Table of contents

Human autologous bioengineered skin has been successfully developed and used to treat skin injuries in a growing number of cases. In current clinical studies, the biomaterial used is fabricated via plastic compression of collagen hydrogel to increase the density and stability of the tissue. To further facilitate clinical adoption of bioengineered skin, the fabrication technique needs to be improved in terms of standardization and automation. Here, we present a one-step mixing technique using highly concentrated collagen and human fibroblasts to simplify fabrication of stable dermal equivalents. As controls, we prepared cellularized dermal equivalents with three varying collagen compositions. We found that the dermal equivalents produced using the simplified mixing technique were stable and pliable, showed viable fibroblast distribution throughout the tissue, and were comparable to highly concentrated manually produced collagen gels. Because no subsequent plastic compression of collagen is required in the simplified mixing technique, the fabrication steps and production time for dermal equivalents are consistently reduced. The present study provides a basis for further investigations to optimize the technique, which has significant promise in enabling efficient clinical production of bioengineered skin in the future.

Despite the advancements in bone transplantation operations, inflammation is still a serious problem that threatens human health at the post-implantation period. Conventional antibiotic therapy methods may lead to some side effects such as ototoxicity and nephrotoxicity, especially when applied in high doses. Therefore, local drug delivery systems play a vital role in bone disorders due to the elimination of the disadvantages introduced by conventional methods. In the presented study, it was aimed to develop Vancomycin (VC) and Gentamicin (GC) loaded chitosan-montmorillonite nanoclay composites (CS/MMT) to provide required antibiotic doses to combat post-implantation infection. CS/MMT nanocomposite formation was supplied by microfluidizer homogenization and spherical drug carrier nanoparticles were obtained by electrospraying technique. Three factors; voltage, distance and flowrate were varied to fabricate spherical nanoparticles with uniform size. Emprical model was developed to predict nanosphere size by altering process variables. Nanospheres were characterized in terms of morphology, hydrodynamic size, zeta potential, drug encapsulation efficiency and release profile. Drug loaded nanospheres have been successfully produced with a size range of 180–350 nm. Nanocomposite drug carriers showed high encapsulation efficiency (80%–95%) and prolonged release period when compared to bare chitosan nanospheres. The drug release from nanocomposite carriers was monitored by diffusion mechanism up to 30 d. The in vitro release medium of nanospheres showed strong antimicrobial activity against gram-positive S. aureus and gram-negative E. coli bacteria. Furthermore, it was found that the nanospheres did not show any cytotoxic effect to fibroblast (NIH/3T3) and osteoblast (SaOS-2) cell lines. The results demonstrated that the prepared composite nanospheres can be a promising option for bone infection prevention at the post implantation period.

Tissue grafts achieve high levels of compositional and mechanical integrity biomimicry and are often considered as the gold standard in clinical practice. Herein, we assessed the potential of decellularised porcine peritoneum (XenoMEM) as a tendon protector sheet and correlated its properties to a commercially available product (TenoGlide^®). XenoMEM presented lower cross-linking ratio (p < 0.05), higher mechanical properties (p < 0.01), lower coefficient of friction (p < 0.01) and higher (p < 0.05) cytocompatibility with human tenocytes than TenoGlide^®. In addition, XenoMEM exhibited lower (p < 0.05) immune response than TenoGlide^® with macrophages. Collectively, these data support the use of XenoMEM in tendon tissue engineering.

Hydrogels are normally not robust enough to meet the repairing requirements of bone defects, therefore, cryogels of higher mechanical properties are developed as the more proper candidates for the purpose. In view of the organic–inorganic composition of natural bone tissues, hydroxyapatite (HA) is envisioned as a good additive for protein cryogels to achieve biomimetic compositions, additionally, as an excellent reinforcement to increase the mechanical properties of cryogels. In this study, methacrylated gelatin (GelMA) was synthesized and corresponding 3D-structured cryogel was fabricated, followed by the incorporation of HA nanowires (HANWs) at different amounts as reinforcements. The results showed that the GelMA/HANW composite cryogels possessed highly porous structure with HANWs being homogeneously distributed. The compressive strengths and mechanical stability of the composite cryogels were improved alongside the increasing contents of HANWs. These composite cryogels were proven non-cytotoxic, able to support cell proliferation and promote osteogenic differentiation of bone mesenchymal stromal cells. More importantly, their porous structure allowed cell migration within the matrix, which was normally hard to be achieved in GelMA hydrogel. With improved performance, GelMA/HANW composite cryogels were thus possibly serving as a new type of bone repair materials.

A composite based on poly (propylene fumarate) (PPF) was investigated as a potential bone repair material for clinical use and it showed low heat release, suitable mechanical property and good biocompatibility. The in situ curing process would finish in less than 10 min. Compared with PMMA, PPF/TCP showed great decrease in heat release as the maximum temperature during curing process was 54.7 °C ± 1.69 °C. The compressive strength was between 109 ± 2 and 133 ± 6 MPa and the compressive modulus was 146 ± 11 to 161 ± 27 MPa, which were believed to be compatible and further supportive to surrounding bone. Besides, the surface morphology and hydrophilicity could be tailored by adjusting the content of β-calcium phosphate (β-TCP). Relatively stable pH value during degradation in PBS solution implied that it would not bring about acidification when implanted in vivo. In addition, PPF/TCP would boost mineralization and the apatite-like deposits on surface may advance the integrity of bone and materials. Moreover, the PPF/TCP obviously degraded and new bone formed especially when loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) in vivo. In summary, PPF/TCP composites showed suitable physical and chemical properties as well as good bioactivity and may therefore be a promising material for bone repair.

Biodegradable polymers have been broadly used as agents that can complex with and deliver osteoinductive agents, but osteoinductivity of the polymers themselves has been rarely studied. Here we report the osteoinductivity of poly(4-hydroxy-L-proline ester) (PHPE), a biodegradable cationic polymer with cell penetrating properties. Under physiological conditions, PHPE degrades into trans-4-hydroxy-L-proline (trans-Hyp), a non-coded amino acid with essential functions in collagen fibril formation and fibril stability. Treatment of SaOS-2 osteoblast-like cells and hFOB 1.19 primary osteoblast cells with PHPE promoted earlier collagen nodule formation and mineralization of the extracellular matrix compared to untreated cells, even when mineralization activators were absent in the growth medium. Our results indicate that PHPE is a potential osteoinductive agent in vitro that can favor bone regeneration. Moreover, this osteoinductive property could be partly attributed to the degradation product trans-Hyp, which could recapitulate some, but not all of the osteogenic activity. The primary findings of this study can be summarized as follows: treatment of cells with PHPE led to (1) the induction of COL1A1 expression, collagen synthesis and secretion in osteoblast-like cells, (2) mineralization of the ECM in both SaOS-2 and hFOB 1.19 primary osteoblasts, and (3) induction of BMP2 gene and protein expression in osteoblast-like cells, which can promote mineralization of the ECM and regeneration of the bone tissue. Our results suggest that PHPE is a non-cytotoxic polymer and can be potentially used to overcome collagenopathies such as osteogenesis imperfecta.

The properties of vaterite-containing tricalcium silicate (referred as V-C₃S) bone cement can be significantly affected by the addition of graphene oxide (GO). The composited bone cement can overcome the problems of calcium silicate-based bone cement, such as poor mechanical properties and low cellular activity. The material properties, mineralization and cell compatibility were characterized. The results demonstrated that GO/V-C₃S had good curing ability, mechanical properties and high injectability. In general, V-C₃S adding with 0.04 wt% GO is better due to its advanced compressive strength (17.14% higher than pure V-C₃S) and lower pH value. In vitro immersion experiments could show its admirable mineralization ability. Cell experiments confirmed its low cytotoxicity and favorable ability of cell proliferation. In addition, enhanced antibacterial property (2–3 times higher compared with pure V-C₃S) was also verified by antibacterial experiments. These results suggest that GO/V-C₃S bone cement is a promising material for biomedical applications.

Astrogilosis is the response of astrocytes to brain trauma which manifest opposite roles on brain injury repair. On the one hand, astrocytes undergoing astrogliosis inhibit tissue regeneration by forming scar tissue, but, on the other hand, they enhance damage repair through secretion of neuro-protecting and neurotrophic factors. Therefore, identifying means that regulate astrogliosis can provide a control over progression and repair of brain damage. We have previously shown that the calcium carbonate skeleton of corals upregulates two manifestations of astrogliosis in astrocytes in culture—expression of the Glial Fibrillay Acidic Protein (GFAP), and shape conversion from non-spiky to reactive spiky cell morphology. Here, we investigated if the surface topography of the coralline skeleton plays a role in GFAP expression and the morphogenesis of reactive astrocytes. To address that, we utilized the non-porous exoskeleton of the coral Trachyphyllia geoffroyi, having three topographies of distinct heights on its surface: rough surface (made of <30 μm height bumps), protrusions (50–250 μm) and ridges (>250 μm). We observed that astrocytes reacted similarly to all three structures in terms of adhesion, acquisition of a spiky morphology and organization in networks. By contrast, the extent by which these cells expressed GFAP was structure-dependent. The expression was 2-fold higher on protrusions and ridges than on the rough surface and acquired. Accordingly, the distribution pattern of the GFAP overexpressing astrocytes followed that of the protrusions and ridges. Hence, fabricating coralline scaffolds with designed flatness/protrusions/ridges ratios can serve to control astrogliosis-derived regeneration in TBI wounds, and as a result improve the capacity to repair brain damage.

Chronic wounds are a global health problem, and their treatments are difficult and long lasting. The development of medical devices through tissue engineering has been conducted to heal this type of wound. In this study, it was demonstrated that the combination of natural and synthetic polymers, such as poly (D-L lactide-co-glycolide) (PLGA) and gelatin (Ge), were useful for constructing scaffolds for wound healing. The aim of this study was to evaluate the influence of different PLGA/gelatin ratios (9:1, 7:3 and 5:5 (v/v)) on the physical, chemical and biological properties of electrospun scaffolds for wound dressings. These PLGA/Ge scaffolds had randomly oriented fibers with smooth surfaces and exhibited distances between fibers of less than 10 μm. The 7:3 and 5:5 PLGA/Ge scaffolds showed higher swelling, hydrophilicity and degradation rates than pure PLGA and 9:1 (v/v) PLGA/Ge scaffolds. Young's moduli of the scaffolds were 72 ± 10, 48 ± 6, 58 ± 6 and 6 ± 1 MPa for the pure PLGA scaffold and the 9:1, 7:3 and 5:5 (v/v) PLGA/Ge scaffolds, respectively. Mesenchymal stem cells (MSCs) seeded on all the PLGA/Ge scaffolds were viable, and the cells were attached to the fibers at the different analyzed timepoints. The most significant proliferation rate was observed for cells on the 7:3 PLGA/Ge scaffolds. Biocompatibility analysis showed that all the scaffolds produced inflammation at the first week postimplantation; however, the 7:3 and 5:5 (v/v) PLGA/Ge scaffolds were degraded completely, and there was no inflammatory reaction observed at the fourth week after implantation. In contrast, the 9:1 PLGA/Ge scaffolds persisted in the tissue for more than four weeks; however, at the eighth week, no traces of the scaffolds were found. In conclusion, the scaffolds with the 7:3 PLGA/Ge ratio showed suitable physical, chemical and biological properties for applications in chronic wound treatments.

Radical mediated photochemical thiol-yne click polymerization of thiol-terminated polyurethane prepolymers, with poly(ethylene glycol) soft segment at two different molecular weights, a propargyl terminated urethane crosslinker and silver salt was utilized to prepare versatile wound dressings containing well-dispersed Ag° nanoparticles produced via in situ reduction of Ag⁺ ions. The dressings with optimized chemical structure showed desirable fluid handling capacity (up to 4.84 g/10 cm² d⁻¹) to provide moist environment over damaged tissue. They were permeable to oxygen and carbon dioxide, therefore, the processes related to tissue regeneration of wound bed could be continued without problem. Their appropriate tensile strength (up to 3.87 MPa) and suitable conformability (less than 0.1% permanent set) enabled protection of damaged skin tissue from external physical forces during the healing process, even for wounds present at organs with a high degree of freedom. The proper cytocompatibility of the prepared dressings and their ability to support growth and proliferation of fibroblast cells as determined by wound scratch healing assay showed the potential utility of the dressings to motivate wound healing progression by migration of cells to the damaged area. In addition, these dressings with in situ formed silver nanoparticles exhibited promising antimicrobial activity against different bacterial and fungal strains, and consequently could encourage wound healing process by prevention from infection in the wound site.

Traditional broad-spectrum antibacterial agents are limited by high toxicity and the destruction to the bacterial flora balance in the wound site. Herein, we propose an opinion that one or several especial antibacterial peptides are adopted to kill the target bacteria in order to precisely manage the bacteria infected chronic wound under the premise of biobalance, and specially employ nisin to treat S. aureus infected chronic wound as model with positive effects. The results showed that without cytotoxicity to the normal cells, only 25 ppm nisin could contrapuntally kill S. aureus and have little inhibitory to other bacteria. Mechanism of antibacterial selectivity indicated the superior biomolecular interaction between nisin and S. aureus compared with E. coli and normal cells. Furthermore, nisin significantly accelerated the healing process of S. aureus-infected rabbit full thickness burn wound, but had no effect on the E. coli-infected wound as a comparison. Therefore, it has been demonstrated that special peptides with antibacterial selectivity can be adopted to precise management for bacteria infected chronic wound under good biobalance.

β-TCP bioceramic, as a kind of biocompatible and biodegradable artificial bone scaffolds, is increasingly used to supplement lamina autografts when performing instrumented or non-instrumented spinal fusion, clinically, although solid fusion is not always achieved. The addition of collagen to β-TCP appears to be a potential strategy to improve bone regeneration, thereby enhancing the rate of spinal fusion. This study aimed to compare the fusion in collagen/β-TCP composite, β-TCP and autologous bone in a posterior spinal fusion model. The fusion grade evaluated radiography was greater in the collagen/β-TCP group than in the β-TCP group (p < 0.05). Stiffness and yield strength of the fused segments in collagen/β-TCP group were comparable to that in autogenous bone group. Histological analysis revealed that the proportion of new bone formation in collagen/β-TCP group were significantly greater than in β-TCP group (p < 0.05). In addition, bone deposition rate in the collagen/β-TCP group was greater than in the β-TCP group (p < 0.05) and comparable to that in the autogenous bone group. We therefore concluded that collagen/β-TCP is superior to β-TCP alone in facilitating posterior spinal fusion. The addition of collagen to β-TCP represents a simple strategy that couples the osteogenic effect, providing a promising alternative to autologous bone in the clinical treatment of spinal disorders.

To reflect the rapidly growing interest in producing tissue sealants using various chemical/physical processes, we report an approach using visible light to control the crosslinking of 3D printable hydrogels as in situ tissue sealant. Gelatin-hydroxyphenylpropionic acid conjugate (Gtn-HPA) is shown to crosslink effectively within 30 s under visible light in the presence of [RuII(bpy)₃]²⁺ and sodium persulphate, which is sufficiently rapid for surgery use. Porous structure can be also introduced by including carboxylmethyl cellulose-tyramine (CMC-Tyr) as a precursor. The detailed parameters involved in the hydrogel formation, including irradiation time and distance, are investigated in this study. The results suggested that a longer exposure time would result in a hydrogel with higher crosslinking density, while sufficient photocrosslinking can be achieved using a routine visible light source at a distance of 100 mm. Surface morphology of the photocrosslinked hydrogels are studied using scanning electron microscopy (SEM) and environmental SEM with results confirming the expected porosity. The tensile strength of the photocrosslinked hydrogels has been tested for both non-porous and porous samples. Notably, the adhesive strengths (adhesion) of the photocrosslinked hydrogels was demonstrated to be significantly higher compared to that of commercial fibrin glue. Finally, a prototype of hand-held applicator has been developed and demonstrated to print out Gtn-HPA/CMC-Tyr hydrogel of designed properties with controlled spatial resolution. The development of both material and applicator in this study provides a promising tissue sealant solution for wound closure in future surgical procedures.

Magnesium containing calcium silicates have recently shown that they are promising materials for various biomedical application with potential use in the form of bulk ceramic, composite scaffold or coatings on metallic substrates. A novel akermanite (AK; Ca₂MgSi₂O₇)/dicalcium phosphate dihydrate (DCPD, CaHPO₄. H₂O) cement mixture was tested in this work in order to produce an alternative AK/DCPD biocement for orthopedic applications. For comparison, we have prepared two cements mixed with 2.5 wt% NaH₂PO₄ solution (labeled as NaH₂PO₄ cement) and with the solution composed of organic 2.5 wt% citric acid a 2.5 wt% trisodium citrate (citrate cement) respectively. The results demonstrated only a partial dissolution of AK, regardless of the type of liquid used. On the other hand, the DCPD was completely hydrolyzed much faster in the citrate cement. The final hydration product was an amorhous quarternary phase of CaO–MgO–SiO₂–P₂O₅ composition with the remaining unreacted akermanite embeded in the cement matrix. The highest early compressive strength was observed in the citrate cement (33 MPa), but much lower value was measured in NaH₂PO₄ cement (7 MPa) after 1 d setting. Different cell responses have been observed when the cells were cultured on the surfaces of cement substrates. While the NaH₂PO₄ cement demonstrated high proliferation activity of osteoblast, the citrate cement showed strong cytotoxic cell response, probably as a result of higher concentration of citrates on the cement surface, which can negatively affect the attachment and proliferation of osteoblastic cells.

In the present study, sythetic biodegradable polymer poly(ε-caprolactone) (PCL) and graphene oxide (GO) were combined together to prepare 3D, composite tissue scaffolds (PCL/GO scaffolds) by using electrospinning technique. Also, the influence of Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) and/or thiophene (Th) modified GO on the composite PCL/GO mats (PCL/GO, PCL/GO-GRGDSP, PCL/GO-Th, PCL/GO-GRGDSP-Th) was further investigated. Characteristic examinations of the scaffolds were carried out by scanning electron microscope (SEM), contact angle (CA) measurements, x-ray photoelectron spectroscopy, TGA, electrical conductivity tests, phosphate buffer saline absorption and shrinkage tests and mechanical tests. All of the scaffolds were exhibited suitable bead-free and uniform morphology according to SEM images. With the addition of GO, better hydrophilicity and a slight CA decrease (∼5°) for the PCL/GO scaffolds were observed. Mechanical properties were reinforced drastically with the addition and well-dispersion of GO into PCL matrix. The incorporation of PCL and GO exhibited enhanced electrical conductivity and the highest value was found for PCL/GO-GRGDSP-Th (2%) as 15.06 μS cm⁻¹. The MG-63 osteoblast cell culture studies (MTT assay, ALP activity, Alizarin-Red staining, fluorescence and SEM analyses) showed that PCL/GO-GRGDSP-Th (1%) scaffolds exhibited the highest biocompatibility performance (1.87 fold MTT absorbance value comparing with neat PCL) due to the advanced properties of GO and the biological interfaces.

Magnetic resonance imaging (MRI), as a diagnostic tool in tissue engineering, has received widespread attention because of its ability to consistently provide degradation and absorption of implants in vivo. For some specific human tissues and organs, such as nerves, muscles and myocardium, their regeneration requires tissue engineering scaffolds have a good electrical conductivity. Graphene oxide (GO) has been extensively studied as a conductive biomaterial having mechanical reinforcement. Based on the above, we propose an MRI conductive scaffold containing gelatin (Gel)/gelatin-polycaprolactone (Gel-PCL)/ultra-small paramagnetic iron oxide (USPIO)/graphene oxide (GO) (Gel/Gel-PCL/USPIO/GO). Their physical and chemical properties as well as biocompatibility are measured in vitro. The purpose of doping USPIO was developed for non-invasive monitoring of tissue engineered implants and tissue reconstruction. Functional modification of GO to match electrophysiological requirement. Co-culture with bone marrow mesenchymal stem cells showed good biocompatibility. Blood experiments have also demonstrated the feasibility of scaffolds as tissue engineered implants. The USPIO-labeled conductive scaffold, as an effective image-guided and electrically stimulating implant, appears to be a reconstruction platform for specific tissues and organs.

Background. Decellularized xenogenic vascular tissue has potential applications in small-diameter tissue engineering vascular grafts. Decellularization removes most xenogenic antigen and leaves most of the extracellular matrix for cell adhesion, migration and proliferation. Recellularization is recognized as an important step to improve the endothelialization of decellularized vascular grafts in vivo and most studies used endothelial cells for recellularization. However, there have been no studies on applying undifferentiated adipose stem cells (ASCs) in recellularization. Material and methods. In this study, we evaluated the feasibility of decellularized porcine coronary artery (DPCA) with ASC recellularization as tissue-engineered vascular grafts by in vitro cell biocompatibility and in vivo aorta repair tests. Porcine coronary artery was decellularized with the enzyme-detergent method and characterized by histology and biochemical methods. In vitro biocompatibility was tested by human and rat adipose stem cells (hASCs/rASCs). In vivo, potential for endothelialization of ASC-seeded DPCA scaffolds were evaluated by rat aorta patch repair model. Results. In vitro, hASCs and rASCs could adhere and maintain cell viability on DPCA scaffold. In vivo, rat abdominal aorta repair model revealed that DPCA with rat ASC seeding had a 100% patency rate. Grossly, there was integration between host tissue and graft tissue, and no leakage or rupture was observed. Histologically, DPCA with rat ASC seeding displayed endothelialization on the luminal side. In addition, the layer structure was preserved with collagen deposition. However, intimal hyperplasia was noted. Conclusion. This preliminary study indicates that DPCA with undifferentiated ASC seeding exhibited cell biocompatibility in vitro and endothelialization in vivo. DPCA with ASC recellularization has potential for use in the development of small-diameter tissue engineering vascular grafts.

Current treatment options for repairing volumetric muscle loss injury involve the use of existing host tissue like muscular flaps or grafts. However, host muscle tissue may not be available and donor site morbidity, such as functional loss and volume deficiency, is often present. In this study, we developed a biofunctionalized muscle-derived decellularized extracellular matrix scaffolding system to utilize endogenous stem/progenitor cells for in situ muscle tissue regeneration. We optimized the decellularization process to enhance cellular infiltration and fabricated an insulin-like growth factor-binding protein 3 (IGFBP-3)-conjugated scaffold for controlled delivery of IGF-I. We then tested in vitro characterization including IGF-I release kinetics and cellular infiltration. In addition, we have analyzed the bioactivities of skeletal muscle cells (C2C12) to assess the indirect effect of released IGF-1 from the scaffold. The IGFBP-3 conjugated scaffolds demonstrated showed sustained release of IGF-1 and 1% SDS decellularized scaffold with IGF-1 showed higher cellular infiltration compared to control scaffolds (no conjugation). In indirect bioactivity assay, IGF-1 conjugated scaffold showed 2.1-fold increased cell activity compared to control (fresh media). Our results indicate that IGFBP-3/IGF-I conjugated scaffold has the potential to be used for in situ muscle tissue regeneration.

To simultaneously impart excellent biological activity and antibacterial function to titanium-based metal materials, rubidium-doped titanium surfaces were prepared via alkali heat treatment, subsequent hydrothermal treatment and final heat treatment. The alkali heat treatment was employed to fabricate an amorphous sodium titanate hydrogel layer on titanium substrates. Thereafter, rubidium was introduced through the hydrothermal process. After final heat treatment, crystallized rubidium titanate and sodium titanate were obtained on titanium surfaces. The viability of MC3T3-E1 cells was inhibited on rubidium-doped titanium surfaces for short-term (day 1). With prolonged duration, the viability and alkali phosphatase (ALP) activity were comparable for various surfaces with different amounts of rubidium (day 5). With further increased culture duration, the collagen synthesis (day 10) and in vitro mineralization of osteoblasts were found to be significantly enhanced on rubidium-doped titanium surfaces. The Rb-doped Ti surfaces showed antibacterial capacity against Staphylococcus aureus at both 12 and 24 h. The results indicate that doping rubidium into titanium surfaces could simultaneously endow materials with favorable osteogenic and antibacterial capacity.

Chitosan fibers were processed using the Net-Shape-Nonwoven (NSN) technique in order to create porous scaffolds which were functionalized in two bioinspired ways: collagen type I coating and unique mineralization with organically modified hydroxyapatite (ormoHAP). While collagen is common to enhance cell attachment on surfaces, the electric-field assisted migration and deposition of ormoHAP on the surface of the NSN-scaffolds is a novel technique which enables sub-micrometer sized mineralization while maintaining the original pore structure. Microscopy revealed fast attachment and morphological adaptation of the cells on both, the pure and the functionalized NSN-scaffolds. Remarkably, the cell number of osteogenically induced hBMSC on ormoHAP-modified NSN-scaffolds increased 3.5–5 fold compared to pure NSN-scaffolds. Osteogenic differentiation of hBMSC/osteoblasts was highest on collagen-functionalized NSN-scaffolds. RT-PCR studies revealed gene expression of ALP, BSP II, and osteocalcin to be high for all NSN-scaffolds. Overall, the NSN-scaffold functionalization with collagen and ormoHAP improved attachment, proliferation, and differentiation of hBMSC and therefore revealed the remarkable potential of their application for the tissue engineering of bone.

Bone regeneration requires porous and mechanically stable scaffolds to support tissue integration and angiogenesis, which is essential for bone tissue regeneration. With the advent of additive manufacturing processes, production of complex porous architectures has become feasible. However, a balance has to be sorted between the porous architecture and mechanical stability, which facilitates bone regeneration for load bearing applications. The current study evaluates the use of high resolution digital light processing (DLP) -based additive manufacturing to produce complex but mechanical stable scaffolds based on β-tricalcium phosphate (β-TCP) for bone regeneration. Four different geometries: a rectilinear Grid, a hexagonal Kagome, a Schwarz primitive, and a hollow Schwarz architecture are designed with 400 μm pores and 75 or 50 vol% porosity. However, after initial screening for design stability and mechanical properties, only the rectilinear Grid structure, and the hexagonal Kagome structure are found to be reproducible and showed higher mechanical properties. Micro computed tomography (μ-CT) analysis shows <2 vol% error in porosity and <6% relative deviation of average pore sizes for the Grid structures. At 50 vol% porosity, this architecture also has the highest compressive strength of 44.7 MPa (Weibull modulus is 5.28), while bulk specimens reach 235 ± 37 MPa. To evaluate suitability of 3D scaffolds produced by DLP methods for bone regeneration, scaffolds were cultured with murine preosteoblastic MC3T3-E1 cells. Short term study showed cell growth over 14 d, with more than two-fold increase of alkaline phosphatase (ALP) activity compared to cells on 2D tissue culture plastic. Collagen deposition was increased by a factor of 1.5–2 when compared to the 2D controls. This confirms retention of biocompatible and osteo-inductive properties of β-TCP following the DLP process. This study has implications for designing of the high resolution porous scaffolds for bone regenerative applications and contributes to understanding of DLP based additive manufacturing process for medical applications.

Insulin-producing and -secreting cells derived from mouse pluripotent stem cells (PSCs) are useful for pancreatic development research and evaluating drugs that may induce insulin secretion. Previously, we have established a differentiation protocol to derive insulin-secreting cells from mouse embryonic stem cells (ESCs) using a combination of growth factors, recombinant proteins, and a culture substratum with net-like fibers. However, it has not been tested which materials and diameters of these fibers are more effective for the differentiation. Therefore, the present study aimed to produce net-like culture substratum formed from polyamide (PA) and polyacrylonitrile (PAN) fibers. Substrata were delineated into PA100, 300, 600, PAN100, 300, and 600 groups based on fiber diameters. The differentiation efficiencies of mouse ESCs cultured on the substrata were then examined by insulin 1 (Ins1) expression. Expression was found to be highest in PA300 differentiated cells, indicating the potential to produce high levels of insulin. To understand any differences in substratum properties, the adsorption capacities of laminin were measured, revealing that PA300 had the highest for it. We next examined the stage of differentiation affected by incubation with PA300. This showed that Sox17- and Pdx1-GFP-positive cells increased during the first step of differentiation. To show the production of insulin without absorption from the medium, we confirmed the expression of insulin C-peptide after differentiation. Finally, we tested the effects of PA300 on the differentiation of human-induced PSC, and found more Sox17-positive cells with the PA300 substratum at the definitive endoderm stage. Furthermore, these cells expressed insulin C-peptide and had glucose-responsive C-peptide secretion. In summary, our study identified and validated a novel substratum which is suitable for pancreatic differentiation of mouse and human PSCs.

A novel composite biomaterial for bone-soft tissue fixation applications was developed. MgO-Silk-PCL, Silk-PCL and MgO-PCL composites were prepared with variable filler concentrations (0, 10, 20 and 30% w/w of MgO nanoparticles and 0%, 5%, 10%, 20% and 30% of degummed silk fiber) in PCL polymer. The highest mechanical properties were obtained with 10% MgO and 20% Silk composite (MSP) wih 1.7× better tensile strength and 7.5× tensile modulus over PCL. It exhibited good cell viability, adhesion and hemocompatibility, increased cell proliferation and differentiation. MgO filler contributed more in increasing tensile strength, whereas silk fiber towards modulus, imparting a synergistic effect on mechanical performance. Prototype bone screws were molded using the MSP composite in a custom-designed mold. It showed significantly increased degradation (2.7 fold after 60 days) in PBS attributable to binary filler phase as compared to PCL. In vivo biosafety studies of MgO-silk-PCL composite screw in SD rats by subcutaneous implantation showed moderate inflammation at 2 weeks which subsided after 4th week. No toxic effect was seen in histopathology of vital organs and in blood parameters. Composite screw showed 2× pull-out strength of PCL in synthetic bone, therefore a potential candidate for bone-soft fixation applications like resorbable orthopedic screws for ACL reconstruction.