, and then they were decellularized. The biological performance of these constructs was tested in vitro and in vivo. Mesenchymal progenitors were seeded on PCL-only and biohybrid scaffolds. Cells not only showed improved attachment on biohybrid scaffolds but also exhibited a significantly higher rate of cell growth and osteogenic activity. The chick chorioallantoic membrane (CAM) assay was used to explore the angiogenic potential of the biohybrid scaffolds. The CAM assay indicated that the presence of the in vitro generated ECM on polymeric scaffolds resulted in higher angiogenic potential and a high degree of tissue infiltration. This study demonstrated that multiscale porous biohybrid scaffolds present a promising approach to improve bioactivity, encourage precursors to differentiate into mature bones, and to induce angiogenesis.

Abstract P3-01-07: Early detection of metastatic breast cancer through gene expression and cell heterogeneity at an engineered metastatic niche

Abstract Background: Current technology limits the detection of metastatic lesions until the patient becomes symptomatic or radiological evidence is identified, and thus invasive cancer cells are not detectable prior to their colonization in distant organs. We have developed a biomaterial implant that serves as a synthetic metastatic niche that recruits tumor cells prior to their accumulation in the lung, a natural metastatic niche. We aimed to dissect the heterogeneity of stromal and immune cells at the synthetic and lung metastatic niches over time, thereby identifying changes in cell populations, phenotypes, and gene expression that could be used for the molecular staging of metastatic disease. Methods: Porous polycaprolactone scaffolds (5 mm diameter x 2 mm height) were implanted subcutaneously into Balb/c mice two weeks before triple negative murine breast cancer cells (4T1) were inoculated into the mammary fat pad. We tracked disease progression in the lung from a pre-metastatic niche (7 days post-inoculation) to micrometastases (14 days) to overt metastatic disease (21 days). Ten genes were selected from the TaqManTM Open Array Mouse Inflammation Panel to develop a PCR-based signature and single value decomposition (SVD) score in the scaffold that successfully delineated metastatic staging. Furthermore, the scaffold and lung were analyzed through single cell RNA-sequencing (Drop-seq) to identify heterogeneity and contributions of each cell type. Results: Ten genes were identified from Open Array of the scaffolds that directly correlated to disease stage in the lungs – S100a8, S100a9, Pglyrp1, Ltf, Camp, Ela2, and Chi3l3 increased with metastatic progression, while Bmp15, Ccl22 and Ccr7 decreased. SVD was applied to establish a single score from the scaffold that could be used for accurate molecular staging. Scaffolds were scored on a scale from 0 to 1, where a value of 0.2 – 0.3 generally correlated to pre-metastatic disease, 0.4 - 0.8 indicated micrometastases and 0.8 – 1.0 demarked metastasis. This model successfully predicted disease stage with an area under the curve of 0.878. When the scaffold-derived gene signature was applied to the lung metastases (21 days), the output was comparable to the scaffold, further demonstrating the physiological relevance of the implants. Single cell analysis indicated that the genes in the signature were primarily expressed by macrophages and neutrophils. After 7 days, 22% of macrophages and 83% of neutrophils were classified as tumor associated cells in the scaffold. In contrast, only 16% of macrophages and 44% of neutrophils in the lung were tumor associated at this time point. These findings support the hypothesis that the scaffolds could be used as early surrogates for the pre-metastatic niche, becoming responsive prior to their biological metastatic counterpart. Moreover, this finding was demonstrated through changes in T cell phenotype, specifically loss of cytotoxic Cd8+ T cells. After 7 days, Cd8+ T cells in the scaffolds decreased by 35%. Whereas Cd8+ T cells in the lungs decreased by 35% after 14 days. Conclusions: A biomaterial scaffold was employed as a synthetic niche that recruits metastatic tumor cells. These scaffolds established a molecular signature of metastatic breast cancer using a 10-gene panel. Additionally, single cell analysis identified the cell types that induce this signature and the disease-associated changes that occur in the scaffold earlier than the lung. In summary, we have shown the potential of a biomaterial scaffold to act as a surrogate metastatic niche that can be used to delineate disease progression and understand the role of specific cell types in the formation of this niche. Mechanistic investigation into the synthetic niche may identify novel therapeutic targets that could be activated to inhibit the advent of metastasis. Citation Format: Sophia M Orbach, Robert S Oakes, Michael D Brooks, Grace G Bushnell, Pridvi Kandagatla, Max S Wicha, Jacqueline S Jeruss, Lonnie D Shea. Early detection of metastatic breast cancer through gene expression and cell heterogeneity at an engineered metastatic niche [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-07.

The effect of melt electrospun writing fiber orientation onto cellular organization and mechanical properties for application in Anterior Cruciate Ligament tissue engineering

The effect of melt electrospun writing fiber arrangement on cellular behavior has not yet been thoroughly investigated. Cellular orientation is particularly important in the context of ligament tissue engineering for orthopedic applications whereby a high degree of cell alignment is present in the native tissue. The aim of this study was to investigate the response of human mesenchymal stem cells (hMSC) to three different patterned porous polycaprolactone scaffolds (aligned, crimped and random) fabricated by melt electrospinning writing, resulting in 20 μm diameter electrospun fibers. Cell orientation was investigated over 4 weeks in vitro and it was demonstrated that the aligned pattern was capable of orientating the hMSCs towards the main direction of the fibers and this feature was maintained over the entire culture period whereas the orientation was rapidly lost in the crimped pattern. In order to fabricate a functional scaffold for ligament tissue engineering, the scaffolds were rolled in three bundles, subsequently braided and combined with a bone compartment (consisting of a melt electrospun scaffold seeded with osteogenically induced hMSCs) for the development of a Bone-Ligament-Bone (BLB) construct. The mechanical properties of non-cellularized and cellularized BLB constructs were assessed under both quasi-static and cyclic conditions. This revealed that the in vitro maturation significantly softened the BLB constructs and that the mechanical properties were several fold lower than those of native tissue. The cyclic testing demonstrated that the presence of cell sheets resulted in increased resilience and elasticity, even though the global mechanical properties were decreased for the in vitro matured constructs (regardless of the pattern). In conclusion, we demonstrated that melt electrospinning writing fiber organization can induce spontaneous cell alignment and that large cellularized BLB constructs with complex geometry can achieve mechanical resilience under cyclic stretching.

Substrate-independent polymer coating with stimuli-responsive dexamethasone release for on-demand fibrosis inhibition.

Tissue fibrosis caused by implantation of tissue engineering scaffolds is an urgent problem in clinical research. In this work, a substrate-independent coating with on-demand release of an antifibrotic drug has been fabricated to effectively address this issue. This coating was formed through a substrate-independent layer-by-layer (LBL) technique via a cationic polyelectrolyte (poly-diallyldimethylammonium, PDDA) and an anionic polyelectrolyte (poly-styrenesulfonate, PSS), where parts of PSS and PDDA were physically replaced by carboxyl functionalized polyethylene glycol grafted onto antifibrotic drug dexamethasone (DEX-PEG-COOH). Considering the easy generation of local inflammation after implantation, an ester bond was designed between PEG-COOH and DEX. Therefore, the overexpressed esterase under inflammatory conditions hydrolyzes the ester bond and thereby releases DEX from the film to inhibit fibrosis occurring in the tissue repair process. The in vivo capacity of this coating to restrain tissue fibrosis was investigated by a skin defect model using porous polycaprolactone (PCL) scaffolds as substrates. The experimental results showed that the fibrosis-related proteins (Col-I, TGF-β and fibronectin) and the infiltration of myofibroblasts (α-SMA) of skin tissues in the coated PCL scaffold group were significantly lower than those in the blank control group and pure PCL scaffold group. Moreover, the histological evaluations showed that the coating group could significantly decrease the deposition of collagen and meanwhile promote the partial regeneration of skin appendages. These results successfully demonstrate that the universal coating prepared with a simple protocol would be an effective strategy to address the fibrosis issues during tissue engineering.

Fabrication and improvement of PCL/alginate/PAAm scaffold via selective laser sintering for tissue engineering

In this work, porous polycaprolactone (PCL) scaffolds were designed and fabricated using selective laser sintering (SLS). The critical processing parameters of the SLS for PCL were optimised. This work post-processed these PCL scaffolds to produce PCL/alginate/polyacrylamide (PAAm) scaffolds to improve their performance. The experiment mechanical property assessment showed that the sample's average elastic modulus increases from 6.99 MPa (PCL) to 12.67 MPa (PCL/alginate/PAAm), and the elongation at break of samples increases from 59% (PCL) to 112.9% (PCL/alginate/PAAm). Cell seeding and in vitro culture showed that cell viability remained above 94% over 5 days. Thus, the current study suggests that a promising strategy for the improvement of the characteristics of PCL/alginate/PAAm scaffolds and advances the potential application of SLS technique towards skeletal tissue repair.

Fabrication and characterization of porous polycaprolactone scaffold via extrusion-based cryogenic 3D printing for tissue engineering

Earlier reports of fabricating 3D porous PCL scaffolds for tissue engineering applications were overshadowed by several limitations such as additional molds cost, relatively low efficiency, and lacking process control. In present study, combined extrusion-based cryogenic 3D printing (ECP) (−20 °C) and subsequent freeze-drying approaches were employed to facilely fabricate polycaprolactone (PCL) scaffolds, with high porosity and fidelity. Freeze-drying caused shrinkage of the scaffolds along X, Y, and Z-axes to some extent. The porosities of CP600, CP800, and CP1000 were found to be 64.0 ± 1.2%, 70.1 ± 1.3%, and 74.3 ± 0.6%, respectively. The fabricated scaffolds were characterized for various structural features and compared with the ones fabricated through traditional extrusion-based melt 3D printing (EMP). The crystallinity of PCL in ECP scaffolds was lower (57.1 ± 2.2%) than EMP scaffolds (69.8 ± 1.3%). The ECP scaffolds showed high alkaline degradation, but low compression properties. The ECP scaffolds promoted the adhesion and proliferation of MCT3T-E1 cells with well-spread morphology on the porous filaments. Together, these features justify the suitability of printed PCL scaffolds for potential TE applications.

Fabrication and Characterization of Bioresorbable Drug-coated Porous Scaffolds for Vascular Tissue Engineering.

Bioresorbable polymers have been studied for several decades as attractive candidates for promoting the advancement of medical science and bio-technology in modern society. In particular, with a well-defined architecture, bioresorbable polymers have prominent advantages over their bulk counterparts for applications in biomedical and implant devices, such as cell delivery, scaffolds for tissue engineering, and hydrogels as well as in the pharmaceutical fields. Biocompatible implant devices based on bioresorbable materials (for instance, bioresorbable polymers that combine the unique advantages of biocompability and easy handling) have emerged as a highly active field due to their promising applications in artificial implant systems and biomedical devices. In this paper, we report an approach to fabricate porous polycaprolactone (PCL) scaffolds using a 3D printing system. And its surface was treated to a hydrophilic surface using plasma treatment. Then, the aspirin and atorvastatin calcium salt mixture was dip coated onto the surface. The drug coating technology was used to deposit the drug material onto the scaffold surface. Our porous PCL scaffold was coated with aspirin and atorvastatin calcium salt to reduce the blood LDL cholesterol and restenosis. These results suggest that our approach may provide a promising scaffold for developing bioresorbable drug-delivery-biomaterials. We further demonstrate that our bioresorbable medical device can be used as vascular scaffolds to provide a wide range of applications for the design of medical devices.

Highly porous polycaprolactone scaffolds doped with calcium silicate and dicalcium phosphate dihydrate designed for bone regeneration

Polycaprolactone (PCL), dicalcium phosphate dihydrate (DCPD) and/or calcium silicates (CaSi) have been used to prepare highly porous scaffolds by thermally induced phase separation technique (TIPS). Three experimental mineral-doped formulations were prepared (PCL-10CaSi, PCL-5CaSi-5DCPD, PCL-10CaSi-10DCPD); pure PCL scaffolds constituted the control group.Scaffolds were tested for their chemical-physical and biological properties, namely thermal properties by differential scanning calorimetry (DSC), mechanical properties by quasi-static parallel-plates compression testing, porosity by a standard water-absorption method calcium release, alkalinizing activity, surface microchemistry and micromorphology by Environmental Scanning electronic Microscopy (ESEM), apatite-forming ability in Hank Balanced Saline Solution (HBSS) by Energy Dispersive X-ray Spectroscopy (EDX) and micro-Raman, and direct contact cytotoxicity.All mineral-doped scaffolds released calcium and alkalinized the soaking medium, which may favor a good biological (osteogenic) response.ESEM surface micromorphology analyses after soaking in HBSS revealed: pure PCL, PCL-10CaSi and PCL-10CaSi-10DCPD kept similar surface porosity percentages but different pore shape modifications. PCL-5CaSi-5DCPD revealed a significant surface porosity increase despite calcium phosphates nucleation (p < 0.05).Micro-Raman spectroscopy detected the formation of a B-type carbonated apatite (Ap) layer on the surface of PCL-10CaSi-10DCPD aged for 28 days in HBSS; a similar phase (but of lower thickness) formed also on PCL-5CaSi-5DCPD and PCL; the deposit formed on PCL-10CaSi was mainly composed of calcite.All PCL showed bulk open porosity higher than 94%; however, no relevant brittleness was observed in the materials, which retained the possibility to be handled without collapsing.The thermo-mechanical properties showed that the reinforcing and nucleating action of the inorganic fillers CaSi and DCPD improved viscoelastic properties of the scaffolds, as confirmed by the increased value of storage modulus and the slight increase in the crystallization temperature for all the biomaterials.A detrimental effect on the mechanical properties was observed in samples with the highest amount of inorganic particles (PCL-10CaSi-10DCPD).All the scaffolds showed absence of toxicity, in particular PCL-10CaSi-10DCPD.The designed scaffolds are biointeractive (release biologically relevant ions), nucleate apatite, possess high surface and internal open porosity and can be colonized by cells, creating a bone forming osteoblastic microenvironment and appearing interesting materials for bone regeneration purposes.

Supercritical fluid-assisted controllable fabrication of open and highly interconnected porous scaffolds for bone tissue engineering.

Recently tremendous progress has been evidenced by the advancements in developing innovative three-dimensional (3D) scaffolds using various techniques for addressing the autogenous grafting of bone. In this work, we demonstrated the fabrication of porous polycaprolactone (PCL) scaffolds for osteogenic differentiation based on supercritical fluid-assisted hybrid processes of phase inversion and foaming. This eco-friendly process resulted in the highly porous biomimetic scaffolds with open and interconnected architectures. Initially, a 23 factorial experiment was designed for investigating the relative significance of various processing parameters and achieving better control over the porosity as well as the compressive mechanical properties of the scaffold. Then, single factor experiment was carried out to understand the effects of various processing parameters on the morphology of scaffolds. On the other hand, we encapsulated a growth factor, i.e., bone morphogenic protein-2 (BMP-2), as a model protein in these porous scaffolds for evaluating their osteogenic differentiation. In vitro investigations of growth factor loaded PCL scaffolds using bone marrow stromal cells (BMSCs) have shown that these growth factor-encumbered scaffolds were capable of differentiating the cells over the control experiments. Furthermore, the osteogenic differentiation was confirmed by measuring the cell proliferation, and alkaline phosphatase (ALP) activity, which were significantly higher demonstrating the active bone growth. Together, these results have suggested that the fabrication of growth factor-loaded porous scaffolds prepared by the eco-friendly hybrid processing efficiently promoted the osteogenic differentiation and may have a significant potential in bone tissue engineering.

3D Printed Polymer Scaffolds for Bone Tissue Regeneration

The regeneration of bone defects is a significant clinical challenge for patients around the world. The ideal scaffolds for bone tissue repair should provide biocompatibility, pore architecture, biodegradability, mechanical support, and cell attachment sites. Conventionally fabricated polymer scaffolds are still unable to make ideal scaffolds for bone tissue repair due to the lack of all the above mentioned properties. The investigated hypothesis was that increasing pore sizes of the scaffolds would cause an increase in the porosity and a decrease in the compressive modulus. In order to test the hypothesis, we designed three different pore sizes (200, 400, and 800 μm) in three different scaffolds using computer software. Relatively new 3D printing technology was used to print the three different types of porous scaffolds using polycaprolactone (PCL) polymer. These scaffolds were characterized for percent porosity, pore architecture, morphology, mechanical properties, and evaluated for biocompatibility and cell attachment with murine pre-osteoblasts. The percent porosity of these scaffolds (n=7) significantly increased from 13.31 to 61.66 (p&amp;lt;0.001) with the increase in pore size. The average compressive modulus of scaffolds (n=7) significantly decreased with the increase in pore size (p&amp;lt;0.001). The averaged compressive modulus of scaffolds with 200, 400, and 800 μm pores is 82.98 ± 2.02, 61.60 ± 2.59, and 47.16 ± 1.73 MPa, respectively. In addition, PCL scaffolds show biocompatibility as determined by an in vitro cell study. These results have shown that the hypothesis is validated, and these 3D printed porous PCL scaffolds can be potentially used for bone regeneration applications.

Homogeneous cell printing on porous PCL/F127 tissue engineering scaffolds

In tissue engineering, cell-laden scaffold has gradually replaced cell-less scaffold due to better biological performance. However, manual pipetting, the traditional cell seeding for cell-laden scaffold, leads to an imprecise and inhomogeneous cell distribution. As an alternative, micro-extrusion of cell-laden hydrogel achieves homogenous cell distribution, but causes high shear stress which is harmful to cells. To address this challenge, the objective of this study is to print cells on porous scaffold precisely without causing high shear stress to produce homogeneous cell-laden hybrid scaffold. Porous polycaprolactone scaffold fabricated through electro-hydrodynamic jetting was used as a representation. To improve scaffold hydrophilicity for better cell adhesion, 6% (w/w) Pluronic F127 was blended with polycaprolactone. HeLa cells, as a demonstration, were ejected on the scaffold fibers through piezoelectric inkjet printing. As a result, inkjet printing showed a more precise and homogeneous cell distribution and enhanced cell proliferation compared to manual pipetting (1.34- fold increase after 7 days). Furthermore, due to the low viscosity of cell solution, the average shear stress caused during inkjet printing was 1.79 kPa as opposed to 18 kPa of micro-extrusion, which is friendly to cells. In summary, through inkjet printing, homogeneous cell-laden hybrid scaffold could be fabricated with lower shear stress.

Acrylic acid plasma coatings for enhanced cell migration in PCL 3D additive manufactured scaffolds

This study describes the additive manufacturing of 3D polycaprolactone (PCL) scaffolds and their subsequent surface modification using non-thermal plasma technology (NTP) for musculoskeletal applications. 100% interporous scaffolds with porosities ranging from 37 to 66% were manufactured and characterized using scanning electron microscopy (SEM). 2 NTP-based modification strategies were employed: 1) He activation and 2) Acrylic acid NTP polymerization. Gas flow, system pressure and scaffold porosity were systematically varied and their influence on functional group incorporation efficiency and distribution were studied using X-ray Photoelectron Spectroscopy (XPS). The NTP-coated samples with optimal operational parameters were conducted to a series of in-vitro experiments involving MC3T3-E1 osteoblasts and adipose derived mesenchymal stem cells (ADSCs) and the influence on cell adhesion, proliferation and migration was analyzed and compared to untreated and plasma-activated samples. The NTP-based acrylic acid coating was found to effectively stimulate cell migration throughout the porous PCL scaffolds for the MC3T3 cells and was also responsible for better ADSC cell adhesion and migration, making them promising structures for musculoskeletal applications.

Growth and Differentiation of Dental Stem Cells of Apical Papilla on Polycaprolactone Scaffolds.

Biodegradable scaffolds are useful tools in the field of tissue engineering and regenerative medicine. The aim of this study was to test the potential of the human stem cells of apical papilla (SCAP) to attach, proliferate and differentiate on a polycaprolactone (PCL)-based scaffolds. SCAP were extracted from the root apical papillae of freshly extracted immature premolar teeth by using enzymatic digestion. Porous PCL scaffolds were fabricated using particle leaching method and NaCl or mannitol as porogens. SCAP of passage 3 were seeded on non-porous and porous PCL scaffolds for up to 14days. For control, cells were cultured on glass coverslips. Picogreen DNA quantification was used to assay for cell proliferation. Cell differentiation and development of calcification nodules were examined using scanning electron microscopy and alizarin red staining. SCAP showed a comparable attachment, growth and proliferation patterns on PCL scaffolds and coverslips. Cell proliferation was enhanced on mannitol scaffolds at all time points. Calcification nodules were detected in all PCL scaffolds while it was not present on glass coverslips. These nodules were detected on NaCl-scaffolds by day 7 and on mannitol and non-porous scaffolds by day 14. In conclusion, SCAP were able to attach, proliferate and differentiate on PCL scaffolds without using any inductive media, indicating their potential application for dental tissue regeneration.

Endoluminal laser-assisted vascular anastomosis-an in vivo study in a pig model.

Microvascular surgery is time consuming and requires high expertise. Laser-assisted vascular anastomosis (LAVA) is a promising sutureless technique that has the potential to facilitate this procedure. In this study, we evaluate the handling of our soldering material and the 1-week patency rate in a porcine model. Six pigs were subjected to LAVA. For each pig, the saphenous artery on one side was transected while the contralateral side was used as control. A porous polycaprolactone scaffold soaked in 40% (w/w) bovine serum albumin solution in combination with 0.1% (w/w) indocyanine green was wrapped at the anastomosis site and at the control site. Both sides were then soldered with a diode laser coupled into a light diffuser fiber emitting radiation with a wavelength of 808nm and a power of 2-2.2W. Vessels were successfully soldered with a 100% immediate patency rate. The 1-week patency rate was 83% for the anastomoses versus 67% for the control side. Vessels irradiated for 80 to 90s tended to maintain the highest patency rate. Macroscopically, there was no difference between the two sides. The patch was easy to handle provided that the environment could be kept dry. This study shows the potential and the limitations of endoluminal LAVA as a one-step procedure without the use of stay sutures. Further studies are needed to improve the soldering material, the long-term patency rate, and standardized irradiation parameters. The long-term effects of laser soldering on the vessel wall remain to be determined.

Effect of CO2 plasma exposure on physico-chemical properties of porous polycaprolactone scaffold

Carbon dioxide plasma was employed for the surface modification of tubular poly(caprolactone) porous matrix. Effect of plasma exposure over surface functionality, mechanical properties, surface morphology, pore size and crystal structure was investigated. Plasma modification is useful in achieving desired surface chemical properties without much alteration in the bulk properties of material. This process yields a porous tubular structure of 40 ± 11 MPa tensile strength, 464 ± 101 % tensile strain, 226 ± 41 g f suture retention strength and 52 % crystallinity. Profound influence of plasma exposure time over pore size and crystal form transition has been observed. Surface became hydrophilic in nature as evident from the low contact angle with 60 s exposure. The resulted surface-modified PCL tubular porous matrix with –COOH density of 40 nM/cm2 can anchor maximum of 2.67 mg/cm2 gelatin on the surface.

Supercritical CO2 foamed polycaprolactone scaffolds for controlled delivery of 5-fluorouracil, nicotinamide and triflusal

The manufacture of porous polycaprolactone (PCL) scaffolds containing three different drugs, namely 5-fluorouracil, nicotinamide and triflusal, was investigated in this work with the aim of obtaining bioactive systems with controlled drug delivery capabilities. The scaffolds were prepared by means of a supercritical CO2 (scCO2) foaming technique by optimizing the drug loading process. This was achieved by dissolving the drugs in organic solvents miscible with scCO2 and by mixing these drug/solvent solutions with PCL powder. The as prepared mixtures were further compressed to eliminate air bubbles and finally processed by the scCO2 foaming technique. ScCO2 saturation and foaming conditions were optimized to create the porosity within the samples and to allow for the concomitant removal of the organic solvents. Physical and chemical properties of porous scaffolds, as well as drug content and delivery profiles, were studied by HPLC. The results of this study demonstrated that the composition of the starting PCL/drug/solvent mixtures affected polymer crystallization, scaffold morphology and pore structure features. Furthermore, it was found that drug loading efficiency depended on both initial solution composition and drug solubility in scCO2. Nevertheless, in the case of highly scCO2-soluble drugs, such as triflusal, loading efficiency was improved by adding a proper amount of free drug inside of the pressure vessel. The drug delivery study indicated that release profiles depended mainly upon scaffolds composition and pore structure features.

Improvement of Distribution and Osteogenic Differentiation of Human Mesenchymal Stem Cells by Hyaluronic Acid and β-Tricalcium Phosphate-Coated Polymeric Scaffold In Vitro.

Bone tissue engineering requires a well-designed scaffold that can be biodegradable, biocompatible, and support the stem cells to osteogenic differentiation. Porous polycaprolactone (PCL) scaffold prepared by fused deposition modeling is an attractive biomaterial that has been used in clinic. However, PCL scaffolds lack biological function and osteoinductivity. In this study, we functionalized the PCL scaffolds by embedding them with a matrix of hyaluronic acid/β-tricalcium phosphate (HA/TCP). Human mesenchymal stem cells (MSCs) were cultured on scaffolds with and without coating to investigate proliferation and osteogenic differentiation. The DNA amount was significantly higher in the HA/TCP-coated scaffold on day 21. At the gene expression level, HA/TCP coating significantly increased the expression of ALP and COLI on day 4. These data correlated with the ALP activity peaking on day 7 in the HA/TCP-coated scaffold. Scanning electron microscope and histological analysis revealed that the cell matrix and calcium deposition were distributed more uniformly in the coated scaffolds compared to scaffolds without coating. In conclusion, the HA/TCP coating improved cellular proliferation, osteogenic differentiation, and uniform distribution of the cellular matrix in vitro. The HA/TCP-PCL scaffold holds great promise to accommodate human bone marrow-derived MSCs for bone reconstruction purposes, which warrants future in vivo studies.