Fabrication of Fish Scale-Based Gelatin Methacryloyl for 3D Bioprinting Application.

Gelatin methacryloyl (GelMA) is an ideal bioink that is commonly used in bioprinting. GelMA is primarily acquired from mammalian sources; however, the required amount makes the market price extremely high. Since garbage overflow is currently a global issue, we hypothesized that fish scales left over from the seafood industry could be used to synthesize GelMA. Clinically, the utilization of fish products is more advantageous than those derived from mammals as they lower the possibility of disease transmission from mammals to humans and are permissible for practitioners of all major religions. In this study, we used gelatin extracted from fish scales and conventional GelMA synthesis methods to synthesize GelMA, then tested it at different concentrations in order to evaluated and compared the mechanical properties and cell responses. The fish scale GelMA had a printing accuracy of 97%, a swelling ratio of 482%, and a compressive strength of about 85 kPa at a 10% w/v GelMA concentration. Keratinocyte cells (HaCaT cells) were bioprinted with the GelMA bioink to assess cell viability and proliferation. After 72 h of culture, the number of cells increased by almost three-fold compared to 24 h, as indicated by many fluorescent cell nuclei. Based on this finding, it is possible to use fish scale GelMA bioink as a scaffold to support and enhance cell viability and proliferation. Therefore, we conclude that fish scale-based GelMA has the potential to be used as an alternative biomaterial for a wide range of biomedical applications.

Fabrication and biological properties of artificial tendon composite from medium chain length polyhydroxyalkanoate.

Medium chain length polyhydroxyalkanoate (MCL-PHA), a biodegradable and biocompatible material, has a mechanical characteristic of hyper-elasticity, comparable to elastomeric material with similar properties to human tendon flexibility. These MCL-PHA properties gave rise to applying this material as an artificial tendon or ligament implant. In this study, the material was solution-casted in cylinder and rectangular shapes in the molds with the designated small holes. A portion of the torn human tendon was threaded into the holes as a suture to generate a composite tendon graft. The tensile testing of the three types of MCL-PHA/tendon composite shows that the cylinder material shape with the zigzag threaded three holes has the highest value of maximum tensile strength at 56 MPa, closing to the ultimate tendon tensile stress (50-100 MPa). Fibroblast cells collected from patients were employed as primary tendon cells for growing to attach to the surface of the MCL-PHA material to prove the concept of the composite tendon graft. The cells could attach and proliferate with substantial viability and generate collagen, leading to chondrogenic induction of tendon cells. An in vivo biocompatibility was also conducted in a rat subcutaneous model in comparison with medical-grade silicone. The MCL-PHA material was found to be biocompatible with the surrounding tissues. For surgical application, after the MCL-PHA material is decomposed, tendon cells should develop into an attached tendon and co-generated as a tendon graft.

Improved polarized light microscopic detection of gouty crystals via dissolution with formalin and ethylenediamine tetraacetic acid.

Conventional polarized light microscopy has been widely used to detect gouty crystals, but its limited sensitivity increases the risk of misidentification. In this study, a number of methods were investigated to improve the sensitivity of polarized light microscopy for the detection of monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD) crystals. We found that coating glass slides with poly-L-lysine, a positively charged polymer, improved the attachment of crystals to the glass surface, resulting in clearer crystal images compared to non-coated slides. Additionally, the sensitivity of detection was further enhanced by selective dissolution, in which 40% v/v formalin phosphate buffer was employed to dissolve MSUM crystals but not CPPD while 10% ethylenediamine tetraacetic acid (EDTA) was employed to dissolved CPPD but not MSUM. The other possible interferences were dissolved in both EDTA and formalin solution. These methods were successfully applied to detect gouty crystals in biological milieu, including spiked porcine synovial fluid and inflamed rat subcutaneous air pouch tissues.

Short-Term Growth Hormone Administration Mediates Hepatic Fatty Acid Uptake and De Novo Lipogenesis Gene Expression in Obese Rats.

Obesity has been linked to metabolic syndrome, type 2 diabetes, and non-alcoholic fatty liver disease (NAFLD). Obesity causes a decrease in growth hormone (GH) levels and an increase in insulin levels. Long-term GH treatment increased lipolytic activity as opposed to decreasing insulin sensitivity. Nonetheless, it is possible that short-term GH administration had no impact on insulin sensitivity. In this study, the effect of short-term GH administration on liver lipid metabolism and the effector molecules of GH and insulin receptors were investigated in diet-induced obesity (DIO) rats. Recombinant human GH (1 mg/kg) was then administered for 3 days. Livers were collected to determine the hepatic mRNA expression and protein levels involved in lipid metabolism. The expression of GH and insulin receptor effector proteins was investigated. In DIO rats, short-term GH administration significantly reduced hepatic fatty acid synthase (FASN) and cluster of differentiation 36 (CD36) mRNA expression while increasing carnitine palmitoyltransferase 1A (CPT1A) mRNA expression. Short-term GH administration reduced hepatic FAS protein levels and downregulated gene transcription of hepatic fatty acid uptake and lipogenesis, while increasing fatty acid oxidation in DIO rats. DIO rats had lower hepatic JAK2 protein levels but higher IRS-1 levels than control rats due to hyperinsulinemia. Our findings suggest that short-term GH supplementation improves liver lipid metabolism and may slow the progression of NAFLD, where GH acts as the transcriptional regulator of related genes.

Effectiveness of losartan on infrapatellar fat pad/synovial fibrosis and pain behavior in the monoiodoacetate-induced rat model of osteoarthritis pain.

Infrapatellar fat pad (IFP)/ synovial fibrosis is closely associated with the clinical symptoms of joint pain and stiffness, which contribute to locomotor restriction in osteoarthritis (OA) patients. Hence, this study was designed to gain insight on whether losartan, a selective angiotensin II type 1 receptor (AT1R) antagonist, has therapeutic benefit to reverse IFP/synovial fibrosis and secondarily to attenuate pain behavior. In male Wistar rats with monoiodoacetic acid (MIA)-induced IFP/synovial fibrosis, a possible role for increased AT1R expression in the pathogenesis of IFP/synovial fibrosis was assessed over an 8-week period. Pain behavior comprised static weight bearing and von Frey paw withdrawal thresholds (PWTs), which were assessed once or twice weekly, respectively. Groups of MIA-rats received oral losartan (30-mg/kg; n = 8 or 100-mg/kg; n = 9) or vehicle (n = 9) for 28-days according to a prevention protocol. Animals were euthanized on day 28 and various tissues (IFP/synovium, cartilage and lumbar dorsal root ganglia (DRGs)) were collected for histological, immunohistochemical and western blot analyses. Administration of once-daily losartan for 28-days dose-dependently attenuated the development of static weight bearing. This was accompanied by reduced IFP/synovial fibrosis and suppression of TGF-ß1 expression. Chronic treatment of MIA-rats with losartan had an anti-fibrotic effect and it attenuated pain behavior in this animal model.

Cefazolin Loaded Oxidized Regenerated Cellulose/Polycaprolactone Bilayered Composite for Use as Potential Antibacterial Dural Substitute.

Oxidized regenerated cellulose/polycaprolactone bilayered composite (ORC/PCL bilayered composite) was investigated for use as an antibacterial dural substitute. Cefazolin at the concentrations of 25, 50, 75 and 100 mg/mL was loaded in the ORC/PCL bilayered composite. Microstructure, density, thickness, tensile properties, cefazolin loading content, cefazolin releasing profile and antibacterial activity against S. aureus were measured. It was seen that the change in concentration of cefazolin loading affected the microstructure of the composite on the rough side, but not on the dense or smooth side. Cefazolin loaded ORC/PCL bilayered composite showed greater densities, but lower thickness, compared to those of drug unloaded composite. Tensile modulus was found to be greater and increased with increasing cefazolin loading, but tensile strength and strain at break were lower compared to the drug unloaded composite. In vitro cefazolin release in artificial cerebrospinal fluid (aCSF) consisted of initial burst release on day 1, followed by a constant small release of cefazolin. The antibacterial activity was observed to last for up to 4 days depending on the cefazolin loading. All these results suggested that ORC/PCL bilayered composite could be modified to serve as an antibiotic carrier for potential use as an antibacterial synthetic dura mater.

Fluorescence identification of arthropathic calcium pyrophosphate single crystals using alizarin red S and a xanthene dipicolylamine ZnII complex.

Calcium pyrophosphate deposition disease, previously known as pseudogout, is a type of chronic and painful joint arthropathy. Accurate identification of calcium pyrophosphate dihydrate (CPPD) single crystals is crucial for determining the best course of treatment. In this study, a two-step method involving alizarin red S (ARS) and a xanthene dipicolylamine ZnII (XDZ) complex was employed for the identification of CPPD single crystals in both triclinic and monoclinic forms using a fluorescence microscope and a microplate reader. The accurate identification method proposed in this study has the potential to advance the diagnosis and treatment of patients suffering from painful gouty arthritis.

Scaffold-Free Cartilage Construct from Infrapatellar Fat Pad Stem Cells for Cartilage Restoration.

Once damaged, the articular cartilage has a very limited intrinsic capacity for self-renewal due to its avascular nature. If left untreated, damaged cartilage can lead to progressive degeneration of bone and eventually causes pain. Infrapatellar fat pad adipose-derived mesenchymal stromal cells (IPFP-ASCs) has a potential role for cartilage restoration. However, the therapeutic role for IPFP-ASCs remains to be evaluated in an appropriate osteochondral defect model. Thus, this study aimed to investigate the potential of using a three-dimensional (3D) cartilage construct of IPFP-ASCs as a promising source of cells to restore articular cartilage and to attenuate pain associated with the cartilage defect in an osteochondral defect model. The chondrogenic differentiation potential of the 3D cartilage construct derived from IPFP-ASCs was determined before implantation and postimplantation by gene expression and immunohistochemistry analysis. Pain-related behavior was also assessed by using a weight-bearing test. A significant pain-associated with the osteochondral defect was observed in this model in all groups postinduction; however, this pain can spontaneously resolve within 3 weeks postimplantation regardless of implantation of IPFP-ASCs constructs. The expression of SOX9 and COL2A1 genes in addition to protein expression were strongly expressed in 3D construct IPFP-ASCs. The existence of mature chondrocytes, along with significant (p < 0.05) positive immunostaining for type II collagen and aggrecan, were identified in the implanted site for up to 12 weeks compared with the untreated group, indicating hyaline cartilage regeneration. Taken together, this study demonstrated the successful outcome of osteochondral regeneration with scaffold-free IPFP-ASCs constructs in an osteochondral defect rat model. It provides novel and interesting insights into the current hypothesis that 3D construct IPFP-ASCs may offer potential benefits as an alternative approach to repair the cartilage defect. Impact statement This study provides evidence of using the human 3D scaffold-free infrapatellar fat pad adipose-derived mesenchymal stromal cells (IPFP-ASCs) construct to restore the full-thickness osteochondral defect in a rat model. This study showed that chondrogenic features of the construct could be retained for up to 12 weeks postimplantation. The results of this proof-of-concept study support that human 3D scaffold-free IPFP-ASCs construct has potential benefits in promoting the hyaline-like native cartilage restoration, which may be beneficial as a tissue-specific stem cell for cell-based cartilage therapy. There are several clinical advantages of IPFP-ASC including ease and minimal invasive harvesting, chondrogenic inducible property, and tissue-specific progenitors in the knee.

Knee osteoarthritis in young growing rats is associated with widespread osteopenia and impaired bone mineralization.

Osteoarthritis (OA) leads to joint pain from intraarticular inflammation with articular cartilage erosion, deterioration of joint function and abnormal subchondral bone structure. Besides aging, chronic repetitive joint injury is a common risk factor in young individuals. Nevertheless, whether OA is associated with bone loss at other skeletal sites is unclear. Since OA-associated proinflammatory cytokines-some of which are osteoclastogenic factors-are often detected in the circulation, we hypothesized that the injury-induced knee OA could result in widespread osteopenia at bone sites distant to the injured knee. Here we performed anterior cruciate ligament transection (ACLT) to induce knee OA in one limb of female Sprague-Dawley rats and determined bone changes post-OA induction by micro-computed tomography and computer-assisted bone histomorphometry. We found that although OA modestly altered bone density, histomorphometric analyses revealed increases in bone resorption and osteoid production with impaired mineralization. The bone formation rate was also reduced in OA rats. In conclusions, ACLT in young growing rats induced microstructural defects in the trabecular portion of weight-bearing (tibia) and non-weight-bearing bones (L5 vertebra), in part by enhancing bone resorption and suppressing bone formation. This finding supports the increasing concern regarding the repetitive sport-related ACL injuries and the consequent bone loss.

In vivo evaluation of bilayer ORC/PCL composites in a rabbit model for using as a dural substitute.

OBJECTIVE: After a neurosurgical procedure, dural closure is commonly needed to prevent cerebrospinal fluids (CSF) leakage and to reduce the risk of complications, including infections and chronic inflammatory reactions. Although several dural substitutes have been developed, their manufacturing processes are complicated and costly and that many of them have been implicated in causing postoperative complications. This study aimed to assess the effectiveness and safety of new bilayer ORC/PCL composites in a rabbit model. METHODS: Two formulations of bilayer oxidized regenerated cellulose (ORC)/poly ε-caprolactone (PCL) knitted fabric-reinforced composites and an autologous graft (pericranium) were employed for dural closure in forty-five male rabbits. Systemic reaction and the local reaction of the samples were assessed and compared at one-, three- and six-months post-implantation by blood chemistry and gross, and microscopic assessment using hematoxylin-eosin and Masson's trichrome stains. RESULTS: No signs of CSF leakage or systemic infection were seen for all samples. All samples demonstrated minimal adhesion to adjacent tissues. The degree of host fibrous connective tissue ingrowth into both composites was comparable to that of the autologous group, but bone formation and osteoclast activities were significantly greater. Both composites progressively degraded over times and the residual thickness of the nonporous layer was 50% of the initial thickness at six months post-implantation. DISCUSSION: Bilayer ORC/PCL composites were successfully employed for dural closure in the rabbit model. They were biocompatible and could support dural regeneration comparable to that of the autologous group, but induced greater osteogenesis.

Performance evaluation of bilayer oxidized regenerated cellulose/poly ε-caprolactone knitted fabric-reinforced composites for dural substitution.

Ideally, alloplastic dural substitute should have functional properties resembling human dura mater and retain a watertight closure to prevent cerebrospinal leakage. Therefore, functional properties for successful dural closure application of newly developed bilayer oxidized regenerated cellulose knitted fabric/poly ε-caprolactone knitted fabric-reinforced composites were studied and compared with human cadaveric dura mater and three commercial dural substitutes including two collagen matrices and one synthetic poly-L-lactide patch. It was found that oxidized regenerated cellulose knitted fabric/poly ε-caprolactone knitted fabric-reinforced composites uniquely contained a bilayer structure consisting of micropores distributed within the relatively dense microstructure. Density, tensile properties and stitch tear strength of oxidized regenerated cellulose knitted fabric/poly ε-caprolactone knitted fabric-reinforced composites were found to be closed to human cadaveric dura mater than those of dense-type and porous-type dural substitutes. Water tightness performance in both sutured and non-sutured forms of oxidized regenerated cellulose knitted fabric/poly ε-caprolactone knitted fabric-reinforced composites was slightly inferior to human cadaveric dura mater, but still better than those of commercial dural substitutes. This study revealed that oxidized regenerated cellulose knitted fabric/poly ε-caprolactone knitted fabric-reinforced composite showed better functional properties than typical dural substitutes and was found to be a good candidate for being employed as a dural substitute. The role and relationship of both microstructure and the type of materials on the functional properties and water tightness of the dural substitutes were also elucidated.