Evaluation of Hyphecan (1‐4,2‐acetamide‐deoxy‐B‐D‐glucan polymer) on wound healing in a rodent model

Objective: To evaluate the effect of an artificial skin Hyphecan (1‐4,2‐acetamide‐deoxy‐B ‐D ‐glucan polymer) on wound healing in a rodent model. Materials and Method: The prospective study was conducted at a basic science laboratory at a tertiary teaching hospital. Two 4 cm × 4 cm full‐thickness wounds were created on the dorsal surface of 10 Spraque–Dawley rats and covered with Hyphecan and Kaltostat, respectively. Wounds were examined and measured on days 4, 10, 21 and 28, and would continue after day 28 until healed up completely. Punch biopsies (3 mm) were taken on days 4, 10 and 28 for histological examination of the response of healing and repair. Results: Despite the fact that the wound healing rate was similar for both groups on days 4, 10, 21 and 28, the average healing time for the Hyphecan group (29.1 ± 1.7 days) was significantly shorter statistically (P = 0.03) than the Kaltostat group (30.7 ± 2.8 days). Conversely, the marked healing response elicited by Hyphecan on day 4 persisted on days 10 and 28 in contrast to Kaltostat, which had only a mild degree of healing response on days 10 and 28. The study suggests that wounds treated by Hyphecan heal faster than Kaltostat. Conclusion: The findings provide basic scientific evidence supporting the clinical use of Hyphecan in different wounds and might also reduce the cost of wound management as Hyphecan is cheaper than Kaltostat and requires a shorter treatment time.      

“Inside‐Out” PEGylation of Bovine β‐Cross‐Linked Hemoglobin

The development of a blood substitute is urgent due to blood shortages and potential communicable diseases. A novel method, inside‐out PEGylation, has been used here to conjugate a multiarm maleimide‐PEG (Mal‐PEG) to β‐cross‐linked (βXL‐Hb) hemoglobin (Hb) tetramers through the Cys β93 residues. This method produces a polymer with a single PEG backbone that is surrounded by multiple proteins, rather than coating a single protein with multiple PEG chains. Electrophoresis under denaturing conditions showed a large molecular weight species. Gel filtration chromatography and analytical ultracentrifugation determined the most prevalent species had three βXL‐Hb to one Mal‐PEG. Thermal denaturation studies showed that the cross‐linked and PEGylated species were more stable than native Hb. Cross‐linking under oxy‐conditions produced a high oxygen affinity Hb species (P₅₀ = 9.18 Torr), but the oxygen affinity was not significantly altered by PEGylation (P₅₀ = 9.67 Torr). Inside‐out PEGylation can be used to produce a hemoglobin‐based oxygen carrier and potentially for other multiprotein complexes.     

Anti‐gal antibodies in α1,3‐galactosyltransferase gene‐knockout pigs

Fang J, Walters A, Hara H, Long C, Yeh P, Ayares D, Cooper DKC, Bianchi J. Anti‐gal antibodies in α1,3‐galactosyltransferase gene‐knockout pigs. Xenotransplantation 2012; 19: 305–310. © 2012 John Wiley & Sons A/S. Abstract Serum anti‐galactose‐α1,3‐galactose (Gal) IgM and IgG antibody levels were measured by ELISA in α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs (78 estimations in 47 pigs). A low level of anti‐Gal IgM was present soon after birth, and rose to a peak at 4–6 m, which was maintained thereafter even in the oldest pigs tested (at >2 yr). Anti‐Gal IgG was also present at birth, peaked at 3 m, and after 6 m steadily decreased until almost undetectable at 20 m. No differences in this pattern were seen between pigs of different gender. Total IgM followed a similar pattern as anti‐Gal IgM, but total IgG did not decrease after 6m. The data provide useful baseline data for future experimental studies in GTKO pigs, e.g., relating to the antibody response to WT pig allografts.     

Expression of human β‐defensin‐2 in the prostate

What’s known on the subject? and What does the study add? We found the expression of human β‐defensin‐2 (HBD‐2) in the prostate for the first time and LPS, a gram negative bacterial component, upregulated HBD‐2 in prostate epithelial cells. We are looking for other antimicrobial peptides expressed in the prostate besides human β‐defensin‐2. Also, we are studying the relationship between antimicrobial peptides and the development or progression of prostate diseases.

OBJECTIVE

To investigate the expression and regulation of human β‐defensin‐2 (HBD‐2) in the prostate.

PATIENTS AND METHODS

Normal human prostate epithelial cell line (RWPE‐1), human prostate cancer cell lines (DU‐145, PC‐3), and paraffin‐embedded prostate tissue from patients with benign prostatic hyperplasia (BPH) were analysed by RT‐PCR and immunohistochemical staining. HBD‐2 expression was also analysed by RT‐PCR and ELISA in RWPE‐1 cells treated with lipopolysaccharide (LPS). Nuclear factor‐κB (NF‐κB) activation was assessed by IκBα immunoblotting and electrophoretic mobility shift assay (EMSA).

RESULTS

BPH tissue and all of the tested prostate cell lines other than PC‐3 constitutively express HBD‐2 mRNA. HBD‐2 protein was strongly detected in prostate gland tissue surrounded by inflammatory cells including macrophages. Exposure to LPS induced HBD‐2 upregulation and NF‐κB activation, as assessed by IκBα phosphorylation and degradation in RWPE‐1 cells. Bay11‐7082, an NF‐κB inhibitor prevented LPS‐induced HBD‐2 production in RWPE‐1 cells.

CONCLUSIONS

Prostate epithelial cells may constitutively express HBD‐2, and its expression was upregulated by LPS. Our data indicate that HBD‐2 may be an important immunomodulatory factor in prostate function. Expression of HBD‐2 in normal prostates and the potential role of HBD‐2 in prostatitis and BPH should be addressed in the future.     

Hemorrhage‐Induced Interleukin‐1 Receptor Pathway in Lung Is Suppressed by 3,5‐Bis(2‐Fluorobenzylidene)‐4‐Piperidone in a Rat Model of Hypovolemic Shock

Severe blood loss in victims of trauma creates an exaggerated inflammatory background that contributes to the development of intravascular coagulopathy and multiple organ dysfunction syndrome. We hypothesized that treatment with diphenyldifluoroketone EF24, an inhibitor of nuclear factor kappa‐B, would have salutary effects in hemorrhagic shock. The objective of this study was to investigate the effect of EF24 on the expression of the interleukin‐1 receptor (IL‐1R) superfamily in a rat model of hypovolemic shock. Hypovolemia was induced by gradually withdrawing approximately 50% of circulating blood, and EF24 was administered intraperitoneally (0.2 mg/kg) in 50 μL of saline. After 6 h of shock, lung tissue was probed immunohistochemically and by immunoblotting to study the expression of Toll‐like receptor 4 (TLR4), IL‐1R, suppression of tumorigenicity 2 (ST2), and single immunoglobulin IL‐1R‐related (SIGIRR). The tissue‐associated pro‐inflammatory cytokines, tumor necrosis factor alpha (TNF‐α) and IL‐6, were measured by enzyme‐linked immunosorbent assay. We observed a reduction in immunoreactive TLR4 and IL‐1R1 in lung tissue of rats treated with EF24. Simultaneously, the pulmonary expression of ST2 and SIGIRR (the putative down‐regulators of the pro‐inflammatory IL‐1R pathway) was increased in EF24‐treated hemorrhaged rats. The concentration of hemorrhage‐induced TNF‐α and IL‐6 in lung tissue homogenates was also reduced by EF24 treatment. These results confirm our previous in vitro observations in lipopolysaccharide‐stimulated dendritic cells that EF24 beneficially modulates the IL‐1R pathway and suggest that it could be investigated as an adjunct therapeutic in managing inflammation associated with hemorrhagic shock.     

In Vivo Evaluation of CaO‐SiO2‐P2O5‐B2O3 Glass‐Ceramics Coating on Steinman Pins

Surface coating using ceramics improves the bone bonding strength of an implant. We questioned whether a new type of glass‐ceramics (BGS‐7) coating (CaO‐SiO₂‐P₂O₅‐B₂O₃) would improve the osseointegration of Steinman pins (S‐pins) both biomechanically and histomorphometrically. An in vivo study was performed using rabbits by inserting three S‐pins into each iliac bone. The pins were 2.2‐mm S‐pins with a coating of 30‐μm‐thick BGS‐7 and 550‐nm‐thick hydroxyapatite (HA), as opposed to an S‐pin without coating. A tensile strength test and histomorphometrical evaluation was performed. In the 2‐week group, the BGS‐7 implant showed a significantly higher tensile strength than the S‐pin. In the 4‐ and 8‐week groups, the BGS‐7 implants had significantly higher tensile strengths than the S‐pins and HA implants. The histomorphometrical study revealed that the BGS‐7 implant had a significantly higher contact ratio than the S‐pin and HA implants in the 4‐week group. The biomechanical and histomorphometrical tests showed that the BGS‐7 coating had superior bone bonding properties than the groups without the coating from the initial stage of insertion. The BGS‐7 coating of an S‐pin will enhance the bone bonding strength, and there might also be an advantage in human bone bonding.     

Bone mineral accrual in 4‐ to 10‐year‐old precompetitive,recreational gymnasts: A 4‐year longitudinal study

Competitive female gymnasts have greater bone mineral measures than nongymnasts. However, less is known about the effect of recreational and/or precompetitive gymnastics participation on bone development. The purpose of this study was to investigate whether the differences previously reported in the skeleton of competitive female gymnasts are also demonstrated in young children with a current or past participation history in recreational or precompetitive gymnastics. One hundred and sixty‐three children (30 gymnasts, 61 ex‐gymnasts, and 72 nongymnasts) between 4 and 6 years of age were recruited and measured annually for 4 years (not all participants were measured at every occasion). Total‐body (TB), lumbar spine (LS), and femoral neck (FN) bone mineral content (BMC) were measured by dual‐energy X‐ray absorptiometry (DXA). Multilevel random‐effects models were constructed and used to predict differences in TB, LS, and FN BMC between groups while controlling for differences in body size, physical activity, and diet. Gymnasts had 3% more TB and 7% more FN BMC than children participating in other recreational sports at year 4 (p < .05). No differences were found at the LS between groups, and there were no differences between ex‐gymnasts' and nongymnasts' bone parameters (p > .05). These findings suggest that recreational and precompetitive gymnastics participation is associated with greater BMC. This is important because beginner gymnastics skills are attainable by most children and do not require a high level of training. Low‐level gymnastics skills can be implemented easily into school physical education programs, potentially affecting skeletal health. © 2011 American Society for Bone and Mineral Research.     

Self‐Assembled Infrapatellar Fat‐Pad Progenitor Cells on a Poly‐ε‐Caprolactone Film For Cartilage Regeneration

Cartilage defects resulting from osteoarthritis (OA) or physical injury can severely reduce the quality of life for sufferers. Current treatment options are costly and not always effective in producing stable hyaline cartilage. Here we investigated a new treatment option that could potentially repair and regenerate damaged cartilage tissue. This novel approach involves the application of infrapatellar fat‐pad derived chondroprogenitor cells onto a mechanically stable biodegradable polymer film that can be easily implanted into a defect site. Poly‐ε‐caprolactone (PCL) films were fabricated via solvent casting in either acetone or chloroform. The hydrophobicity, mechanical properties, and surface morphology of the films were examined. Progenitor cells from infrapatellar fat‐pad were isolated, expanded, and then seeded onto the films. The cells were allowed to self‐assemble on films, and these were then cultured in a chemically defined chondrogenic media for 28 days. The self‐assembled tissue was characterized via histological staining, gene expression analysis, immunohistochemistry, and biochemical analysis. Chondrogenic differentiation was induced to generate a cartilaginous matrix upon the films. Despite differences between in the appearance, surface morphology, and mechanical properties of the films cast in chloroform or acetone, both methods produced tissues rich in sulfated glycosaminoglycan and collagen, although the extracellular matrix produced on chloroform‐cast films appeared to contain more collagen type II and less collagen type I than acetone‐cast films. These self‐assembled constructs have the potential to be implanted into defect sites as a potential treatment for cartilage defect regeneration.     

Proof of Concept for the Inhibition of Foot‐and‐Mouth Disease Virus Replication by the Anti‐Viral Drug 2′‐C‐Methylcytidine in Severe Combined Immunodeficient Mice

Recent European contingency plans envisage emergency vaccination as an animal‐friendly control strategy for foot‐and‐mouth disease (FMD). Anti‐viral drugs may be used as an alternative or complementary measure. We here demonstrate that the nucleoside analogue 2′‐C‐methylcytidine (2′CMC) protects severe combined immunodeficient (SCID) mice against lethal FMD virus infection. In brief, SCID mice were inoculated with serotype A FMD virus and treated for five consecutive days with 2′CMC. All 15 treated mice remained healthy until the end of the study at 14 days post‐infection (dpi). At that time, viral RNA was no longer detected in 13 of 15 treated mice. All eight untreated mice suffered from an acute generalized disease and were euthanized for ethical reasons on average at 4 dpi. These results illustrate the potential of small molecules to control FMD.     

Effects of Pulsatile Bioreactor Culture on Vascular Smooth Muscle Cells Seeded on Electrospun Poly (lactide‐co‐ε‐caprolactone) Scaffold

Electrospun nanofibrous scaffolds have several advantages, such as an extremely high surface‐to‐volume ratio, tunable porosity, and malleability to conform over a wide variety of sizes and shapes. However, there are limitations to culturing the cells on the scaffold, including the inability of the cells to infiltrate because of the scaffold's nano‐sized pores. To overcome the limitations, we developed a controlled pulsatile bioreactor that produces static and dynamic flow, which improves transfer of such nutrients and oxygen, and a tubular‐shaped vascular graft using cell matrix engineering. Electrospun scaffolds were seeded with smooth muscle cells (SMCs), cultured under dynamic or static conditions for 14 days, and analyzed. Mechanical examination revealed higher burst strength in the vascular grafts cultured under dynamic conditions than under static conditions. Also, immunohistology stain for alpa smooth muscle actin showed the difference of SMC distribution and existence on the scaffold between the static and dynamic culture conditions. The higher proliferation rate of SMCs in dynamic culture rather than static culture could be explained by the design of the bioreactor which mimics the physical environment such as media flow and pressure through the lumen of the construct. This supports regulation of collagen and leads to a significant increase in tensile strength of the engineered tissues. These results showed that the SMCs/electrospinning poly (lactide‐co‐ε‐caprolactone) scaffold constructs formed tubular‐shaped vascular grafts and could be useful in vascular tissue engineering.     

The Pharmacological Profile of a Novel Highly Potent Bisphosphonate,OX14 (1‐Fluoro‐2‐(Imidazo‐[1,2‐α]Pyridin‐3‐yl)‐Ethyl‐Bisphosphonate)

Bisphosphonates are widely used in the treatment of clinical disorders characterized by increased bone resorption, including osteoporosis, Paget's disease, and the skeletal complications of malignancy. The antiresorptive potency of the nitrogen‐containing bisphosphonates on bone in vivo is now recognized to depend upon two key properties, namely mineral binding affinity and inhibitory activity on farnesyl pyrophosphate synthase (FPPS), and these properties vary independently of each other in individual bisphosphonates. The better understanding of structure activity relationships among the bisphosphonates has enabled us to design a series of novel bisphosphonates with a range of mineral binding properties and antiresorptive potencies. Among these is a highly potent bisphosphonate, 1‐fluoro‐2‐(imidazo‐[1,2 alpha]pyridin‐3‐yl)‐ethyl‐bisphosphonate, also known as OX14, which is a strong inhibitor of FPPS, but has lower binding affinity for bone mineral than most of the commonly studied bisphosphonates. The aim of this work was to characterize OX14 pharmacologically in relation to several of the bisphosphonates currently used clinically. When OX14 was compared to zoledronate (ZOL), risedronate (RIS), and minodronate (MIN), it was as potent at inhibiting FPPS in vitro but had significantly lower binding affinity to hydroxyapatite (HAP) columns than ALN, ZOL, RIS, and MIN. When injected i.v. into growing Sprague Dawley rats, OX14 was excreted into the urine to a greater extent than the other bisphosphonates, indicating reduced short‐term skeletal uptake and retention. In studies in both Sprague Dawley rats and C57BL/6J mice, OX14 inhibited bone resorption, with an antiresorptive potency equivalent to or greater than the comparator bisphosphonates. In the JJN3‐NSG murine model of myeloma‐induced bone disease, OX14 significantly prevented the formation of osteolytic lesions (p < 0.05). In summary, OX14 is a new, highly potent bisphosphonate with lower bone binding affinity than other clinically relevant bisphosphonates. This renders OX14 an interesting potential candidate for further development for its potential skeletal and nonskeletal benefits. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.