A novel controlled drug-delivery system for growth hormone applied to healing skin wounds in diabetic rats

Controlled release systems for drugs, hormones and growth factors can be particularly useful in tissue repair processes. These systems act as a biodegradable support containing the substance to be delivered, allowing their gradual release. In the past years, the local application of growth factors has acquired special relevance as a therapeutic option for use in subjects who show deficient tissue scarring, the hormone dose being the limiting factor for its success. In this study, the in vitro biocompatibility of a copolymer formed by vinylpyrrolidone and 2-hydroxyethyl methacrylate, used as an administration vehicle for hGH, was evaluated. The system was then tested in vivo in terms of its capacity for healing incisional wounds in healthy and diabetic rats. For the in vitro studies, polymer and hormone degradation rates were determined, and polymer biocompatibility was evaluated in fibroblast cultures. In the in vivo experiments, an incision was made in the back of the animals, and polymers discs with/without hGH, were introduced in the aperture. Morphological, immunohistochemical and morphometric evaluations were performed on wound tissue specimens 3-10 days after surgery. In vitro, the polymer was found to be biodegradable and showed no toxic effects on fibroblasts, the hormone being slowly released to the culture medium. In untreated diabetic rats, a delayed skin scarring and cell response were observed, compared to that noted in healthy animals. Skin closure, keratinisation and fibrosis occurred earlier in the presence of the polymer-hGH system. The use of this co-polymer as an administration vehicle for hGH improves the wound scarring process in the pathological setting of diabetes.     

Bioactive functionalized polymer of malic acid for bone repair and muscle regeneration

A bioactive poly(β-hydroxyalkanoate) derived from malic acid was prepared and tested on bone repair and muscle regeneration. This functionalized and hydrolyzable polymer was obtained after several steps, the first one being the anionic copolymerization of three malolactonic acid esters. Chemical modifications were carried out on the terpolymer to turn benzyl-protecting groups into carboxyl groups and allyl groups into sulfonate groups. The resulting polymer bore carboxylate, sulfonate, and sec-butyl pendent groups in 65/25/10 molar proportions and were aimed at interacting with heparan binding growth factors. This polymer did not present any toxic effect in cell viability of HepG₂ cells, over a large range of concentrations (0.01-0.25 mg l^-1). Its ability to improve wound healing was tested in vivo and positive results are reported. Furthermore, the bioactivity of this polymer was evaluated using the regeneration model of Extensor digitorum longus (EDL) rat muscle. The study displayed a significant increase in the muscle regeneration and maturation.     

Tacrolimus (FK506) and cyclosporin A reduce macrophage recruitment to the rat brain injured at perinatal and early postnatal periods

Abstract

Objective: The present article presents an overview of the literature, and analyses the methods and the primary questions related to assessment of proliferation index using the Ki-67/MIB-1 labeling index in pituitary adenomas. Although atypical adenomas are characterized by their atypical morphological features by an elevated mitotic index, a Ki-67 (MIB-1) labeling index greater than 3% and extensive nuclear staining for p53, use of the proliferation index (LI) of pituitary adenomas in assessing the degree of tumor aggressiveness is a controversial topic in the literature, and there are disparate results involving many studies.

Methods: A review of literature was carried out to correlate the role of Ki-67 LI and its correlation with clinical findings, tumor size, invasiveness, recurrence, adenoma subtype, adenoma doubling time, and pituitary carcinomas is addressed.

Results: The prognosis cannot be predicted on the basis of the Ki-67 LI alone. Although there is no direct relation between Ki-67 LI and some of these variables and controversial data were found regarding some topics, our review justify the use of Ki-67 in the analysis of pituitary adenomas as an additional information for clinical decision.

Conclusion: Although assessment of proliferative may be helpful in predicting subsequent tumor recurrence or invasiveness, there are many other important and as yet unidentified factors pituitary tumors. It is clear that further research is needed to clarify these molecular mechanisms to predict those with a potentially poor clinical outcome.     

Surgical Correction of Patent Ductus Venosus in Three BroThers

We report the presence of a patent ductusvenosus in three brothers who underwent surgicalcorrection. Patent ductus venosus was demonstrated byultrasonography. Portosystemic venous shunt ratios asevaluated by [¹²³I]iodoamphetamine per rectalportal scintigraphy were 67%, 50%, and 77%,respectively. Histologic examination of liver biopsyspecimens revealed fatty degeneration in all cases.Portal vein pressure before and after temporarily occluding thepatent ductus venosus was estimated by an Anthron P-Ucatheter introduced into the portal vein via theligament teres hepatis. Portal venous pressure increased from 10 to 17 cm H₂O, 16 to 23 cmH₂O, and 14 to 27 cm H₂O,respectively. Therefore, banding of the ductus venosuswith Teflon tape was attempted to prevent portalhypertension following complete ligation. As a result, portal venous pressures afterstricture of the ductus venosus were 12, 21, and 20 cmH2), respectively. Bile acid and liver enzymes decreasedand returned to normal within 14 days after surgery. Interestingly, serum concentrations ofhepatocyte growth factor (HGF) increased significantlyafter restoration of the portal blood flow and thengradually decreased, but remained persistently elevated for at least two weeks in two cases measuredafter surgical correction. One month after correction,liver function returned to normal as assessed byserological and histological parameters in all cases. These results suggest that it is important todetermine whether stricture or complete ligation isindicated for a patent ductus venosus during surgicalcorrection, based on the portal venous pressure after temporal test occlusion of the duct. Inaddition, HGF may be a useful marker for normalizationof hepatic microcirculation after surgery.     

The Effect of Aligned Core–Shell Nanofibres Delivering NGF on the Promotion of Sciatic Nerve Regeneration

Recent bioengineering strategies for peripheral nerve regeneration have been focusing on the development of alternative treatments for nerve repair. In this study, we incorporated nerve growth factor (NGF) into aligned core–shell nanofibres by coaxial electrospinning, and reeled the scaffold into aligned fibrous nerve guidance conduits (NGCs) for nerve regeneration study. This aligned PLGA/NGF NGC combined physical guidance cues and biomolecular signals to closely mimic the native extracellular matrix (ECM). The effect of this aligned PLGA/NGF NGC on the promotion of nerve regeneration was evaluated in a 13-mm rat sciatic nerve defect using functional and morphological analysis. After 12 weeks implantation, the results of electrophysiological and muscle weight examination demonstrated that the functional recovery of the regenerated nerve in the PLGA/NGF NGC group was significantly better than that in the PLGA group, yet had no significant difference compared with the autograft group. The toluidine blue staining study showed that more nerve fibres were regenerated in the PLGA/NGF group, while the electron microscopy study indicated that the regenerated nerve in the PLGA/NGF group was more mature than that in the PLGA group. This study demonstrated that the aligned PLGA/NGF could greatly promote peripheral nerve regeneration and have a potential application in nerve regeneration.     

The Effects of One-Step Self-Etch Adhesives on the Induction of Oxidative Stress and Production of TGF-β1 and BMP-2 by Human Gingival Fibroblasts

The aim of this study was to evaluate and compare the effects of two self-etch adhesive materials on the induction of oxidative stress and production of transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) by cultured human gingival fibroblasts (HGF). Inflammation-free attached gingiva was obtained from healthy donors under informed consent. Following 24- and 72-h exposure of HGF to two different elutes of the test materials, cell viability was determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Lipid peroxidation, a major indicator of oxidative stress, was measured by the thiobarbituric acid reactive substance (TBARS) assay. TGF-β1 and BMP-2 levels in cell-free culture media were determined by enzyme-linked immunosorbent assay (ELISA). Cell viability of the test groups was significantly lower than those of control at 24 and 72 h (P < 0.001), but showed an increase at 72 h (P < 0.001). The TBARS levels of both test groups were significantly greater than that of control (P < 0.05), and displayed similar values at 72 h (P > 0.05). For both materials, the levels of TGF-β1 and BMP-2 were significantly greater than that of control (P < 0.05). Both test groups showed increased TGF-β1 levels. These results indicate that the tested self-etch adhesives might be capable of inducing production of TGF-β1 and BMP-2 in cultured HGF, despite their cytotoxic and oxidative stress-producing potential.     

Novel Degradable Co-polymers of Polypyrrole Support Cell Proliferation and Enhance Neurite Out-Growth with Electrical Stimulation

Synthetic polymers such as polypyrrole (PPy) are gaining significance in neural studies because of their conductive properties. We evaluated two novel biodegradable block co-polymers of PPy with poly(ε-caprolactone) (PCL) and poly(ethyl cyanoacrylate) (PECA) for nerve regeneration applications. PPy–PCL and PPy–PECA co-polymers can be processed from solvent-based colloidal dispersions and have essentially the same or greater conductivity (32 S/cm for PPy–PCL, 19 S/cm for PPy–PECA) compared to the PPy homo-polymer (22 S/cm). The PPy portions of the co-polymers permit electrical stimulation whereas the PCL or PECA blocks enable degradation by hydrolysis. For in vitro tests, films were prepared on polycarbonate sheets by air brushing layers of dispersions and pressing the films. We characterized the films for hydrolytic degradation, electrical conductivity, cell proliferation and neurite extension. The co-polymers were sufficient to carry out electrical stimulation of cells without the requirement of a metallic conductor underneath the co-polymer film. In vitro electrical stimulation of PPy–PCL significantly increased the number of PC12 cells bearing neurites compared to unstimulated PPy–PCL. For in vivo experiments, the PPy co-polymers were coated onto the inner walls of nerve guidance channels (NGCs) made of the commercially available non-conducting biodegradable polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV). The NGCs were implanted in a 10 mm defect made in the sciatic nerve of rats, and harvested after 8 weeks. Histological staining showed axonal growth. The studies indicated that these new conducting degradable biomaterials have good biocompatibility and support proliferation and growth of PC12 cells in vitro (with and without electrical stimulation) and neurons in vivo (without electrical stimulation).     

Conditional Deletion of Indian Hedgehog in Limb Mesenchyme Results in Complete Loss of Growth Plate Formation but Allows Mature Osteoblast Differentiation

Indian hedgehog (Ihh) is widely recognized as an essential factor for proper skeletal development. Previous in vivo studies using mutant Ihh mouse models were limited by perinatal lethality or carried out after a growth plate formed. Thus the important role of Ihh in mesenchymal cell differentiation has not been investigated. In this study, we established Prx1‐Cre;Ihh^fl/fl mice to ablate Ihh specifically in limb mesenchyme to allow us to observe the phenotype continuously from prenatal development to 3 weeks of age. Mutant mice displayed severe limb abnormalities characterized by complete lack of secondary ossification center and growth plate, indicating an essential role for Ihh in the development of these structures. Interestingly, we discovered that osteoblast differentiation and bone formation could occur in conditions of deficient Ihh. This is a novel finding that has not been observed because of the early lethality of previous Ihh mutants. Mature osteoblasts expressing osteocalcin could be detected in the center of mutant bones at postnatal day 10 (P10). Osteoclasts and blood vessel formation were also present, suggesting active bone remodeling. Histomorphometric analyses show a significant increase in osteoclast number with no major changes in bone formation rate at 3 weeks of age. Mutant long bones in the limbs were deformed, with cortices comprised of irregular woven bone. Also, there was a marked decrease in gene expression of osteoblastic and osteocytic markers. Moreover, mutant long bones displayed bone dysplasia in which we observed increased osteoclast activity and partially reduced osteoblastic and osteocytic differentiation that lead ultimately to loss of bone structures at 3 weeks of age. In summary, our data show for the first time, the presence of mature osteoblasts in long bones of the limbs despite the complete loss of growth plate formation due to Ihh deficiency. These data indicate an important function for Ihh in regulating limb mesenchymal cell differentiation. © 2015 American Society for Bone and Mineral Research.     

ER Stress During the Pubertal Growth Spurt Results in Impaired Long‐Bone Growth in Chondrocyte‐Specific ERp57 Knockout Mice

Long‐bone growth by endochondral ossification is cooperatively accomplished by chondrocyte proliferation, hypertrophic differentiation, and appropriate secretion of collagens, glycoproteins, and proteoglycans into the extracellular matrix (ECM). Before folding and entering the secretory pathway, ECM macromolecules in general are subject to extensive posttranslational modification, orchestrated by chaperone complexes in the endoplasmic reticulum (ER). ERp57 is a member of the protein disulfide isomerase (PDI) family and facilitates correct folding of newly synthesized glycoproteins by rearrangement of native disulfide bonds. Here, we show that ERp57‐dependent PDI activity is essential for postnatal skeletal growth, especially during the pubertal growth spurt characterized by intensive matrix deposition. Loss of ERp57 in growth plates of cartilage‐specific ERp57 knockout mice (ERp57 KO) results in ER stress, unfolded protein response (UPR), reduced proliferation, and accelerated apoptotic cell death of chondrocytes. Together this results in a delay of long‐bone growth with the following characteristics: (1) enlarged growth plates; (2) expanded hypertrophic zones; (3) retarded osteoclast recruitment; (4) delayed remodeling of the proteoglycan‐rich matrix; and (5) reduced numbers of bone trabeculae. All the growth plate and bone abnormalities, however, become attenuated after the pubertal growth spurt, when protein synthesis is decelerated and, hence, ERp57 function is less essential. © 2015 American Society for Bone and Mineral Research.     

Hyperactive Transforming Growth Factor‐β1 Signaling Potentiates Skeletal Defects in a Neurofibromatosis Type 1 Mouse Model

Dysregulated transforming growth factor beta (TGF‐β) signaling is associated with a spectrum of osseous defects as seen in Loeys‐Dietz syndrome, Marfan syndrome, and Camurati‐Engelmann disease. Intriguingly, neurofibromatosis type 1 (NF1) patients exhibit many of these characteristic skeletal features, including kyphoscoliosis, osteoporosis, tibial dysplasia, and pseudarthrosis; however, the molecular mechanisms mediating these phenotypes remain unclear. Here, we provide genetic and pharmacologic evidence that hyperactive TGF‐β1 signaling pivotally underpins osseous defects in Nf1^flox/?;Col2.3Cre mice, a model which closely recapitulates the skeletal abnormalities found in the human disease. Compared to controls, we show that serum TGF‐β1 levels are fivefold to sixfold increased both in Nf1^flox/?;Col2.3Cre mice and in a cohort of NF1 patients. Nf1‐deficient osteoblasts, the principal source of TGF‐β1 in bone, overexpress TGF‐β1 in a gene dosage–dependent fashion. Moreover, Nf1‐deficient osteoblasts and osteoclasts are hyperresponsive to TGF‐β1 stimulation, potentiating osteoclast bone resorptive activity while inhibiting osteoblast differentiation. These cellular phenotypes are further accompanied by p21‐Ras–dependent hyperactivation of the canonical TGF‐β1–Smad pathway. Reexpression of the human, full‐length neurofibromin guanosine triphosphatase (GTPase)‐activating protein (GAP)‐related domain (NF1 GRD) in primary Nf1‐deficient osteoblast progenitors, attenuated TGF‐β1 expression levels and reduced Smad phosphorylation in response to TGF‐β1 stimulation. As an in vivo proof of principle, we demonstrate that administration of the TGF‐β receptor 1 (TβRI) kinase inhibitor, SD‐208, can rescue bone mass deficits and prevent tibial fracture nonunion in Nf1^flox/?;Col2.3Cre mice. In sum, these data demonstrate a pivotal role for hyperactive TGF‐β1 signaling in the pathogenesis of NF1‐associated osteoporosis and pseudarthrosis, thus implicating the TGF‐β signaling pathway as a potential therapeutic target in the treatment of NF1 osseous defects that are refractory to current therapies. © 2013 American Society for Bone and Mineral Research.