The aim of this study is to print 3D polycaprolactone (PCL) scaffolds at high and low temperature (HT/LT) combined with salt leaching to induced porosity/larger pore size and improve material degradation without compromising cellular activity of printed scaffolds. PCL solutions with sodium chloride (NaCl) particles either directly printed in LT or were casted, dried, and printed in HT followed by washing in deionized water (DI) to leach out the salt. Micro-Computed tomography (Micro-CT) and scanning electron microscope (SEM) were performed for morphological analysis. The effect of the porosity on the mechanical properties and degradation was evaluated by a tensile test and etching with NaOH, respectively. To evaluate cellular responses, human bone marrow-derived mesenchymal stem/stromal cells (hBMSCs) were cultured on the scaffolds and their viability, attachment, morphology, proliferation, and osteogenic differentiation were assessed. Micro-CT and SEM analysis showed that porosity induced by the salt leaching increased with increasing the salt content in HT, however no change was observed in LT. Structure thickness reduced with elevating NaCl content. Mass loss of scaffolds dramatically increased with elevated porosity in HT. Dog bone-shaped specimens with induced porosity exhibited higher ductility and toughness but less strength and stiffness under the tension in HT whereas they showed decrease in all mechanical properties in LT. All scaffolds showed excellent cytocompatibility. Cells were able to attach on the surface of the scaffolds and grow up to 14 days. Microscopy images of the seeded scaffolds showed substantial increase in the formation of extracellular matrix (ECM) network and elongation of the cells. The study demonstrated the ability of combining 3D printing and particulate leaching together to fabricate porous PCL scaffolds. The scaffolds were successfully printed with various salt content without negatively affecting cell responses. Printing porous thermoplastic polymer could be of great importance for temporary biocompatible implants in bone tissue engineering applications

Bioactive glasses, such as 45S5 Bioglass (BG), have been shown to promote bone ingrowth both in vitro and in vivo. The goal of this study was to analyze the effect of a high dose of BG (20%) in Direct Ink Writing (DIW)-produced controlled-geometry PCL-BG composite scaffolds in both their mechanical and biological performance. Porous cubes of 5 × 5 × 5 mm, 50% porosity and pore size and strut diameter of 400 µm were fabricated in a 3D-Bioplotter (EnvisionTec) to investigate their biological performance (n = 3). Additionally, cylindrical specimens (10 mm diameter; 15 mm height) with same internal structure were fabricated for mechanical testing (n = 6). The cylindrical specimens were tested by compression in a universal testing machine (ZwickRoell) with a 10 kN load cell. The tests were performed at 1.00 mm/min with extensometers in both sides. For biological characterization, scaffolds were sterilized in 70% ethanol overnight and pre-incubated with DMEM for 1 hour at room temperature. 1×10. 5. human gingival mesenchymal stem cells (hGMSCs) in 50 µl DMEM were seeded on the scaffolds using agarose molds to improve cell adhesion, and cultured in standard cell-culture conditions for 3, 7 and 14 days. To measure cell proliferation, the reagent CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS, Promega) was added to the cell-seeded scaffolds at each time point, using non-seeded scaffolds as blank controls. The OD (490 nm) was measured in a BioTek 800 TS plate reader. Both the apparent elastic modulus and yield stress were significantly lower in the scaffolds with 20% BG than their PCL control counterparts (p < 0.0001 for elastic modulus and p < 0.005 for yield stress, t-test). Cell proliferation in the scaffolds by MTS was variable, with the 20% BG scaffolds showing a significantly higher signal after seven days in culture (p < 0.05 by t-test), but a significantly lower signal after 14 days in culture (p < 0.05 by t-test). In conclusion, scaffolds with 20% BG showed a lower mechanical performance than their PCL counterparts in terms of both their apparent elastic modulus and yield stress. Additionally, scaffolds with 20% BG showed variable cell proliferation rates in terms of their metabolic activity over a two-week period. The decrease in proliferation rate after week 2 after an initial increase at the end of week 1 could be due to cytotoxic effects of the BG at this high dose (20%) after long term exposure. These results suggest that a dosage of 20% BG may not necessarily improve the mechanical and biological performance of scaffolds, so future experiments are required in order to characterize the optimum BG dosage in PCL scaffolds for tissue engineering applications

Electrospinning is an advantageous technique for cartilage tissue engineering (CTE) applications due to its ability to produce nanofibers recapitulating the size and alignment of the collagen fibers present within the articular cartilage superficial zone. Moreover, coaxial electrospinning allows the fabrication of core-shell fibers able to encapsulate and release bioactive molecules in a sustained manner. Kartogenin (KTG) is a small heterocyclic molecule, which was demonstrated to promote the chondrogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells(hBMSCs)[1]. In this work, we developed and evaluated the biological performance of core-shell poly(glycerol sebacate)(PGS)/poly(caprolactone)(PCL) aligned nanofibers (core:PGS/shell:PCL) mimicking the native articular cartilage extracellular matrix(ECM) and able to promote the sustained release of the chondroinductive drug KTG[2]. The produced coaxial aligned PGS/PCL scaffolds were characterized in terms of their structure and fiber diameter, chemical composition, thermal properties, mechanical performance under tensile testing and in vitro degradation kinetics, in comparison to monoaxial PCL aligned fibers and respective non-aligned controls. KTG was incorporated into the core PGS solution to generate core-shell PGS-KTG/PCL nanofibers and its release kinetics was studied by HPLC analysis. KTG-loaded electrospun aligned scaffolds capacity to promote hBMSCs chondrogenic differentiation was evaluated by assessing cell proliferation, typical cartilage-ECM production (sulfated glycosaminiglycans(sGAG)) and chondrogenic marker genes expression in comparison to non-loaded controls. All the scaffolds fabricated showed average fiber diameters within the nanometer-scale and the core-shell structure of the fibers was clearly confirmed by TEM. The coaxial PGS-KTG/PCL nanofibers evidenced a more sustained drug release over 21 days. Remarkably, in the absence of the chondrogenic cytokine TGF-β3, KTG-loaded nanofibers promoted significantly the proliferation and chondrogenic differentiation of hBMSCs, as suggested by the increased cell numbers, higher sGAG amounts and up-regulation of the chondrogenic genes COL2A1, Sox9, ACAN and PRG4 expression. Overall, our results highlight the potential of core-shell PGS-KTG/PCL aligned nanofibers for the development of novel MSC-based CTE strategies. Acknowledgements: The authors thank FCT for funding through the project InSilico4OCReg (PTDC/EME-SIS/0838/2021) and to institutions iBB (UID/BIO/04565/2020) and Associate Laboratory I4HB (LA/P/0140/2020)

Postoperative knee stability is critical in determining the success after reconstruction; however, only posterior and anterior stability is assessed. Therefore, this study investigates medial and lateral rotational knee laxity changes after partial and complete PCL tear and after PCL allograft reconstruction. The extending Lachman test assessed knee instability in six fresh-frozen human cadaveric knees. Tibia rotation was measured for the native knee, after partial PCLT (pPCLT), after full PCLT (fPCLT), and then after PCLR tensioned at 30° and 90°. In addition, tests were performed for the medial and lateral sides. The tibia was pulled with 130N using a digital force gauge. A compression load of 50N was applied to the joint on the universal testing machine (MTS Systems) to induce contact. Three-dimensional tibial rotation was measured using a motion capture system (Optotrak). On average, the tibia rotation increased by 33%-42% after partial PCL tear, and by 62%-75% after full PCL tear when compared to the intact case. After PCL reconstruction, the medial tibia rotation decreased by 33% and 37% compared to the fPCL tear in the case that the allograft was tensioned at 30° and 90° of flexion, respectively. Similarly, lateral tibial rotation decreased by 15% and 2% for allograft tensioned at 30° and 90° of flexion respectively, compared to the full tear. Rotational decreases were statistically significant (p<0.005) at the lateral pulling after tensioning the allograft at 90°. PCLR with the graft tensioned at 30° and 90° both reduced medial knee laxity after PCLT. These results suggest that while both tensioning angles restored medial knee stability, tensioning the Achilles graft at 30° of knee flexion was more effective in restoring lateral knee stability throughout the range of motion from full extension to 90° flexion, offering a closer biomechanical resemblance to native knee function

Critical-sized bone defects can result from trauma, inflammation, and tumor resection. Such bone defects, often have irregular shapes, resulting in the need for new technologies to produce suitable implants. Bioprinting is an additive manufacturing method to create complex and individualised bone constructs, which can already include vital cells. In this study, we established an extrusion-based printing technology to produce osteoinductive scaffolds based on polycaprolactone (PCL) combined with calcium phosphate, which is known to induce osteogenic differentiation of stem cells. The model was created in python based on the signed distance functions. The shape of the 3D model is a ring with a diameter of 20 mm and a height of 10 mm with a spongiosa-like structure. The interconnected irregular pores have a diameter of 2 mm +/− 0.2 mm standard deviation. Extrusion-based printing was performed using the BIO X6. To produce the bioink, PCL (80 kDa) was combined with calcium phosphate nanopowder (> 150 nm particle size) under heating. After printing, 5 × 10. 6. hMSC were seeded on the construct using a rotating incubator. We were able to print a highly accurate ring construct with an interconnected pore structure. The PCL combined with calcium phosphate particles resulted in a precise printed construct, which corresponded to the 3D model. The bioink containing calcium phosphate nanoparticles had a higher printing accuracy compared to PCL alone. We found that hMSC cultured on the construct settled in close proximity to the calcium phosphate particles. The hMSC were vital for 22 days on the construct as demonstrated by life/dead staining. The extrusion printing technology enables to print a mechanically stable construct with a spongiosa-like structure. The porous PCL ring could serve as an outer matrix for implants, providing the construct the stability of natural bone. To extend this technology and to improve the implant properties, a biologised inner structure will be integrated into the scaffold in the future

Introduction and Objective. Several in vitro studies have shed light on the osteogenic and chondrogenic potential of graphene and its derivatives. Now it is possible to combine the different biomaterial properties of graphene and 3D printing scaffolds produced by tissue engineering for cartilage repair. Owing to the limited repair capacity of articular cartilage and bone, it is essential to develop tissue-engineered scaffolds for patients suffering from joint disease and trauma. However, chondral lesions cannot be considered independently of the underlying bone tissue. Both the microcirculation and the mechanical support provided with bone tissue must be repaired. One of the distinctive features that distinguish graphene from other nanomaterials is that it can have an inductive effect on both bone and cartilage tissue. In this study, the effect of different concentrations of graphene on the in vivo performance of single-layer poly-ε-caprolactone based-scaffolds is examined. Our hypothesis is that graphene nanoplatelet- containing, robocast PCL scaffolds can be an effective treatment option for large osteochondral defect treatment. For this purpose, different proportions of graphene- containing (1%,3%,5%,10 wt%) PCL scaffolds were studied in a 5mm diameter osteochondral defect model created in the rabbit knee. Materials and Methods. In the study graphene-containing (1, 3, 5, 10 wt%), porous and oriented poly-ε-caprolactone-based scaffolds were prepared by robocasting method to use in the regeneration of large osteochondral defects. Methods: The scaffolds were implanted into the full-thickness osteochondral defect in a rabbit model to evaluate the regeneration of defect in vivo. For this purpose, twenty female New Zealand white rabbits were used and they were euthanized at 4 and 8 weeks of implantation. The reparative osteochondral tissues were harvested from rabbit distal femurs and then processed for gross appearance assessment, radiographic imaging, histopathological and immunohistochemical examinations. Results. Results revealed that, graphene- containing graft materials caused significant amelioration at the defect areas. Graphene-containing graft materials improved the fibrous, chondroid and osseous tissue regeneration compared to the control group. The expressions of bone morphogenetic protein-2 (BMP-2), collagen-1 (col-1), vascular endothelial growth factor (VEGF) and alkaline phosphatase (ALP) expressions were more prominent in graphene- containing PCL implanted groups. Results also revealed that the ameliorative effect of graphene increased by the elevation in concentration. The most prominent healing was observed in 10 wt% graphene-containing PCL based composite scaffold implanted group. Conclusions. This study demonstrated that graphene- containing, robocast PCL scaffolds has efficacy in the treatment of large osteochondral defect. Subchondral new bone formation and chondrogenesis were observed based on immunohistochemical examinations. 3D printed PCL platforms have great potential for the investigation of the osteochondral regeneration mechanism. The efficacy of graphene-containing PCL scaffolds on osteogenesis, vascularization, and mineralization was shown at different graphene concentrations at 4th and 8th weeks. Immunohistochemical studies showed statistical significance in the 5wt% and 10 wt% graphene-containing groups compared to the 1wt% and 3 wt% graphene-containing groups at the end of the eighth week

Abstract. Objectives. Direct ink writing (DIW) has gained considerable attention in production of personalized medical implants. Laponite nanoclay is added in polycaprolactone (PCL) to improve printability and bioactivity for bone implants. The 3D structure of DIW printed PCL/Laponite products was qualitatively evaluated using micro-CT. Methods. PCL/LP composite ink was formulated by dissolving 50% m/v PCL in dichloromethane with Laponite loading of up to 30%. The rheological properties of the inks were determined using Discovery HR-2 rheometer. A custom-made direct ink writer was used to fabricate both porous scaffold with 0°/90° lay-down pattern, and solid dumbbell-shaped specimens (ASTM D638 Type IV) with two printing orientations, 0° and 90° to the loading direction in tensile testing. The 3D structure of specimens was assessed using a micro-CT. Independent t-tests were performed with significance level at p<0.05. Results. The addition of Laponite in PCL ink has significantly enhanced viscosity for shape fidelity and shear-thinning property facilitating extrusion for DIW. Uniform distribution of Laponite was illustrated by micro-CT. For the 32-layer scaffold, interconnectivity of pores is observed at all 3 planes. The variation of height and width of layers is within 6% except the bottom 2 layers which are significantly lower and wider than other layers for mechanical support. For solid specimens, no ditches/interfaces between filaments are observed in 90° orientation while they are distinctive in 0° orientation because deposited filaments contact each other sooner in 90° orientation. 90° specimens also have lower air gap fraction (0.8 vs 5.4 %) and significantly higher Young's modulus (235 vs 195 MPa) and tensile strength (12.0 vs 9.5 MPa). Conclusions. The mechanical properties and printability of PCL/Laponite composites can be improved by controlling printing parameters; Micro-CT is an important tool to investigate the structure and properties of 3D printed products for bone tissue engineering

Tendon-to-bone multi-tissue transition exhibits a hierarchical and continuous gradient of matrix composition and alignment, allowing for efficient transmission of mechanical loading between tendon and bone. Upon injury, main problems associated with tendon-to-bone regeneration include disorganized matrix deposition, with a gradual loss of mineral content resulting in poor mechanical properties, limiting tissue integration and the formation of a graded interface. Therefore, we propose to assembly two types of continuous microfibres with distinct topological and compositional features tailored to guide cell alignment and matrix deposition while matching the mechanical requirements of the native tissue. Wet-spinning was used to produce textured composite microfibres using different flow rates and two polymer blends to replicate the anisotropic architecture of tendon (PCL/Gelatin, 22/9%, w/v) and the isotropic organization together with mineral composition of bone (PCL/Gelatin/Hydroxyapatite, 22/9% w/v and 7.7% w/w HAp). Obtained microfibres morphology, chemical and mechanical properties were evaluated. Biological performance was studied using human adipose-derived stem cells (hASCs). Cytoskeleton alignment, nuclei elongation and matrix mineralization were evaluated. Textile techniques were used to create a 3D fibrous scaffold. Morphological features were analyzed by micro-CT. PCL/Gelatin fibers produced at 1 mL/h extrusion rate exhibited the highest anisotropic alignment, in opposition to PCL/Gelatin/HAp fibers produced under the same condition. Micro-CT analysis of PCL/Gelatin/HAp fibers demonstrated variations within pore diameter and particles size between the different flow rates. Herein, PCL/Gelatin fibers induced a higher cytoskeleton alignment and nuclei elongation (p < 0.0001) in seeded hASCs. In contrast, significantly higher mineralization was found in PCL/Gelatin/HAp fibres (day 7, p < 0.04; day 14, p < 0.0001) as observed by alizarin red staining and quantification, suggesting the induction of an osteogenic-like phenotype. As proof of concept, textile techniques were used to assemble the two types of fibers and create a 3D scaffold presenting a continuous gradient in HAp content, as well as topological cues. After 14 days of culture with hASCs, a gradient of collagen deposition and matrix mineralization was found (p < 0.014, p < 0.0001). Higher deposition of collagen type II was observed in the tendon and interface parts of the fibrous scaffold and collagen type X in the interface. Overall, the wet-spinning method was efficiently used to engineer continuous textured composite microfibers. PCL/Gelatin fibers supported cell alignment mimicking tendon one, while PCL/Gelatin/HAp fibers induced mineral deposition and a possible phenotypic change without additional medium supplementation. Textile techniques allowed fibres assemblage and 3D scaffolds fabrication envisioning tendon-to-bone applications

Purpose. To compare postoperative clinical outcomes between posterior cruciate ligament (PCL) retaining and resecting total knee arthroplasty (TKA) using same cruciate-substituting (CS) inserts, and to elucidate the clinical relevance of the residual PCL in cruciate-retaining TKA, considering intraoperative influence factors, such as the posterior tibial slope, posterior condylar offset, joint gap, joint balance, and joint laxity. Methods. A total of 64 consecutive knees (44 patients) were enrolled in this study and divided into following two groups: 39 knees underwent PCL-retaining TKA group (CR group), and 25 underwent PCL-resecting TKA group (CS group). Preoperative patients’ demographic data and one-year postoperative clinical outcomes including range of motion, the Knee Injury and Osteoarthritis Outcome Score (KOOS), the Japanese Orthopaedic Association (JOA) score, and Forgotten Joint Score-12 (FJS-12) were compared between two groups. Results. Regarding range of motion, the average preoperative ROM was -14.3/120.0 degrees in the CR group and improved to -2.4/118.9 degrees postoperatively. In the CS group, the average preoperative ROM was -7.5/130 degrees and changed to -2.2/122.4 degrees postoperatively. There was no significant difference in the postoperative ROM between the groups (P=0.16). The KOOS (from 47.1 to 69.5 in CR group; from 41.1 to 70.8 in CS group) and JOA scores (from 59.2 to 76.9 in CR group; from 55.6 to 80.8 in CS group) were significantly improved postoperatively in both groups (P < 0.01). However, there was no significance in these postoperative scores between two groups (P = 0.09). There was also no significance in FJS-12 between two groups (70.3 in CR group and 66.9 in CS group; P=0.53). Conclusions. Residual PCL in TKA with a CS insert would not impact one-year postoperative clinical outcomes including KOOS, JOA, and FJS-12

The extracellular matrix (ECM) is the non-cellular structural support that provides cells with a network of biochemical and biomechanical factors for cellular processes. The ECM regulates cell function, differentiation and homeostasis. Here, we present a proteomics characterization of three commonly used additive manufactured polymers: polylactic acid (PLA), polyactive (PEOT/PBT) and polycaprolactone (PCL). We cultured human mesenchymal stromal cells (hMSCs) and make them undergo chondrogenic and osteogenic differentiation on 3D printed PCL, PEOT/PBT and PLA scaffolds. hMSCs were cultured in basal, chondrogenic and osteogenic media (200000 cells/scaffold) and analyzed after 35 days of culture. Differentiation was proved through biochemical assays, immunofluorescence and histology. The protein content was explored using label free liquid chromatography mass spectrometry (LC-MS), which revealed upregulated proteins and their related pathways. A higher difference was found among different media compared to the scaffold type through principal component analysis (PCA). Interestingly, in all three materials, chondrogenesis was characterized by a lower but more diverse amount of proteins. PCL induced ECM production in both differentiation media, but it led to more apoptosis and GAG degradation in the chondrogenic medium compared to the osteogenic one. During chondrogenesis in PEOT/PBT and PLA, cell differentiation resulted in the activation of stress response cascades, collagen formation and ECM remodelling. On the other hand, in osteogenesis, PCL enhanced insulin-like growth factor pathway and fibrin clot related pathways

Conventional 3D printing by itself is incapable of creating pores on a micro scale within deposited filaments throughout 3D scaffolds. These pores and hence larger surface areas are needed for cells to be adhered, proliferated, and differentiated. The aim of this work was to fabricate 3D polycaprolactone (PCL) scaffolds with internal multiscale porosity by using two different 3D printing techniques (ink/pellet of polymer-salt composite in low/high temperature printing) combined with salt leaching to improve cell adhesion, and cell proliferation besides to change degradation rate of PCL scaffolds:. 1. Non-solvent phase separation integrated 3D printing of polymer-salt inks with various salt content (i.e., low temperature ink-based printing, LT). 2. FDM printing of composite polymer-salt pellets which will be obtained by casting and evaporating of prepared ink (i.e., high temperature composite-pellet-based printing, HT). Further, the two approaches were followed by post salt leaching. Stem cells were able to attach on the surface and grow up to 14 days based on increasing cellular activities

A spine compression fracture is a very common form of fracture in elderly with osteoporosis. Injection of polymethyl methacrylate (PMMA) to fracture sites is a minimally invasive surgical treatment, but PMMA has considerable clinical risks. We develop a novel type thermoplastic injectable bone substitute contains the proprietary composites of synthetic ceramic bone substitute and absorbable thermoplastic polymer. We used thermoplastic biocompatible polymers Polycaproactone (PCL) to encapsulate calcium-based bone substitutes hydroxyapatite (Ca10(PO4)6(OH)2, HA) and tricalcium phosphate (TCP) to form a biodegradable injectable bone composite material. The space occupation ration PCL:HA/TCP is 1:9. After heating process, it can be injected to fracture site by specific instrument and then self-setting to immediate reinforce the vertebral body. The thermoplastic injection bone substitute can obtain good injection properties after being heated by a heater at 90˚C for three minutes, and has good anti-washout property when injected into normal saline at 37˚C. After three minutes, solidification is achieved. Mechanical properties were assessed using the material compression test system and the mechanical support close to the vertebral spongy bone. In vitro cytotoxicity MTT assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was performed and no cell cytotoxicity was observed. In vivo study with three New Zealand rabbits was performed, well bone growth into bone substitute was observed and can maintain good mechanical support after three months implantation. The novel type thermoplastic injection bone substitute can achieve (a) adequate injectability and viscosity without the risk of cement leakage; (b) adequate mechanical strength for immediate reinforcement and prevent adjacent fracture; (c) adequate porosity for new bone ingrowth; (e) biodegradability. It could be developed as a new option for treating vertebral compression fractures

Abstract. Objectives. The objective of this study is to investigate the effect of solvents and rheological properties of PCL/Hydroxyapatite ink on the shape fidelity of the 3D printed scaffolds for bone tissue engineering. Methods. A series of inks were made consisting of 50% (w/v) of polycaprolactone (PCL) filled with 0%, 3.5% and 12.5% (w/V) of hydroxyapatite (HA) in dichloromethane (DCM) and chloroform (CHF). Steady and oscillatory shear rheological tests were performed on a rheometer (Discovery HR-3). Solvent-cast direct ink writing was performed with a custom-made 3D printer for the fabrication of PCL/HA scaffold structures with 2–8 layers. Optical microscope and scanning electron microscopy (SEM) were used to assess the shape fidelity. Results. Shape fidelity of the inks was quantitively assessed on the 3D printed scaffold structures allowing subjective comparisons. The addition of HA particles increased zero-shear viscosity by up to 900%. For oscillatory tests, plateau of storage modulus was observed in the low-frequency region which is attributed to good dispersion of the HA particles inside the matrix that leads to the formation of filler networks, resulting in pseudo-solid behavior and shape fidelity improvement. As the HA concentration increases, the plateau becomes more pronounced and the shape fidelity increases. With the same concentration, all DCM inks also show higher viscosity (from 10% to 200%) and better shape fidelity than CHF inks. As DCM has a lower boiling point (39.6 °C) than CHF (61.2°C), DCM evaporates quicker reducing the fusion and diffusion of deposited ink filaments before solidification which is observed in SEM images. Conclusions. This study reveals insights into using rheological characterizations as a tool for evaluation of shape fidelity of solvent-based DIW inks and also provides fundamental information on the influence of different solvents on the fidelity of 3D printed scaffolds. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

INTRODUCTION. In the treatment of nonunions, and other complications of bone repair, an attractive alternative to bone autografts would be the use of a combination of autologous mesenchymal progenitors cells (MSCs), biomaterials and growth factors. Our goal was to determine the therapeutic potential and contribution to the repair process of different sources of mesenchymal stem cells for the treatment of nonunions. METHODS. The right femur of Sprague-Dawley (SD) rats was stabilized with an aluminum plate (20 mm long, 4 mm wide, 2 mm thick) and four screws (1.5 mm diameter, 8 mm long). A diaphyseal critical size defect was performed (5 mm). Six groups (n=6–8 animals each) were created. A nonunion group (Control group, empty defect); LBA group, live bone allograft; BMP2 group, rhBMP-2 (2 μg) in collagen sponge; PCL group, polycaprolactone scaffold; PMSCs group, PCL scaffold loaded with 5×10. 6. periosteum-derived MSCs; and BMSCs group, PCL scaffold loaded with 5×10. 6. bone marrow-derived MSCs. For cell tracking purposes, LBA and MSCs were derived from SD-GFP transgenic rats. The repair process was followed up by x-rays up to sacrifice, week 10. After sacrifice, femurs were analyzed by micro computed tomography (μCT), histology and immunohistochemistry. For multiple comparisons one-way ANOVA followed by Dunnett”s test for single comparisons was used. Statistical significance was established for p<0.05. RESULTS. Control group did not show healing during follow up or by μCT and histological analysis. Treatment groups BLA and BMP2 showed full healing by week 10 (LBA, 6 out of 6 animals; BMP2, 4 out of 6 animals). The repair callus was quantified by mCT, Control group showed limited formation of bone (11.47±2.01 mm. 3. ) while both LBA and BMP2 groups showed increased bone formation by week 10 when compared with control group (LBA, 35.36±2.24 mm. 3. , p=0.0022; BMP2, 33.32±1.84 mm. 3. , p=0.0022). Histological and μCT analysis confirmed the experimental nonunion model. In PCL treated groups a low number of animals showed radiographic healing: PCL group 1 out of 8 animals; PMSCs group, 2 out of 6 animals; BMSCs group, 0 out of 6 animals. Interestingly, quantification of the repaired callus showed that only PMSCs group produced a significant volume of bone when compared with the Control group (PMSCs, 24.97±6.03 mm. 3. , p=0.0411). PCL and BMSCs groups do not produced significant amount of bone in the repair callus (PCL, 19.00±4.25 mm. 3. , p=0.3095; BMSCs, 12.88±2.38 mm. 3. , p=0.9372). Healing was confirmed by histology and μCT analysis. Finally, the engraftment of transplanted cells was analysed by immunohistochemistry (anti-GFP antibody). Of the three groups receiving cells only the LBA group showed positive signal for GFP at week 10-post surgery. CONCLUSIONS. In conclusion, periosteum-derived progenitor cells are suitable for mimetic autograft design although integration is not yet achieved

Extensive annulus fibrosus (AF) radial tears lead to intervertebral disc (IVD) herniation. While unrepaired defects in the AF are associated with postoperative reherniation and high IVD degeneration prevalence, current surgical strategies are limited to symptomatic treatment of pain and disregard the structural integrity of the AF. For all these reasons, this study is focused on i) designing polycaprolactone (PCL) electrospun implants that mimic the multi-lamellar fibrous structure of the native tissue and ii) assessing their ability to properly close and repair an AF defect in a sheep in vivo model. Oriented PCL mats were produced by electrospinning with average fiber diameters of 1.3µm and a tensile modulus (55±1MPa) matching the one of a native human AF lamella (∼47MPa). In vitro experiments demonstrated a spontaneous colonization of PCL mats by human and ovine AF cells. In vivo study was carried out on 6 sheep in which 5 lumbar discs were exposed using a left retroperitoneal approach. Defects (2×5mm, 2mm depth) were created in the outer annulus, with randomized distribution of conditions including 10-layer oriented or non-oriented mats, untreated and healthy groups. X-ray and MRI examinations were performed every month until explantations at 1, 3 and 6 months, followed by immuno-histological analysis. Data showed no dislocation of the implants, cell infiltration between the PCL mats and within the mats, and a continuous type I collagen tissue formation between the implants and the surrounding AF tissue. These results highlight that multi-layer PCL electrospun mat is a promising biomaterial for AF repair

Summary. Properties of human amniotic membrane are particularly interesting. To use it as an Advanced Therapeutic Medicinal Product in bone surgery, we are evaluating its association with a potentially osteoinductive scaffold. Introduction. The human Amniotic Membrane (hAM) is known to have a good potential to help the regeneration of tissues. It has been used for 100 years in many medical disciplines because of its properties: a membrane containing stem cells and growth factors, with low immunogenicity and anti-microbial, anti-inflammatory, anti-fibrotic and analgesic properties. Moreover, previous published data showed the possibility of in vitro osteodifferenciation of the whole tissue. We aim to use hAM as an Advanced Therapeutic Medicinal Product for bone repair to treat large defects or pseudarthrosis. So we are studying the association of hAM with nanofiber jet sprayed polycaprolactone (PCL) scaffolds and the possibility to induce its osteodifferenciation. Materials and Methods. HAM from cesarean delivery were provided by a local bank of tissue. A biodegradable microfiber PCL scaffold (∼500 μm thick) was produced using a novel jet spraying technique and provided by Biomedical Tissues society (Nantes, France). We cultured hAM in contact with PCL scaffolds either in MSCs expansion medium or in MSCs osteogenic medium. Then we grafted these montages in an ectopic murin model (in subcutaneous implantation) and we explanted grafted tissues after 1, 2, 4 and 8 weeks. Osteogenic potential was evaluated by immunological studies immediately after in vitro cell studies and after explantations from the mice. Control studies were performed with human mesenchymal stromal cells (hMSCs) seeded on PCL scaffold in MSCs osteogenic medium and cultured with/ without dynamic culture, via an orbital rotator at 150 rpm for 28 days. Cell viability, proliferation and osteoblastic differentiation were evaluated at different times of culture until 28 days. Results. To date, in vitro studies showed a macroscopic mineralization of hAM cultured in MSCs osteogenic medium. Osteogenic potential evaluated by immunological investigations are currently underway. Control studies showed that:. - PCL jet sprayed scaffolds supported proliferation and maintained viability of hMSCs. - Scanning electron microscopy analysis and confocal imaging showed cell attachment with a spread cell morphology after just 1.5 hours. Cells attached along the PCL nanofibres.?. - Entire scaffold depth was infiltrated with cells at days 7 and 28, as seen by DAPI and hematoxylin and eosin staining. - Minimal collagen deposition was evident after 7 days but was observed in significant amounts after 28 days. Static conditions had the greatest collagen matrix production. - Alkaline phosphatse gene expression increased in dynamic conditions compared to 2D cultures. Osteocalcin gene expression increased on PCL scaffolds compared to 2D plastic. - Dynamic loading did not appear necessary for proliferation, infiltration or collagen production. Discussion. The association of the hAM with an osteoinductive scaffold could have 2 benefits:. - The handling of the hAM. - The osteodifferentiation of the hAM without the employment of chemical products (osteoinductive supplements that would need to be use with a “good manufacturing products” validation). These preliminary data showed that PCL scaffolds presented osteoinduction properties. The association of hAM with PCL scaffolds seems to be a good compromise answering both our needs

Summary. A promising approach to stimulate in vivo bone formation by using our newly developed magnesium-based bone substitutes, which can be an alternative to treat the patients with bone loss in addition to the anticatabolic drugs and growth factors. Introduction. Bone impairment arising from osteoporosis as well as other pathological diseases is a major health problem. Anti-catabolic drugs such as bisphosphonates and other biological agents such as bone morphogenetic proteins and insulin-like growth factor can theoretically apply to stimulate bone formation. However, the formation of more brittle bone and uncontrolled release rate are still a challenge nowadays. Hence, we propose to stimulate bone formation by using a newly developed magnesium-based bone substitute. Indeed, the presence of magnesium ions can stimulate bone growth and healing by enhancing osteoblastic activity. This study aims to investigate the mechanical, in vitro and in vivo properties of this novel bone substitute. Methods. The bone substitutes were prepared by incorporating 9% TMSPM-treated Mg granules (i.e. 45μm & 150μm) into biodegradable polymer, polycaprolactone (PCL). The TMSPM silane-coupling agent treatment was used to protect the Mg particles from rapid degradation. Compression test was performed to study the mechanical properties of the bone substitute by using the MTS machine. A 7-day stimulated body fluid (SBF) immersion test was conducted to test their bioactivity. The surface composition was checked by energy dispersive x-ray spectroscopy (EDX) after immersion. The cytocompatibility and osteogenic differentiation properties of the bone substitutes were studied by MTT, ALP assays and qRT-PCR with the use of MC3T3-E1 pre-osteoblasts. Finally, the in vivo response of the bone substitutes was evaluated by using rat model of 2 months. Micro-CT was used to monitor the volume change of bone formation. Pure PCL was used as the control. Results. At least 36% higher compressive modulus was found on the new bone substitutes as compared to pure PCL. Calcium and phosphate deposition were detected on the Mg bone substitutes but not on pure PCL after 7-day SBF immersion. Significantly higher cell viabilities and specific ALP activities were found on the new bone substitutes as compared to pure PCL. Additionally, significantly higher ALP, Type I collagen, osteopontin and Runx2 expressions were found on the Mg-based substitutes at different time points. Finally, more than 15% new bone was found on the Mg bone substitutes after 1 week of post-operation and 40% higher after 3 weeks. Discussion/Conclusion. The increased compressive moduli of the Mg-based bone substitutes suggested that the mechanical property of PCL could be enhanced by incorporating Mg granules and the values fall within the range of cancellous bone (50 – 800 MPa). Moreover, the detection of the calcium and phosphate on the bone substitutes showed that they might possess osteoinductivity. The in vitro study showed the enhanced cytocompatibility and osteogenic differentiation properties of the new bone substitutes, which was possibly due to the effect of Mg ions release. Our previous study showed that only a low level of Mg ions (i.e. 50ppm) is able to stimulate the growth and differentiation of osteoblasts. Hence, this suggested the importance of controlling the release of Mg ions. This also explained why more new bone formation was found on the new bone substitutes than pure PCL during animal implantation. Hence, all the data presented here suggested our new bone substitutes maybe a potential candidate to stimulate new bone formation

Introduction and Objective. In the elderly population, chronic rotator cuff tears are often associated with high re-rupture rates after surgical tendon refixation. Implant materials, especially in combination with additives are supposed to positively influence healing outcome. Furthermore, adequate mechanical properties are crucial. In order to realize degradable implants with high specific surface area, polycaprolactone (PCL) was chosen as basic material and processed by electrospinning to achieve a high surface area for growth factor implementation and subsequent cell attachment. Materials and Methods. PCL (M. n. approx. 80,000 g/mol) was used to generate fibre mats by electrospinning (relative collector velocity 8 m/s; flow rate of 4 ml/h). Mechanical analysis was performed according to EN ISO 527–2:2012 with test specimen 1BA (5 mm in diameter). Maximum force at failure (Fmax) as well as stiffness were evaluated. For preclinical in vivo testing, a coating with CS-g-PCL was performed to increase cellular adhesion and biological integration. Native and TGF-ß3 loaded mats were examined in a chronic rat tendon defect model with dissection of the M. infraspinatus, four week latency and following refixation at the humerus with different PCL-fibre mats (approval Nr. 33.12–42502–04–15/2015). After 8 weeks, rats were finalized and tendon-bone insertions were analyzed biomechanically and via histological methods. Results. Electrospun PCL-fibre mats (n = 6) showed maximum forces of 2.19 ± 0.8 N and a stiffness of 0.38 ± 0.12 N/mm. Native rat infraspinatus tendons showed Fmax values of 28.4 ± 7.2 N and a stiffness of 11.8 ± 4.9 N/mm. After implantation, Fmax of the implant-tendon-regenerate was significantly lower in CS-g-PCL - fibre mat groups compared to native control tendons (mean 52 % of native tendon value). Functionalization with TGF-ß3 led to increased Fmax (78 % of the native tendon value). However, differences were not statistically significant. Histological evaluation revealed no differences between non loaded and TGF-ß3 loaded mats. The implants were strongly disintegrated. Granulation tissue and a high number of foreign body giant cells were present. Conclusions. Although mechanical properties of fabricated mats were low, loading of the fibre mats influenced the biomechanical outcome of refixed tendons, presumably due to their high potential for binding biological active substances like TGF-ß3. However, in ongoing studies these cell reactions, especially regarding polarization of macrophages and foreign body cells need to be characterized. This research project has been supported by the German Research Foundation “Graded Implants FOR 2180 – tendon- and bone junctions” WE 4262/6-2 and parts were published in J Tissue Eng Regen Med. 2020 Jan;14(1):186–197. doi: 10.1002/term.2985

More than 250,000 people are suffering from Anterior Cruciate Ligament (ACL) related injuries each year in the US, with a cost of $17–25K/patient. There is an unmet clinical demand for improving grafts/scaffolds to provide biological integration in addition to mechanical support. Currently, no data is available for the utilization of fibrous scaffolds with bimodal distribution for ACL regeneration. The novelty in this study is that it proposes for the first time to investigate the collagen fibril diameter distribution in healthy and injured bovine ACL tissue, and utilization of such structure for scaffold design. Objectives are 1) developing a bovine ACL tear model and measuring the collagen fibril diameter distribution of both healthy and injured ACL tissues, and 2) fabricating scaffolds to mimic the structural properties of healthy and injured ACL tissue. Bovine ACL tissues (1–3 years old) were harvested and characterized for their fibril diameter distribution using Transmission Electron Microscopy (TEM) and biomechanical properties under tension. The electrospun polycaprolactone (PCL) scaffolds were characterized using SEM and mechanical testing. Healthy and injured ACL fibril diameter, and that of PCL scaffolds representing healthy and injured ACL are compared using unpaired student t-test. The proposed fibrous scaffold design represents a significant departure from the conventional unimodal approach, and is expected to have significant contribution to ACL regeneration. These discoveries will serve as the foundation for the development of biomimetic tissue engineering substrates aimed at promoting biological graft fixation

Abstract. 3D printing of synthetic scaffolds mimicking natural bone chemical composition, structure, and mechanical properties is a promising approach for repairing bone injuries. Direct ink writing (DIW), a type of 3D printing, confers compatibility with a wide range of materials without exposing these materials to extreme heat. Optimizing ink properties such as filament formation capabilities, shear-thinning, and high storage modulus recovery would improve DIW fabrication characteristics. In this study, composite inks based on biodegradable polycaprolactone (PCL), reinforced with nano-hydroxyapatite (HAp), and loaded with vancomycin were designed and evaluated for their rheological properties, wettability, mechanical properties, and antimicrobial properties. The formulated composite inks displayed a shear-thinning behaviour exhibited storage modulus recovery percentages above 80% for all formulations, which is essential for extrusion deposition by DIW at room temperature. Ink formulations were able to form fully interconnected lattice scaffolds with porosities ranging from 42% to 65%. Increasing the HAp concentrations from 55% to 85% w/w increased the shear thinning behaviour and reduced the printed filament width to more closely match the nozzle diameter; this indicates higher HAp proportion reduces ink shrinkage. The scaffold had high wettability at HAp proportions above 65% w/w and the compressive elastic modulus of DIW printed scaffolds exhibited within the range of trabecular bone. Antimicrobial activity was apparent from the agar diffusion assay; zones of inhibition ranging from 15.82 ± 0.25 mm and 20.06 ± 0.25 mm were observed after 24 hr for composite scaffolds loaded with 3% and 9% w/w vancomycin respectively. Vancomycin-loaded PCL/HAp composite inks were developed, displaying good printability, wettability, mechanical properties, and antimicrobial properties, making them an attractive choice for bone repair and regeneration. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project