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Bovine serum albumin methacryloyl (BSAMA) is a newly emerging photocurable globular protein-based material whereas gelatin methacryloyl (GelMA) is one of the most popular photocurable fibrous protein-based materials. So far, the influence of their different structural conformations as building blocks on hydrogel properties and mineral deposition has not been investigated. Here, we compared their differences in structures, gelation kinetics, hydrogel properties, mineralization, and cell behaviors. BSAMA maintained a stable globular structure while GelMA exhibited temperature-sensitive conformations (4 - 37 °C). BSAMA displayed slower gelation kinetics and much more retarded enzymatic degradation compared to GelMA. Photocurable BSAMA (6.41 - 390.95 kPa) and GelMA hydrogels (36.09 - 199.70 kPa) exhibited tunable mechanical properties depending on their concentrations (10 - 20%). Interestingly, BSAMA hydrogels mineralized needle-like apatite (Ca/P: 1.409) with higher crystallinity compared to GelMA hydrogels (Ca/P: 1.344). BSAMA and GelMA supported satisfactory cell (MC3T3-L1) viability of 99.43 ± 0.57% and 97.14 ± 0.69%, respectively. However, BSAMA gels were less favorable to cell proliferation and migration than GelMA gels. In serum-free environments, cells on GelMA displayed a higher amount of attachment, a more elongated shape, and a longer protrusion compared to those on BSAMA (p 0.01) during the early adhesion.

Hydrogels have a wide range of applications in tissue engineering, drug delivery, device fabrication for biological studies and stretchable electronics. For biomedical applications, natural polymeric hydrogels have general advantages such as biodegradability and non-toxic by products as well as biocompatibility. However, applications of nature derived hydrogels have been severely limited by their poor mechanical properties. For example, most of the protein derived hydrogels do not exhibit high stretchability like methacrylated gelatin hydrogel has ∼11% failure strain when stretched. Moreover, protein derived elastomeric hydrogels that are fabricated from low molecular weight synthetic peptides require a laborious process of synthesis and purification. Biopolymers like gelatin, produced in bulk for pharma and the food industry can provide an alternative for the development of elastomeric hydrogels. Here, we report the synthesis of ureidopyrimidinone (Upy) functionalized gelatin and its fabrication into soft elastomeric hydrogels through supramolecular interactions that could exhibit high failure strain (318.73 ± 44.35%). The hydrogels were fabricated through a novel method involving co-solvent optimization and structural transformation with 70% water content. It is anticipated that the hydrogel fabrication method involves the formation of hydrophobic cores of ureidopyrimidinone groups inside the hydrogel which introduced elastomeric properties to the resulting hydrogel. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Nanyang Technological University Published version The authors acknowledge the financial support provided by Ministry of Education, Singapore, Singapore Centre for 3D Printing (SC3DP), A*STAR (Singapore Food Story R&D Programme), and School of Material Sciences and Engineering, Nanyang Technological University, Singapore.

Nowadays, hydrogels-based microneedles (MNs) have attracted a great interest owing to their outstanding qualities for biomedical applications. For the fabrication of hydrogels-based microneedles as tissue engineering scaffolds and drug delivery carriers, various biomaterials have been tested. They are required to feature tunable physiochemical properties, biodegradability, biocompatibility, nonimmunogenicity, high drug loading capacity, and sustained drug release. Among biomaterials, human proteins are the most ideal biomaterials for fabrication of hydrogels-based MNs; however, they are mechanically weak and poorly processible. To the best of the knowledge, there are no reports of xeno-free human protein-based MNs so far. Here, human albumin-based hydrogels and microneedles for tissue engineering and drug delivery by using relatively new processible human serum albumin methacryloyl (HSAMA) are engineered. The resultant HSAMA hydrogels display tunable mechanical properties, biodegradability, and good biocompatibility. Moreover, the xeno-free HSAMA microneedles display a sustained drug release profile and significant mechanical strength to penetrate the model skin. In vitro, they also show good biocompatibility and anticancer efficacy. Sustainable processible human albumin-based biomaterials may be employed as a xeno-free platform in vivo for tissue engineering and drug delivery in clinical trials in the future.

Xenogeneic bones are potential templates for bone regeneration. In this study, decellularized porcine bone powder with attenuated immunogenicity was incorporated into a photocurable hydrogel, gelatin methacryloyl (GelMA), to obtain scaffolds with good mechanical properties for bone tissue engineering. The decellularized bone powder (DCB)-GelMA hybrid scaffolds had higher compressive strength and stiffness values when the DCB content was increased. In vitro evaluations revealed the biocompatibility of these scaffolds. The scaffolds could induce human bone marrow mesenchymal stem cells (hMSCs) to undergo osteogenic differentiation even in the absence of an induction medium. The efficiency of the scaffolds for bone regeneration applications was further evaluated using an in vivo cranial bone defect model in rats. Micro-CT images showed that the hybrid scaffolds with 20% DCB content had the best effect in promoting new bone regeneration. Thus, it was concluded that the DCB-GelMA hybrid scaffolds have high potential in bone tissue engineering applications.

Photopolymerization of protein-derived polymers functionalized with methacryloyl groups has been increasingly used to fabricate three-dimensional tissue constructs for biomedical applications because photocurable protein-based polymers (e.g., gelatin and collagen methacryloyl) feature spatial-temporal controllability of engineering complex constructs as well as inherent biological properties. Herein, we report photocurable albumin-based hydrogels. First, photocurable bovine serum albumin methacryloyl (BSA-MA) with different degrees of substitution (DM) was successfully synthesized in a precise manner, without substantially altering BSA native secondary structure. Resultant photocurable BSA-MA hydrogels exhibited tunable physio-biochemical properties over the swelling, degradation, and mechanical properties. Moreover, photo-cross-linked BSA-MA hydrogels provided a permissible environment to support cell viability and functionality both in two- and three-dimensional culture systems. Photocurable BSA-MA hydrogels may be used as a versatile platform for various bioapplications including tissue engineering and 3D bioprinting.

Developing biomaterials-based tissue engineering scaffolds with personalized features and intrinsic biocompatibility is appealing and urgent. Through utilizing various strategies, albumin, as the most abundant protein in plasma, could be fabricated into sustainable, cost-effective, and potentially personalized hydrogels that would display enormous biological applications. To date, much of the albumin-based research is primarily engrossed in using albumin as a therapeutic molecule or a drug carrier, not much as a scaffold for tissue engineering. For this reason, we have come up with a detailed and insightful review of recent progress in albumin-based hydrogels having an emphasis on production techniques, material characteristics, and biological uses. It is envisioned that albumin-based scaffolds would be appealing and useful platforms to meet current tissue engineering needs and achieve the goal of clinical translation to benefit patients. STATEMENT OF SIGNIFICANCE: The creation of autologous material-based scaffolds is a potential method for preventing immunological reactions and obtaining the best therapeutic results. Patient-derived albumin hydrogels may consequently provide improved opportunities for personalized treatment due to their abundant supply and minimal immunogenicity. To provide a detailed and insightful summary on albumin-based hydrogels, this review includes latest comprehensive information on their preparation procedures, features, and applications in 3D printing and other biomedical applications. The challenges, along with the future potential for implementing albumin-based hydrogels in clinics, have also been addressed.

Despite the fact that classic autograft is the gold standard material for periodontal plastic surgery, it has some drawbacks, including the need for a second surgical site and the scarcity of palatal donor tissues. However, only a few research works on the manufacturing of bioengineered intraoral connective tissue grafts have been conducted. In this work, porous bovine serum albumin methacryloyl/gelatin methacryloyl (BG) biohybrid scaffolds were developed for super-elasticity, shape recovery, suturability for persistent stability, sufficient scaffolding function, and convenient manipulating characteristics to fabricate an intraoral graft substitute with superb stability to resist frequent dynamic forces caused by functional movement (speaking, masticating, and swallowing). Furthermore, in a 3D cell culture assay, BG scaffolds demonstrated excellent cell adhesion and proliferation of L929 cells. In addition, the BG scaffolds were able to release Ibuprofen in a controlled manner for postoperative recovery. The use of a low-cost, optimized cryogelation technique for functional biomacromolecules offers up new possibilities to develop promising scaffolds for dental clinical settings.

Cancer cells undergoing epithelial-mesenchymal transition (EMT) acquire stem cell-like phenotype associated with malignant behaviour, chemoresistance, and relapse. Current two-dimensional (2D) in-vitro culture models of tumorigenesis are inadequate to replicate the complexity of in-vivo microenvironment. Therefore, the generation of functional three-dimensional (3D) constructs is a fundamental prerequisite to form multi-cellular tumour spheroids for studying basic pathological mechanisms. In this study, we focused on two major points (i) designing and fabrication of 3D hybrid scaffolds comprising electrospun fibers with cancer cells embedded within hydrogels, and (ii) determining the potential roles of 3D hybrid scaffolds associated with EMT in cancer progression and metastasis. Our findings revealed that 3D hybrid scaffold enhances cell proliferation and induces cancer cells to undergo EMT, as demonstrated by significant up-regulation of EMT associated transcriptional factors including Snail1, Zeb1, and Twist2; and mesenchymal markers whereas epithelial marker, E-Cadherin was downregulated. Remarkably, this induction is independent of cancer cell-type as similar results were obtained for breast cancer cells, MDA-MB-231 and gastric cancer cells, MKN74. Moreover, the hybrid scaffolds enrich aggressive cancer cells with stem cell properties. We showed that our 3D scaffolds could trigger EMT of cancer cells which could provide a useful model for studying anticancer therapeutics against metastasis.

Animal models for studying human breast cancer carcinogenesis and testing drug candidates on human breast cancer have extensively been proposed. Especially, chemically induced breast tumor models have been used because they can mimic the progression of clinical cancer from the beginning and can be generated with a facile procedure. Pristane is a hydrocarbon oil that is used as a chemical carcinogen to induce tumorigenesis in mice as well as arthritis and lupus nephritis in rats. In only a few studies, pristane-induced breast cancer models have been reported. This chapter is designated to describe pristane-induced mammary carcinoma models. Here, we provide a protocol for generating pristane-induced breast tumors in mice models for analyzing and for testing potential therapeutics on them. The given protocol can be applied to other animal models with some changes.

Animal models for studying human breast cancer carcinogenesis and testing drug candidates on human breast cancer have extensively been proposed. Especially, chemically induced breast tumor models have been used because they can mimic the progression of clinical cancer from the beginning and can be generated with a facile procedure. Pristane is a hydrocarbon oil that is used as a chemical carcinogen to induce tumorigenesis in mice as well as arthritis and lupus nephritis in rats. In only a few studies, pristane-induced breast cancer models have been reported. This chapter is designated to describe pristane-induced mammary carcinoma models. Here, we provide a protocol for generating pristane-induced breast tumors in mice models for analyzing and for testing potential therapeutics on them. The given protocol can be applied to other animal models with some changes.

Contains fulltext : 238919.pdf (Publisher’s version ) (Closed access) In the sphere of liver tissue engineering (LTE), 3D bioprinting has emerged as an effective technology to mimic the complex in vivo hepatic microenvironment, enabling the development of functional 3D constructs with potential application in the healthcare and diagnostic sector. This review gears off with a note on the liver's microscopic 3D architecture and pathologies linked to liver injury. The write-up is then directed towards unmasking recent advancements and prospects of bioprinting for recapitulating 3D hepatic structure and function. The article further introduces available stem cell opportunities and different strategies for their directed differentiation towards various hepatic stem cell types, including hepatocytes, hepatic sinusoidal endothelial cells, stellate cells, and Kupffer cells. Another thrust of the article is on understanding the dynamic interplay of different hepatic cells with various microenvironmental cues, which is crucial for controlling differentiation, maturation, and maintenance of functional hepatic cell phenotype. On a concluding note, various critical issues and future research direction towards clinical translation of bioprinted hepatic constructs are discussed.

Gelatin methacryloyl (GelMA; GM) contains impurities, including hydrolabile photosensitive methacrylate groups or soluble methacrylic acid (MA), which could be potentially detrimental to its in vitro and in vivo applications. To date, the influence of GM photocurable side chains on the cytotoxicity and ambient structural stability has remained to be investigated. Here, we successfully separated highly substituted decoupled gelatin methacrylamide (DGM) from GM via removing methacrylate impurities in order to evaluate its stability, cell viability, and cell toxicity, compared to GM, DGM plus soluble MA, and soluble MA. The photocurable methacrylate groups in GM were hydrolytically labile in neutral solutions, changing into soluble MA over time; on the other hand, the photocurable methacrylamide groups in DGM remained intact under the same conditions. Soluble MA was found to decrease cell viability in a dose dependent manner and caused severe cell toxicity at above 10 mg/mL. DGM plus MA started to impair cell viability at a 25 mg/mL concentration. DGM exhibited excellent cell viability and little cell toxicity across the treated concentrations (0.1–25 mg/mL). DGM without hydrolabile methacrylate and cytotoxic MA impurities could be a better choice for long term stability and good cell compatibility for bioapplications including bioprinting and cell encapsulation.

The capacity of a biomaterial to innately modulate cell behavior while meeting the mechanical property requirements of the implant is a much sought-after goal within bioengineering. Here we covalently incorporate soluble elastin into a gelatin&ndash, poly (ethylene glycol) (PEG) hydrogel for three-dimensional (3D) cell encapsulation to achieve these properties. The inclusion of elastin into a previously optimized gelatin&ndash, PEG hydrogel was then evaluated for effects on entrapped fibroblasts, with the aim to assess the hydrogel as an extracellular matrix (ECM)-mimicking 3D microenvironment for cellular guidance. Soluble elastin was incorporated both physically and covalently into novel gelatin/elastin hybrid PEG hydrogels with the aim to harness the cellular interactivity and mechanical tunability of both elastin and gelatin. This design allowed us to assess the benefits of elastin-containing hydrogels in guiding fibroblast activity for evaluation as a potential dermal replacement. It was found that a gelatin&ndash, PEG hydrogel with covalently conjugated elastin, supported neonatal fibroblast viability, promoted their proliferation from 7.3% to 13.5% and guided their behavior. The expression of collagen alpha-1(COL1A1) and elastin in gelatin/elastin hybrid gels increased 16-fold and 6-fold compared to control sample at day 9, respectively. Moreover, cells can be loaded into the hydrogel precursor solution, deposited, and the matrix cross-linked without affecting the incorporated cells adversely, thus enabling a potential injectable system for dermal wound healing.

A bilayer swellable drug-eluting ureteric stent (BSDEUS) is engineered and implemented, as a sustained drug delivery platform technology that enhances localized drug delivery to the highly impermeable urothelium, for the treatment of urothelial diseases such as strictures and carcinomas. On deployment, the device swells to co-apt with the ureteric wall and ensure drug availability to these tissues. BSDEUS consists of a stent spray-coated with a polymeric drug containing polylactic acid-cocaprolactone (PLC) layer which is overlaid by a swellable polyethylene glycol diacrylate (PEGDA) based hydrogel. In-vitro quantification of released drug demonstrated a tunable time-profile, indicating sustained delivery over 1-month. The PEGDA hydrogel overlayer enhanced drug release and transport into explanted porcine ureteric tissues ex-vivo, under a simulated dynamic fluid flow. A preliminary pilot invivo feasibility study, in a porcine model, demonstrated that the swollen hydrogel co-apts with the urothelium and thus enables localized drug delivery to the target tissue section. Kidney functions remained unaffected and device did not result in either hydronephrosis or systemic toxicity. This successful engineering of a bilayer coated stent prototype, demonstrates its feasibility, thus offering a unique solution for drug-based urological therapy. ASTAR (Agency for Sci., Tech. and Research, S’pore) MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) Accepted version

Treating large acute or chronic wounds remains a challenging task because of a lack of effective methods to accelerate wound healing. Though growth factor-based wound products are found to be effective, they are costly and are potentially associated with increased cancer mortality. Effective, safe, and affordable strategies to tackle large or chronic wounds need to further be designed and developed. Here, we designed a new antioxidant-embedded hydrogel system for speeding up the wound healing process. We prepared multifunctional poly (vinyl alcohol)/sodium alginate (PVA/SA) hydrogels with the incorporation of 5-hydroxymethylfurfural (5-HMF) and silver nanoparticles (Ag-NPs), and investigated their physiochemical and biological properties in vitro and in vivo. PVA, SA, 5-HMF, and Ag-NPs were employed for good mechanical properties, good biocompatibility, anti-inflammation, and anti-bacterial activity, respectively. 5-HMF is commonly found in many food products (e.g. honey, coffee, and black garlic) and is known as an antioxidant. In our in vitro study, 5-HMF was found to effectively facilitate the proliferation and migration of human skin fibroblasts (HSF), and collagen production. Besides, 5-HMF-embedded PVA/SA hybrid hydrogels supported controlled release and good cell compatibility, and more importantly accelerated wound healing in vivo through ameliorated inflammation, enhanced angiogenesis/vascularization, increased collagen production, and promoted re-epithelialization.

Biosynthetic poly(ethylene glycol) (PEG)-based hydrogels have been extensively investigated as extracellular matrix (ECM) mimicking gels as they retain the benefits of both ECM (biological cues) and synthetic hydrogels (tunable mechanical properties). In this article, we developed and characterized a new gelatin-PEG (GP) hydrogel that retains the benefits of gelatin and synthetic hydrogels. In this strategy, the thiolation of gelatin was accomplished by reacting with Traut's reagent; the thiolated gelatin was then conjugated to one end of PEG diacrylate (PEGDA) by Michael-type addition reaction. Two kinds of GP precursors, GP30 and GP60, were synthesized by changing the amount of Traut's reagent, while the weight ratio between thiolated-gelatin and PEGDA of GP30 and GP60 was 1.451:1 and 0.785:1, respectively. Finally, neonatal human dermal fibroblasts were encapsulated into the hydrogel by cross-linking the remaining double bonds of precursor under ultraviolet light. These GP hydrogels can encapsulate the fibroblasts in situ with high cell viability. Moreover, the behaviors of cells within the GP hydrogels can be modulated by varying the cross-linking density of GP hydrogel (storage modulus from 40 to 2000 Pa). In particular, this article showed that a minimum amount of cell-binding motifs (gelatin >2.30 wt/vol % and 44.0% dry weight percentage) are required for attachment; and appropriate initial rheological and structural properties (storage modulus ∼150 nm) can accelerate the attachment of cells and improve cell viability. Hence, this mixed-hydrogel platform allows an easily control hydrogel structure and modulates cell behavior to reconstruct new tissue in the three-dimensional microenvironments. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2401-2411, 2016.

Biomaterials act as the most important components in various treatments of cardiovascular diseases aiming at myocardial regeneration. To provide a functional engineered cardiac tissue for potential therapeutic intervention needs successful integration of biomaterials into native tissue. This chapter presents a general description on biomaterials from synthetic to natural polymers in use for cardiac tissue engineering. An introduction on the replacement and repair of the cardiovascular system using engineered biomaterials (e.g. injectable hydrogels) is shown here. The goal of this chapter is to familiarize the readers with the basic concept on materials selection and design for cardiovascular tissue engineering strategies.

Gelatin methacryloyl (GelMA) is a versatile material for a wide range of bioapplications. There is an intense interest in developing effective chemical strategies to prepare GelMA with a high degree of batch-to-batch consistency and controllability in terms of methacryloyl functionalization and physiochemical properties. Herein, we systematically investigated the batch-to-batch reproducibility and controllability of producing GelMA (target highly and lowly substituted versions) via a one-pot strategy. To assess the GelMA product, several parameters were evaluated, including the degree of methacryloylation, secondary structure, and enzymatic degradation, along with the mechanical properties and cell viability of GelMA hydrogels. The results showed that two types of target GelMA with five batches exhibited a high degree of controllability and reproducibility in compositional, structural, and functional properties owing to the highly controllable one-pot strategy.

Hydrogels are three-dimensional (3D) polymeric networks that can confine a substantial fraction of water within their structure. Photopolymerization is one of the efficient methods to prepare hydrogels, by which polymers undergo a photocrosslinking process upon light exposure for a few minutes in the presence of an initiator. In comparison to other crosslinking methods (e.g. physical crosslinking and chemical addition/condensation crosslinking), photopolymerization has many advantages including fast and effective polymerization and good spatio-temporal control of hydrogel formation, and can occur under ambient reaction conditions at room temperature in physiological solutions. For these reasons, photocurable hydrogels have been extensively utilized for biomedical applications such as 3D hydrogel scaffold fabrication, drug delivery systems, and 3D bio-printing. This chapter begins with the introduction of photoinitiators, which play an important role in the preparation of photocurable hydrogels. Subsequently, two types of photocurable hydrogels and their preparation methods are described; the first section focuses on photocurable hydrogels made from synthetic materials, followed by the second section on those from natural materials.

Gelatin methacryloyl (GelMA; GM) is a promising nature-derived photocurable material that can mimic the extracellular matrix because GelMA features tailorable mechanical properties, proteolytic degradation, and good cell adhesion. GelMA contains not only methacrylamide but also methacrylate. However, the hydrolytic stability of methacrylamide and methacrylate groups of GelMA in aqueous solutions has not been scrutinized. Here, the structural change of GelMA through hydrolysis is investigated for the first time. The structural change of hydrolyzed GelMA is quantitatively identified using colorimetric and 1H NMR methods. The methacrylate groups decompose markedly at high pH solutions, but the methacrylamide groups remain stable. Further, pure gelatin methacrylamide is successfully decoupled from GelMA for a better understanding of GelMA structure and future use for biomedical applications. NRF (Natl Research Foundation, S’pore)

We report an effective and novel method to controllably produce highly substituted gelatin-MA with nearly 100% degree of substitution despite the use of a very low concentration of methacrylic anhydride (MAA). The method is based on sequential time-lapse loading of MAA after pH adjustment in a carbonate–bicarbonate buffer system. Rheological studies indicate that hydrogels formed from more highly substituted gelatin-MA exhibit significantly improved mechanical stiffness.

3D bioprinting is an emerging technology that enables fabrication of three-dimensional organised cellular constructs. One of the major challenges in 3D bioprinting is to develop a material to meet the harsh requirements (cell-compatibility, printability, structural stability post-printing and bio-functionality to regulate cell behaviours) suitable for printing. Gelatin methacryloyl (GelMA) has recently emerged as an attractive biomaterial in tissue engineering because it satisfies the requirements of bio-functionality and mechanical tunability. However, the poor rheological property such as low viscosity at body temperature inhibits its application in 3D bioprinting. In this work, an enzymatic crosslinking method triggered by Ca2+-independent microbial transglutaminase (MTGase) was introduced to catalyse isopeptide bonds formation between chains of GelMA, which could improve its rheological behaviours, specifically its viscosity. By combining enzymatic crosslinking and photo crosslinking, it is possible to tune the solution viscosity and quickly stabilize the gelatin macromolecules at the same time. The results showed that the enzymatic crosslinking can increase the solution viscosity. Subsequent photo crosslinking could aid in fast stabilization of the structure and make handling easy. NRF (Natl Research Foundation, S’pore) Published version

Gelatin methacryloyl (GelMA) is a versatile biomaterial that has been shown to possess many advantages such as good biocompatibility, support for cell growth, tunable mechanical properties, photocurable capability, and low material cost. Due to these superior properties, much research has been carried out to develop GelMA as a bioink for bioprinting. However, there are still many challenges, and one major challenge is the control of its rheological properties to yield good printability. Herein, this study presents a strategy to control the rheology of GelMA through partial enzymatic crosslinking. Unlike other enzymatic crosslinking strategies where the rheological properties could not be controlled once reaction takes place, we could, to a large extent, keep the rheological properties stable by introducing a deactivation step after obtaining the optimized rheological properties. Ca2+-independent microbial transglutaminase (MTGase) was introduced to partially catalyze covalent bond formation between chains of GelMA. The enzyme was then deactivated to prevent further uncontrolled crosslinking that would render the hydrogel not printable. After printing, a secondary post-printing crosslinking step (photo crosslinking) was then introduced to ensure long-term stability of the printed structure for subsequent cell studies. Biocompatibility studies carried out using cells encapsulated in the printed structure showed excellent cell viability for at least 7 d. This strategy for better control of rheological properties of GelMA could more significantly enhance the usability of this material as bioink for bioprinting of cell-laden structures for soft tissue engineering. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) Published version

We have been able to control hydrogel compliance and cell spreading in a three-dimensional (3D) cell-laden system (hydrogel) using soluble PEG-OH. This was accomplished by encapsulating smooth muscle cells (SMCs) into poly(ethylene glycol)-fibrinogen (PEG-fibrinogen or PF) with poly(ethylene glycol)-diol (PEG-OH) as a macromolecular leachant. The cell-encapsulating hydrogels were prepared with three concentrations of soluble PEG-OH having a mass of 10 kDa (1, 5 and 10% w/v). Rheology was used to measure the elastic (storage) component of the complex shear modulus of these hydrogels, while quantitative morphometrics were used to characterize SMC morphology. PF hydrogel with a higher amount of PEG-OH displayed a lower storage modulus and a higher elongated cell morphology of SMCs. Structural changes of PF hydrogels mainly owing to gelation-induced phase separation imparted by the soluble PEG-OH in 3D cell-laden hydrogels dramatically affected both the properties of the hydrogel network including the modulus as well as cell spreading.

This study was conducted to investigate the effects of Chromium-Methionine (Cr-Met) chelate feeding for different durations on growth and carcass characteristics in the late fattening stage of Holstein steers. Nine Holstein steers were randomly assigned to three dietary treatments (3 head per treatment) including Non Cr-Met chelate feeding (NCM, av. BW of 433.3 kg), Cr-Met chelate feeding for 2 months (2CM, av. BW of 459.6 kg), and Cr-Met chelate feeding for 4 months (4CM, av. BW of 490.0 kg), respectively. The feeding amount of Cr-Met chelate to an animal was limited to 400 ppb/day. Dry matter intake showed no differences among all the treatments (p>0.05). Average daily gain was also higher in the animals fed Cr-Met chelate diets than NCM (p 0.05). The variations in meat quality were smaller in 4CM compared to other treatments. 4CM showed higher total and net income than other treatments (p

This study was performed on two groups (10 cows) for primiparous Holstein lactating cows (av. 98 days in milk : DIM) which were divided into low forage diet (LF) and high forage diet (HF) groups based on forage : concentrate ratio (F : C ratio). The F : C ratios of LF and HF groups were 37:63 and 62:38, respectively. Concentrate intake was significantly higher in the LF group than the HF group whereas the HF group showed higher forage intake (12.9 kg) compared to the LF group (7.4 kg) (p 0.05), but the HF group tended to be higher. CP, TDN and NEL intake showed no significant difference between the two groups (p>0.05). Though, there was no significant difference on actual milk between the two groups (26 vs. 23.9 kg/d, p>0.05), the LF group tended to be higher. 4% FCMs of LF and HF groups were 22.8 and 22.3 kg/d, respectively, and showed no significant difference (p>0.05). The HF group was higher in fat content and lower in MUN. C14:0, C16:0 and C16:1n7 of milk fatty acid were significantly higher in the HF group (p

Three-dimensional, protein-based hydrogel scaffolds that successfully mimic in vivo extracellular matrix microenvironments are desirable for tissue engineering and regenerative medicine applications, and can provide highly capable in vitro tissue analogues. However, the fabrication of protein-based scaffolds with uniform porosity, thin walls and durable mechanical properties remains a challenging prospect that might be overcome by integrating advances in microfabrication and protein functionalization. Towards this goal, herein, we report the successful fabrication of a highly ordered, gelatin-based inverted colloidal crystal (ICC) hydrogel platform that is robust and supports high levels of cell function. In particular, the utilization of colloidal templating microfabrication strategies together with highly substituted, photocrosslinkable gelatin methacryloyl (GelMA) allowed us to fabricate protein-based three-dimensional scaffolds with uniform pore interconnectivity, structural stability and tailorable degradation properties. The resulting GelMA ICC scaffolds provided cell attachment sites and promoted intercellular interaction of hepatocytes, which resulted in improved cell function compared to a flat 2D system. The results demonstrate the potential of GelMA ICC scaffolds to become an effective tissue engineering platform for drug screening and regenerative medicine. An engineered protein-based material that can be used to replace damaged tissue in humans has been created by scientists in Singapore and China. The cells in our body are supported by a three-dimensional extracellular matrix. Creating artificial tissue that can replace this matrix when it is damaged or diseased requires biomaterials that have uniform porosity, thin walls and mechanical durability. Now, Nam-Joon Cho from Nanyang Technological University in Singapore and co-workers have created a gelatin hydrogel that supports cells while allowing them to maintain their vital functionality. This three-dimensional scaffold has uniform pore interconnectivity, structural stability and tailorable degradation properties. The team created it by using a colloidal templating microfabrication technique and a material called gelatin methacryloyl. The biomaterial provided cells with attachment sites as well as improved cell function over previous two-dimensional approaches. We demonstrate the fabrication of three-dimensional, highly ordered protein-based hydrogel platforms for tissue engineering applications. The combination of colloidal templating microfabrication strategies and highly substituted, photocrosslinkable gelatin methacryloyl (GelMA) protein allowed us to fabricate inverted colloidal crystal (ICC) scaffolds with uniform pore interconnectivity, high structural stability and tailorable degradation properties. The resulting scaffolds provided cell attachment sites and promoted intercellular interaction among hepatocytes, which resulted in improved cell function compared to a flat, two-dimensional system. The results demonstrate the potential of GelMA ICC scaffolds to become an effective tissue engineering platform for drug screening and regenerative medicine applications.

Gelatin methacryloyl (GelMA) has been increasingly considered as an important bioink material due to its tailorable mechanical properties, good biocompatibility, and ability to be photopolymerized in situ as well as printability. GelMA can be classified into two types: type A GelMA (a product from acid treatment) and type B GelMA (a product from alkali treatment). In current literature, there is little research on the comparison of type A GelMA and type B GelMA in terms of synthesis, rheological properties, and printability for bioink applications. Here, we report the synthesis, rheological properties, and printability of types A and B GelMA. Types A and B GelMA samples with different degrees of substitution (DS) were prepared in a controllable manner by a time-lapse loading method of methacrylic anhydride (MAA) and different feed ratios of MAA to gelatin. Type B GelMA tended to have a slightly higher DS compared to type A GelMA, especially in a lower feed ratio of MAA to gelatin. All the type A and type B GelMA solutions with different DS exhibited shear thinning behaviours at 37 °C. However, only GelMA with a high DS had an easy-to-extrude feature at room temperature. The cell-laden printed constructs of types A and B GelMA at 20% w/v showed around 75% cell viability.

Gelatin-methacryloyl (GelMA) is one of the most commonly used photopolymerizable biomaterials in bio-applications. However, GelMA synthesis remains suboptimal, as its reaction parameters have not been fully investigated. The goal of this study is to establish an optimal route for effective and controllable GelMA synthesis by systematically examining reaction parameters including carbonate-bicarbonate (CB) buffer molarity, initial pH adjustment, MAA concentration, gelatin concentration, reaction temperature, and reaction time. We employed several analytical techniques in order to determine the degree of substitution (DS) and conducted detailed structural analysis of the synthesized polymer. The results enabled us to optimize GelMA synthesis, showing the optimal conditions to balance the deprotonation of amino groups with minimizing MAA hydrolysis, which led to nearly complete substitution. The optimized conditions (low feed ratio of MAA to gelatin (0.1 mL/g), 0.25 M CB buffer at pH 9, and a gelatin concentration of 10–20%) enable a simplified reaction scheme that produces GelMA with high substitution with just one-step addition of MAA in one pot. Looking forward, these optimal conditions not only enable facile one-pot GelMA synthesis but can also guide researchers to explore the efficient, high methacrylation of other biomacromolecules. NRF (Natl Research Foundation, S’pore) Published version

In this work, a comparison between two different physical-chemical gels, poly(NIPAAm-co-cysteamine) with poly(NIPAAm-co-cysteamine-vinylsulfone) and poly(NIPAAm-co-cysteamine) with poly(NIPAAm-co-HEMA-acrylate), is made. These hydrogels undergo gelation via dual mechanisms: temperature sensitivity (physical gelation) and chemical crosslinking (chemical gelation). The advantages of using both gelation mechanisms are to reduce the creep experienced by purely physical gels and to increase the elastic modulus of purely chemical gels. Here, the physical-chemical gels were synthesized and characterized for their chemical, structural, thermal, mechanical and morphological properties. The gels were also tested for their gelation kinetics, swelling, degradation and cytotoxicity. The copolymers were successfully synthesized and their phase transition temperatures fall in a feasible range (29-34°C) for use in vivo. With rheology, it was shown that use of simultaneous physical and chemical gelation resulted in improved properties, with increased elastic moduli and reduced frequency dependence. The rates of reaction of thiols to vinyls differ between the two systems, demonstrating a greater effect of chemical gelation in one gelling system over the other, due to the faster rate of thiols consumed into reaction. The morphology of the gels proved to be quite different when analyzed by scanning electron microscopy, showing differences in swelling behaviors. Cell studies illustrated good growth of cells exposed to the gels. Both hydrogels, although possessing slight differences, demonstrate the capability of being injected in vivo for use as embolic agents for occlusion of aneurysms.

A thermosensitive, injectable, and bioresorbable polymer hydrogel, poly(N-isopropylacrylamide-co-dimethyl-γ-butyrolactone acrylate-co-acrylic acid) [poly(NDBA)], was synthesized by radical copolymerization with 7.00 mol % dimethyl-γ-butyrolactone acrylate in tetrahydrofuran. The chemical composition was determined by acid titration in conjunction with (1) H NMR quantification. The molecular weight and polydispersity were determined by gel permeation chromatography in conjunction with static light scattering. The degradation properties of the polymer hydrogel were characterized using differential scanning calorimetry, percentage mass loss, cloud point test, and swelling ratio over time. It was found that the initial lower critical solution temperature (LCST) of the polymer is between room temperature and body temperature and that it takes about 2 weeks for the LCST to surpass body temperature under physiological conditions. An indirect cytotoxicity test indicated that this copolymer has relatively low cytotoxicity as seen with 3T3 fibroblast cells. The in vivo-gelation and degradation study showed good agreement with in vitro-degradation findings, and no detrimental effects to adjacent tissues were observed after the complete dissolution of the polymer. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.

Thermosensitive poly(organophosphazene) gels have been synthesized with a host of side groups, including α-amino-ω-methoxy-poly(ethylene glycol), hydrophobic amino acid esters (PheOEt, LeuOEt, and IleuOEt), depsipeptide ethyl ester (GlyGlycOEt), and lysine ethyl ester (lysOEt). The fraction of the last side group, lysOEt, which possesses two amine functional groups, was designed to be in the range of 0.1–0.3 mol per polymer unit. The poly(organophosphazenes) have been characterized via 1H- and 31P-NMR spectroscopies, GPC, and elemental analysis. The phase transition behavior of the poly(organophosphazenes) in aqueous solution has been determined via viscometry. Some of the poly(organophosphazenes) with amino functional groups exhibit reversible sol–gel transitions at temperatures near those of the human body, when in aqueous solution. These polymers form a sol at lower temperatures, and become gels at higher temperatures. Also, these polymer solutions have been found to behave generally like Newtonian fluids in the sol state, but appear to exhibit pseudoplastic qualities in the gel state. The polymers possessing depsipeptide ethyl esters (ethyl-2-(O-glycyl)glycolate) as a side group tend to exhibit much higher degradation rates under physiological conditions than do those which lack the depsipeptide ethyl ester group. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci 120:998–1005, 2011

A thermosensitive, bioresorbable and in situ gelling co-polymer, poly(N-isopropylacrylamide-co-dimethyl-gamma-butyrolactone acrylate-co-acrylic acid), was synthesized by radical co-polymerization with varying dimethyl-gamma-butyrolactone acrylate (DBA) content. The materials properties were characterized using differential scanning calorimetry, gel-permeation chromatography in conjunction with static light scattering, Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR) and acid titration. The initial lower critical solution temperature (LCST) of the synthesized co-polymer is between room temperature and body temperature. With the increase of DBA content, the LCST decreases, but then increases after the ring-opening hydrolysis of the DBA side-group. The FT-IR and NMR spectra show the co-polymerization of three monomers, as well as the hydrolysis-dependent ring-opening of the DBA side-group. The addition of acrylic acid increases the initial LCST and accelerates the degradation rate of the co-polymer. An indirect cytotoxicity test indicated that this co-polymer has relatively low cytotoxicity as seen with 3T3 fibroblast cells.

BACKGROUND:In situ gelling polymers, like poly(N-isopropylacrylamide) (poly(NIPAAm)), have many potential medical applications due to their biocompatibility and thermosensitivity. RESULTS: Radio-opaque thermosensitive poly(NIPAAm) grafted with 10.7 wt% 2,2′-(ethylenedioxy)bis(ethylamine)-2,3,5-triiodobenzamide was successfully synthesized and characterized. The conjugated polymer showed good visibility with X-ray fluoroscopy. The polymer had a lower critical solution temperature of 30 °C after conjugation with triiodobenzamide as determined by cloud point determination and a transition peak temperature of 33.3 ± 0.57 °C as determined by differential scanning calorimetry. CONCLUSION: The polymer synthesized was highly visible under X-rays, based upon the percentage incorporation of triiodobenzamide. After conjugation of the NIPAAm to the triiodobenzamide through a bis(ethylamine) linkage, the resultant polymer retained lower critical solution temperature characteristics in a temperature region that makes it physiologically useful. Copyright © 2009 Society of Chemical Industry

Novel, bioerodible, thermosensitive poly(NIPAAm-co-dimethyl–γ-butyrolactone), with hydrolysis-dependent thermosensitivity, were synthesized by radical polymerization with varying dimethyl-γ-butyrolactone content and the properties of the copolymers were characterized using Differential Scanning Calorimetry (DSC), High Performance Liquid Chromatography (HPLC) in conjunction with Static Light Scattering, Fourier Transformed Infrared Spectroscopy (FTIR), Nuclear Magnetic Resonance (NMR) and acid titration. The lower critical solution temperature (LCST) of the copolymers decreased with increasing dimethyl-γ-butyrolactone content, but then increased after ring-opening hydrolysis of the dimethyl-γ-butyrolactone side group. FTIR and NMR spectra showed the copolymerization of these two monomers and the hydrolysis-dependent ring-opening of the dimethyl-γ-butyrolactone side group. It was also found that there are no low-molecular-weight by-products but rather dissolution of the polymer chains at 37°C during the time frame of application. Models of the kinetics suggest that the hydrolysis reaction is self-catalytic due to an increase in hydrophilicity, and thus accessible water concentration, caused by ring-opening of the dimethyl-γ-butyrolactone.