MgCa-Based Alloys Modified with Zn- and Ga-Doped CaP Coatings Lead to Controlled Degradation and Enhanced Bone Formation in a Sheep Cranium Defect Model.

Mg-based biodegradable metallic implants are gaining increased attraction for applications in orthopedics and dentistry. However, their current applications are hampered by their high rate of corrosion, degradation, and rapid release of ions and gas bubbles into the physiological medium. The aim of the present study is to investigate the osteogenic and angiogenic potential of coated Mg-based implants in a sheep cranial defect model. Although their osteogenic potential was studied to some extent, their potential to regenerate vascularized bone formation was not studied in detail. We have studied the potential of magnesium-calcium (MgCa)-based alloys modified with zinc (Zn)- or gallium (Ga)-doped calcium phosphate (CaP) coatings as a strategy to control their degradation rate while enhancing bone regeneration capacity. MgCa and its implants with CaP coatings (MgCa/CaP) as undoped or as doped with Zn or Ga (MgCa/CaP + Zn and MgCa/CaP + Ga, respectively) were implanted in bone defects created in the sheep cranium. MgCa implants degraded faster than the others at 4 weeks postop and the weight loss was ca. 50%, while it was ca. 15% for MgCa/CaP and <10% in the presence of Zn and Ga with CaP coating. Scanning electron microscopy (SEM) analysis of the implant surfaces also revealed that the MgCa implants had the largest degree of structural breakdown of all the groups. Radiological evaluation revealed that surface modification with CaP to the MgCa implants induced better bone regeneration within the defects as well as the enhancement of bone-implant surface integration. Bone volume (%) within the defect was ca. 25% in the case of MgCa/CaP + Ga, while it was around 15% for undoped MgCa group upon micro-CT evaluation. This >1.5-fold increase in bone regeneration for MgCa/CaP + Ga implant was also observed in the histopathological examination of the H&E- and Masson's trichrome-stained sections. Immunohistochemical analysis of the bone regeneration (antiosteopontin) and neovascularization (anti-CD31) at the defect sites revealed >2-fold increase in the expression of the markers in both Ga- and Zn-doped, CaP-coated implants. Zn-doped implants further presented low inflammatory reaction, notable bone regeneration, and neovascularization among all the implant groups. These findings indicated that Ga- and Zn-doped CaP coating is an important strategy to control the degradation rate as well as to achieve enhanced bone regeneration capacity of the implants made of Mg-based alloys.

Zn doped CaP coatings used for controlling the degradation rate of MgCa1 alloy: In vitro anticorrosive properties, sterilization and bacteria/cell-material interactions.

The purpose of this study was to investigate the effect of Zn doped CaP coatings prepared by micro-arc oxidation method, as a possible approach to control MgCa1 alloy degradation. All the prepared coatings comprised a calcium deficient CaP phase. The control in this evaluation was performed with undoped CaP coating in SBF solution at body temperature (37 ± 0.5°C). The investigation involved determination of microchemical, mechanical, morphological, properties along with anticorrosive, cytocompatibility and antibacterial efficacy. The effect of sterilization process on the properties of the surfaces was also investigated. The results showed that the addition of Zn into CaP increased the corrosion resistance of MgCa1 alloy. Moreover, the adhesion strength of the coatings to MgCa1 alloy was enhanced by Zn addition. In cytotoxicity testing of the samples, extracts of the samples in MEM were incubated with L929 cells and malformation, degeneration and lysis of the cells were examined microscopically after 72 h. The results showed that all samples were cytocompatible. The degradation of MgCa1 alloy in the simulated body fluids (SBF) or DMEM was decreased by coating with CaP. Moreover, the degradation rate of CaP was further decreased by adding a small amount of Zn into the CaP matrix. The samples having CaP coatings and Zn doped CaP coating demonstrated antibacterial efficacy against E.coli. As a result, coating of magnesium alloy with Zn-doped CaP decreased the degradation rate, increased the corrosion resistance, cytocompatibility and the antibacterial effects of the alloys.

LOCAL-IGF-1 and GH application IMPROVES germ cell histology, spermatogenesis and fertility after experimental testicular torsion and detorsion.

OBJECTIVE: To evaluate the impact of insulin like growth factor-1(IGF-1) and growth hormone (GH) on testis histology, spermatogenesis, and fertility in prepubertal rats exposed to 6 h of testicular torsion (TT) and detorsion. MATERIAL-METHOD: Forty-eight male Wistar-albino rats weighing 30-70g and at 3-week age were allocated into six groups involving eight rats in each group as follows: Group 1:Sham, Group 2:Control, Group 3:Gelatin, Group 4:Local-IGF-1, 5: Local-GH, Group 6: Systemic-GH. Right testis was only exposed and sutured in the sham group, and right testes were rotated clockwise, 720°, fixed, and 6 h later, detorsion on the testis was done in groups 2-6. Unloaded gelatin, 5 µg local-IGF-1 loaded, and 2IU rhGH loaded gelatin were sutured to the right testis after detorsion in groups 3-5. In Group 6, 0.3IU/100gr/d rhGH was given for seven days via subcuticular route after detorsion. Each of the rats cohabited with two female rats five weeks later. Afterward, both right and left testes were removed. Mean diameter of seminiferous tubules (STD), mean biopsy score count of the testis (TBSC), mean percentage of haploid cells (HCP) were assessed, and fertility parameters were evaluated. RESULTS: STD and TBSC of the ipsilateral testes were significantly reduced in control and gelatin groups when compared to sham, local-IGF-1, and local-GH groups. STD and TBSC of the ipsilateral testes of the systemic-GH group were decreased compared to the sham group. HCP of the ipsilateral testes of control, gelatin, and systemic-GH groups were significantly lower than the sham, local-IGF-1, and local-GH groups. STD, TBSC, and HCP of the contralateral testes were significantly reduced in control and gelatin groups when compared separately to sham, local-IGF-1, systemic- GH, and local-GH groups. The difference between groups regarding potency, fertility, fecundity indexes, and mean fetus numbers were not significant. CONCLUSION: Even though there was significant and permanent histologic germ cell damage and reduced HCP in both ipsilateral and contralateral testes, experimental 6 h TT and detorsion in prepubertal rats did not have a negative impact on future fertility. Local-IGF-1and rhGH treatment improved germ cell histology and spermatogenesis in both ipsilateral and contralateral testes of prepubertal rats, subjected to 6 h of TT and detorsion.

Potential of pectin for biomedical applications: a comprehensive review.

Pectin is a polysaccharide extracted from various plants, such as apples, oranges, lemons, and it possesses some beneficial effects on human health, including being hypoglycemic and hypocholesterolemic. Therefore, pectin is used in various pharmaceutical and biomedical applications. Meanwhile, its low mechanical strength and fast degradation rate limit its usage as drug delivery devices and tissue engineering scaffolds. To enhance these properties, it can be modified or combined with other organic molecules or polymers and/or inorganic compounds. These materials can be prepared as nano sized drug carriers in the form of spheres, capsules, hydrogels, self assamled micelles, etc., for treatment purposes (mostly cancer). Different composites or blends of pectin can also be produced as membranes, sponges, hydrogels, or 3D printed matrices for tissue regeneration applications. This review is concentrated on the properties of pectin based materials and focus especially on the utilization of these materials as drug carriers and tissue engineering scaffolds, including 3D printed and 3D bioprinted systems covering the studies in the last decade and especially in the last 5 years.

pH responsive release of curcumin from photocrosslinked pectin/gelatin hydrogel wound dressings.

The aim of this study was to develop hydrogel wound dressings made of photocrosslinkable pectin and gelatin with pH dependent release of curcumin, an antimicrobial agent. Methacrylated forms of pectin and gelatin (PeMA and GelMA, respectively) were synthesized, and hydrogels were prepared with different compositions (1:1, 1:2 and 1:3 v/v ratios of PeMA and GelMA) by UV exposure. Pure GelMA was used as control group. Average pore diameter of hydrogels with the highest PeMA content (P1:G1) was 43 µm. All hydrogels showed about 90% swelling. P1:G3 demonstrated the highest stability (retained about 37% of their initial weight after 21 days incubation in PBS), a reasonable compressive modulus (ca. 22 kPa), oxygen permeability (7.44 mg/mL) and preventing ability for bacterial penetration. Therefore, P1:G3 hydrogels were chosen and loaded with curcumin for further studies. In aqueous medium (10 mM PBS, pH 7.4), about 4 times faster release of curcumin was observed than that in medium with pH 5.0. Since infected wounds have alkaline pH compared to healthy tissue, faster release at basic medium is preferable for wound grafts. Disk diffusion tests proved antibacterial efficacy of the hydrogels against S. aureus and E. coli. Live/Dead and Alamar blue assays conducted with L929 fibroblasts showed cytocompatibility of the hydrogels. It was concluded that curcumin loaded P1:G3 hydrogels are promising candidates as wound dressing materials to be further tested in the treatment of infected and chronic wounds.

Lithocholic acid conjugated mPEG-b-PCL micelles for pH responsive delivery to breast cancer cells.

In this study, micelles composed of methoxy poly(ethylene glycol)-block-poly(ε-caprolactone) copolymer (mPEG-b-PCL), which has ionically conjugated lithocholic acid (LCA) and providing pH sensitive release of LCA in acidic media, were prepared as drug carrier devices for cancer therapy. Micelles were produced by co-solvent evaporation method at two different temperatures (60 °C and 25 °C) and coded as LCA60**M and LCA25**M, respectively). Hydrodynamic diameters were 86.9 nm and 228.2 nm, and zeta potentials were -7.54 mV and -18.83 mV for LCA60**M and LCA25**M, respectively. For all micelles, release of LCA was higher in acidic media (pH 5.0) compared to physiological media (pH 7.4). Micelles loaded with a fluorescent dye, coumarin 6, demonstrated effective internalization into triple negative MDA-MB-231 breast cancer cells in 4 h. LCA60**M (41.7 ± 1.5%) and LCA25**M (44.5 ± 2.2%) had higher inhibitory effect on the cell migration compared to free LCA (64.7 ± 1.3%). Both LCA conjugated micelles decreased lipogenic activity and increased expressions of Bax (1.3 fold) and p53 (1.2 fold) apoptotic genes. Annexin V-FITC results exhibited high apoptotic cell number after the treatment of MDA-MB-231 cells with micelles. Free LCA and LCA conjugated LCA60**M and LCA25**M micelles decreased mitochondrial transmembrane potential of the cells by 41.8 ± 3.0%, 30.4 ± 0.9%, and 57.1 ± 0.5, respectively. Micelles also caused an effective decrease in angiogenesis ability of HUVECs. The novelty of this study is the prepared micelles, which have ionic conjugation of LCA to mPEG-b-PCL, and pH responsive release of LCA demonstrating effective apoptosis on breast cancer cells. These micelles may have great potential for cancer treatment. However, further in vivo studies are needed before clinical translation.

Corrosion Resistance and Cytocompatibility of Magnesium-Calcium Alloys Modified with Zinc- or Gallium-Doped Calcium Phosphate Coatings.

In orthopedic surgery, metals are preferred to support or treat damaged bones due to their high mechanical strength. However, the necessity for a second surgery for implant removal after healing creates problems. Therefore, biodegradable metals, especially magnesium (Mg), gained importance, although their extreme susceptibility to galvanic corrosion limits their applications. The focus of this study was to control the corrosion of Mg and enhance its biocompatibility. For this purpose, surfaces of magnesium-calcium (MgCa1) alloys were modified with calcium phosphate (CaP) or CaP doped with zinc (Zn) or gallium (Ga) via microarc oxidation. The effects of surface modifications on physical, chemical, and mechanical properties and corrosion resistance of the alloys were studied using surface profilometry, goniometry, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), nanoindentation, and electrochemical impedance spectroscopy (EIS). The coating thickness was about 5-8 µm, with grain sizes of 43.1 nm for CaP coating and 28.2 and 58.1 nm for Zn- and Ga-doped coatings, respectively. According to EIS measurements, the capacitive response (Yc) decreased from 11.29 to 8.72 and 0.15 Ω-1 cm-2 sn upon doping with Zn and Ga, respectively. The Ecorr value, which was -1933 mV for CaP-coated samples, was found significantly electropositive at -275 mV for Ga-doped ones. All samples were cytocompatible according to indirect tests. In vitro culture with Saos-2 cells led to changes in the surface compositions of the alloys. The numbers of cells attached to the Zn-doped (2.6 × 104 cells/cm2) and Ga-doped (6.3 × 104 cells/cm2) coatings were higher than that on the surface of the undoped coating (1.0 × 103 cells/cm2). Decreased corrosivity and enhanced cell affinity of the modified MgCa alloys (CaP coated and Zn and Ga doped, with Ga-doped ones having the greatest positive effect) make them novel and promising candidates as biodegradable metallic implant materials for the treatment of bone damages and other orthopedic applications.

Enhancing esophageal repair with bioactive bilayer mesh containing FGF.

We aimed to prepare a bioactive and biodegradable bilayer mesh formed by fibroblast growth factor (FGF) loaded gelatin film layer, and poly ε-caprolactone (PCL) film layer, and to investigate its treatment efficacy on esophageal anastomosis. It is envisaged that the bioactive mesh in in vivo model would improve tissue healing in rats. The full thickness semicircular defects of 0.5 × 0.5 cm2 were created in anterior walls of abdominal esophagus. The control group had abdominal esophagus isolated with distal esophageal blunt dissection, and sham group had primary anastomosis. In the test groups, the defects were covered with bilayer polymeric meshes containing FGF (5 µg/2 cm2), or not. All rats were sacrificed for histopathology investigation after 7 or 28 days of operation. The groups are coded as FGF(-)-7th day, FGF(+)-7th day, and FGF(+)-28th day, based on their content and operation day. Highest burst pressures were obtained for FGF(+)-7th day, and FGF(+)-28th day groups (p < 0.005) and decreased inflammation grades were observed. Submucosal and muscular collagen deposition scores were markedly increased in these groups compared to sham and FGF(-)-7th day groups having no FGF (p = 0.002, p = 0.001, respectively). It was proved that FGF loaded bioactive bilayer mesh provided effective repair, reinforcement and tissue healing of esophageal defects.

3D printed hybrid bone constructs of PCL and dental pulp stem cells loaded GelMA.

Fabrication of scaffolds using polymers and then cell seeding is a routine protocol of tissue engineering applications. Synthetic polymers have adequate mechanical properties to substitute for some bone tissue, but they are generally hydrophobic and have no specific cell recognition sites, which leads to poor cell affinity and adhesion. Some natural polymers, have high cell affinity but are mechanically weak and do not have the strength required as a bone supporting material. In the present study, 3D printed hybrid scaffolds were fabricated using PCL and GelMA carrying dental pulp stem cells (DPSCs), which is printed in the gaps between the PCL struts. This cell loaded GelMA was shown to support osteoinductivity, while the PCL provided mechanical strength needed to mimic the bone tissue. 3D printed PCL/GelMA and GelMA scaffolds were highly stable during 21 days of incubation in PBS. The compressive moduli of the hybrid scaffolds were in the range of the compressive moduli of trabecular bone. DPSCs were homogeneously distributed throughout the entire hydrogel component and exhibited high cell viability in both scaffolds during 21 days of incubation. Upon osteogenic differentiation DPSCs expressed two key matrix proteins, osteopontin and osteocalcin. Alizarin red staining showed mineralized nodules, which demonstrates osteogenic differentiation of DPSCs within GelMA. This construct yielded a very high cell viability, osteogenic differentiation and mineralization comparable to cell culture without compromising mechanical strength suitable for bone tissue engineering applications. Thus, 3D printed, cell loaded PCL/GelMA hybrid scaffolds have a great potential for use in bone tissue engineering applications.

Bioinks-materials used in printing cells in designed 3D forms.

Use of materials to activate non-functional or damaged organs and tissues goes back to early ages. The first materials used for this purpose were metals, and in time, novel materials such as ceramics, polymers and composites were introduced to the field to serve in medical applications. In the last decade, the advances in material sciences, cell biology, technology and engineering made 3D printing of living tissues or organ models in the designed structure and geometry possible by using cells alone or together with hydrogels through additive manufacturing. This review aims to give a brief information about the chemical structures and properties of bioink materials and their applications in the production of 3D tissue constructs.

A two-compartment bone tumor model to investigate interactions between healthy and tumor cells.

We produced a novel three-dimensional (3D) bone tumor model (BTM) to study the interactions between healthy and tumor cells in a tumor microenvironment, the migration tendency of the tumor cells, and the efficacy of an anticancer drug, Doxorubicin, on the cancer cells. The model consisted of two compartments: (a) a healthy bone tissue mimic, made of poly(lactic acid-co-glycolic acid) (PLGA)/beta-tricalcium phosphate (ß-TCP) sponge seeded with human fetal osteoblastic cells (hFOB) and human umbilical vein endothelial cells (HUVECs), and (b) a tumor mimic, made of lyophilized collagen sponge seeded with human osteosarcoma cells (Saos-2). The tumor mimic component was placed into a central cavity created in the healthy bone mimic and together they constituted the complete 3D bone tumor model (3D-BTM). The porosities of both sponges were higher than 85% and the diameters of the pores were 199 ± 52 µm for the PLGA/TCP and 50-150 µm for the collagen scaffolds. The compression Young's modulus of the PLGA/TCP and the collagen sponges were determined to be 4.76 MPa and 140 kPa, respectively. Cell proliferation, morphology, calcium phosphate forming capacity and alkaline phosphatase production were studied separately on both the healthy and tumor mimics. All cells demonstrated cellular extensions and spread well in porous scaffolds indicating good cell-material interactions. Confocal microscopy analysis showed direct contact between the cells present in different parts of the 3D-BTM. Migration of HUVECs from the healthy bone mimic to the tumor compartment was confirmed by the increase in the levels of angiogenic factors vascular endothelial growth factor, basic fibroblast growth factor, and interleukin 8 in the tumor component. Doxorubicin (2.7 µg.ml-1) administered to the 3D-BTM caused a seven-fold decrease in the cell number after 24 h of interaction with the anticancer drug. Caspase-3 enzyme activity assay results demonstrated apoptosis of the osteosarcoma cells. This novel 3D-BTM has a high potential for use in studying the metastatic capabilities of cancer cells, and in determining the effective drug types and combinations for personalized treatments.

3D and 4D Printing of Polymers for Tissue Engineering Applications.

Three-dimensional (3D) and Four-dimensional (4D) printing emerged as the next generation of fabrication techniques, spanning across various research areas, such as engineering, chemistry, biology, computer science, and materials science. Three-dimensional printing enables the fabrication of complex forms with high precision, through a layer-by-layer addition of different materials. Use of intelligent materials which change shape or color, produce an electrical current, become bioactive, or perform an intended function in response to an external stimulus, paves the way for the production of dynamic 3D structures, which is now called 4D printing. 3D and 4D printing techniques have great potential in the production of scaffolds to be applied in tissue engineering, especially in constructing patient specific scaffolds. Furthermore, physical and chemical guidance cues can be printed with these methods to improve the extent and rate of targeted tissue regeneration. This review presents a comprehensive survey of 3D and 4D printing methods, and the advantage of their use in tissue regeneration over other scaffold production approaches.

Biomechanical analysis of a modified suture technique for septal extension grafts: Transloop suture.

BACKGROUND: A successful rhinoplasty procedure requires a well-defined and properly projected nasal tip; however, surgical control of the nasal tip is difficult. The aim of this investigation was to assess the efficacy and safety of a modified suture technique, which can be used to fix the caudal septal extension graft during primary rhinoplasty of the Asian population and revision septorhinoplasties of the Caucasian population, and to compare it with those of other commonly used techniques. METHODS: After peeling of perichondrium of scapular cartilages, cartilage pieces of 3 × 1 cm in size and 2 mm in thickness were divided into two from the midline. These pieces were repaired end-to-end using three different repair techniques: two simple interrupted in Group A (n = 40), vertical figure-of-eight in Group B (n = 40) and modified vertical figure-of-eight (transloop) in Group C (n = 40). All repaired cartilage specimens were subjected to a biomechanical analysis, in which four different forces were applied: tension, lateral bending, shearing and buckling. RESULTS: According to the tensile test, Group C had statistically significantly higher strength than Group A at 2 mm range. The lateral bending test similarly revealed that Group C had statistically significantly higher strength at 1.5 mm and 2 mm range than Group A. However, there was no statistically significant difference between the three groups in the assessment of shearing and buckling forces. CONCLUSION: The modified transloop suture technique provides a more stable repair, and we consider that it can be used as an alternative suture repair method.

Square prism micropillars on poly(methyl methacrylate) surfaces modulate the morphology and differentiation of human dental pulp mesenchymal stem cells.

Use of soluble factors is the most common strategy to induce osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro, but it may raise potential side effects in vivo. The topographies of the substrate surfaces affect cell behavior, and this could be a promising approach to guide stem cell differentiation. Micropillars have been reported to modulate cellular and subcellular shape, and it is particularly interesting to investigate whether these changes in cell morphology can modulate gene expression and lineage commitment without chemical induction. In this study, poly(methyl methacrylate) (PMMA) films were decorated with square prism micropillars with different lateral dimensions (4, 8 and 16 µm), and the surface wettability of the substrates was altered by oxygen plasma treatment. Both, pattern dimensions and hydrophilicity, were found to affect the attachment, proliferation, and most importantly, gene expression of human dental pulp mesenchymal stem cells (DPSCs). Decreasing the pillar width and interpillar spacing of the square prism pillars enhanced cell attachment, cell elongation, and deformation of nuclei, but reduced early proliferation rate. Surfaces with 4 or 8 µm wide pillars/gaps upregulated the expression of early bone-marker genes and mineralization over 28 days of culture. Exposure to oxygen plasma increased wettability and promoted cell attachment and proliferation but delayed osteogenesis. Our findings showed that surface topography and chemistry are very useful tools in controlling cell behavior on substrates and they can also help create better implants. The most important finding is that hydrophobic micropillars on polymeric substrate surfaces can be exploited in inducing osteogenic differentiation of MSCs without any differentiation supplements.

An estradiol releasing, proangiogenic hydrogel as a candidate material for use in soft tissue interposition.

INTRODUCTION AND OBJECTIVES: Soft tissue interposition (STI) using local and/or regional flaps is often necessary in urogenital reconstruction to stimulate wound healing and prevent recurrence. Harvesting STI flaps can cause donor site morbidity and may not be available in some patients. In this study, we designed estradiol (E2) releasing hydrogel that could be used as an alternative to a STI flap and to investigate its ability to stimulate tissue production and angiogenesis. MATERIALS AND METHODS: A hydrogel was constructed by crosslinking a solution of estradiol, methacrylated gelatin (15%, w/v), and methacrylated hyaluronic acid (1%, w/v). The release of estradiol was measured using a UV-spectrophotometer (λmax = 220 nm). Angiogenesis was evaluated by an ex ovo chicken embryo chorioallantoic membrane (CAM) assay. RESULTS: Estradiol was gradually released from the hydrogel over 21 days. The hydrogels could be easily manipulated with surgical forceps without any deformation. The hydrogels significantly increased collagen production of human dermal fibroblasts (HDFs). Scanning electron microscopic examination demonstrated that HDFs produced significantly more extracellular matrix (ECM) on estradiol releasing hydrogels compared with the controls. Estradiol releasing hydrogels doubled the number of blood vessels growing toward the hydrogel compared with the controls (vasculogenic index, 59.6 [±6.4] and 25.6 [±4.0], respectively; [P < 0.05]). CONCLUSION: We present a proangiogenic, degradable hydrogel that can be used as an off-the-shelf available substitute to traditional STI flaps. This is achieved by using estradiol as a potent stimulator of new tissue production and new blood vessel formation.

Hydrogels of agarose, and methacrylated gelatin and hyaluronic acid are more supportive for in vitro meniscus regeneration than three dimensional printed polycaprolactone scaffolds.

In this study, porcine fibrochondrocyte-seeded agarose, methacrylated gelatin (GelMA), methacrylated hyaluronic acid (MeHA) and GelMA-MeHA blend hydrogels, and 3D printed PCL scaffolds were tested under dynamic compression for potential meniscal regeneration in vitro. Cell-carrying hydrogels produced higher levels of extracellular matrix (ECM) components after a 35-day incubation than the 3D printed PCL. Cells on GelMA exhibited strong cell adhesion (evidenced with intense paxillin staining) and dendritic cell morphology, and produced an order of magnitude higher level of collagen (p < 0.05) than other materials. On the other hand, cells in agarose exhibited low cell adhesion and round cell morphology, and produced higher levels of glycosaminoglycans (GAGs) (p < 0.05) than other materials. A low level of ECM production and a high level of cell proliferation were observed on the 3D printed PCL. Dynamic compression at 10% strain enhanced GAG production in agarose (p < 0.05), and collagen production in GelMA. These results show that hydrogels have a higher potential for meniscal regeneration than the 3D printed PCL, and depending on the material used, fibrochondrocytes could be directed to proliferate or produce cartilaginous or fibrocartilaginous ECM. Agarose and MeHA could be used for the regeneration of the inner region of meniscus, while GelMA for the outer region.

Cell behavior on the alginate-coated PLLA/PLGA scaffolds.

Here, we investigated the effect of preparation temperature and alginate-coating on L929 fibroblast behavior on lyophilized microporous PLLA/PLGA (95:5, w/w) scaffolds. The lower freezing temperature used during lyophilization (-80 °C) resulted in smaller pores (around 50 µm) and higher compressive modulus (1500 kPa) than those prepared at the higher temperature (-20 °C) (pore size: 120 µm, compressive modulus: 600 kPa) (p < 0.01). Cell proliferation was significantly lower on the alginate-coated scaffolds (p < 0.05), probably due to weak cell adhesion on alginate, rapid degradation/dissolution of the alginate hydrogel (40% weight loss after 2 weeks of incubation) (p < 0.05), which resulted in loss of material and cells, and the decrease in the pH (p < 0.05), which probably resulted in decreased cell metabolic activity. Cells tended to get less round on the scaffolds prepared at -20 °C, which had lower compressive modulus and larger pores, and upon coating with alginate, which resulted in a hydrophilic surface that had lower stiffness. When the scaffolds had closer stiffness to the cells, the cells tended to get more branched. The most branched morphology of the fibroblasts was obtained in the presence of alginate, a natural polymer having a similar stiffness with that of the L929 fibroblasts (4 kPa).

Preparation and characterization of poly(ε-caprolactone) scaffolds modified with cell-loaded fibrin gel.

Poly(ε-caprolactone) (PCL) is one of the most commonly used polymers in the production of tissue engineered scaffolds for hard tissue treatments. Incorporation of cells into these scaffolds significantly enhances the healing rate of the tissue. In this study, PCL scaffolds were prepared by wet spinning technique and modified by addition of fibrinogen in order to form a fibrin network between the PCL fibers. By this way, scaffolds would have micro- and nanofibers in their structures. Drying of the wet spun constructs was achieved by application of ethanol dehydration or freeze drying techniques. Fibrinogen solutions (as low: 2 mg/mL; or high: 10 mg/mL concentrations) were added onto the scaffolds and fibrin formation was achieved via fibrinogen crosslinking. Results showed that ethanol dehydration led to film-like coating on the fibers while freeze-drying led to nanofiber bridges between PCL fibers establishing an interconnected web in the structure. Mechanical properties of the scaffolds were improved in the presence of the fibrin net. After the seeding of Saos-2 cells, higher attachment and homogeneous distribution of the cells was achieved on the samples modified with high concentration of fibrinogen. These scaffolds can be good candidates for the treatment of problematic bone defects.

A 3D printed PCL/hydrogel construct with zone-specific biochemical composition mimicking that of the meniscus.

Engineering the meniscus is challenging due to its bizonal structure; the tissue is cartilaginous at the inner portion and fibrous at the outer portion. Here, we constructed an artificial meniscus mimicking the biochemical organization of the native tissue by 3D printing a meniscus shaped PCL scaffold and then impregnating it with agarose (Ag) and gelatin methacrylate (GelMA) hydrogels in the inner and outer regions, respectively. After incubating the constructs loaded with porcine fibrochondrocytes for 8 weeks, we demonstrated that presence of Ag enhanced glycosaminoglycan (GAG) production by about 4 fold (p < 0.001), while GelMA enhanced collagen production by about 50 fold (p < 0.001). In order to mimic the physiological loading environment, meniscus shaped PCL/hydrogel constructs were dynamically stimulated at strain levels gradually increasing from the outer region (2% of initial thickness) towards the inner region (10%). Incorporation of hydrogels protected the cells from the mechanical damage caused by dynamic stress. Dynamic stimulation resulted in increased ratio of collagen type II (COL 2) in the Ag-impregnated inner region (from 50% to 60% of total collagen), and increased ratio of collagen type I (COL 1) in the GelMA-impregnated outer region (from 60% to 70%). We were able to engineer a meniscus, which is cartilage-like at the inner portion and fibrocartilage-like at the outer portion. Our construct has a potential for use as a substitute for total meniscus replacement.

A bilayer scaffold prepared from collagen and carboxymethyl cellulose for skin tissue engineering applications.

Treatment of chronic skin wound such as diabetic ulcers, burns, pressure wounds are challenging problems in the medical area. The aim of this study was to design a bilayer skin equivalent mimicking the natural one to be used as a tissue engineered skin graft for use in the treatments of problematic wounds, and also as a model to be used in research related to skin, such as determination of the efficacy of transdermal bioactive agents on skin cells and treatment of acute skin damages that require immediate response. In this study, the top two layers of the skin were mimicked by producing a multilayer construct combining two different porous polymeric scaffolds: as the dermis layer a sodium carboxymethyl cellulose (NaCMC) hydrogel on which fibroblasts were added, and as the epidermis layer collagen (Coll) or chondroitin sulfate-incorporated collagen (CollCS) on which keratinocytes were added. The bilayer construct was designed to allow cross-talk between the two cell populations in the subsequent layers and achieves paracrine signalling. It had interconnected porosity, high water content, appropriate stability and elastic moduli. Expression of vascular endothelial growth factor (VEGF), basic-fibroblast growth factor (bFGF) and Interleukin 8 (IL-8), and the production of collagen I, collagen III, laminin and transglutaminase supported the attachment and proliferation of cells on both layers of the construct. Attachment and proliferation of fibroblasts on NaCMC were lower compared to performance of keratinocyte on collagen where keratinocytes created a dense and a stratified layer similar to epidermis. The resulting constructs succesfully mimicked in vitro the natural skin tissue. They are promising as grafts for use in the treatment of deep wounds and also as models for the study of the efficacy of bioactive agents on the skin.