Estudio de biocompatibilidad de matrices poliméricas combinadas, responsivas al pH, con aplicación en Ingeniería de Tejido Óseo / Biocompatibility study of combined pH-responsive polymeric matrices with application in Bone Tissue Engineering

El presente trabajo muestra la obtención de un material a partir de un polímero sintético (TerP) y otro natural, mediante entrecruzamiento físico y su caracterización fisicoquímica y biológica, con el fin de emplearlos para regeneración de tejido óseo. Las membranas fueron obtenidas por la técnica de evaporación del solvente y caracterizadas por espectroscopia FTIR, ensayos de hinchamiento, medidas de ángulo de contacto y microscopia electrónica de barrido (SEM). Se encontró que la compatibilidad entre los polímeros que la constituyen es estable a pH fisiológico y que, al incorporar mayor cantidad del TerP a la matriz, esta se vuelve más hidrofóbica y porosa. Además, teniendo en cuenta la aplicación prevista para dichos materiales, se realizaron estudios de biocompatibilidad y citotoxicidad con células progenitoras de médula ósea (CPMO) y células RAW264.7, respectivamente. Se evaluó la proliferación celular, la producción y liberación de óxido nítrico (NO) al medio de cultivo durante 24 y 48 horas y la expresión de citoquinas proinflamatorias IL-1ß y TNF-α de las células crecidas sobre los biomateriales variando la cantidad del polímero sintético. Se encontró mayor proliferación celular y menor producción de NO sobre las matrices que contienen menos proporción del TerP, además de poseer una mejor biocompatibilidad. Los resultados de este estudio muestran que el terpolímero obtenido y su combinación con un polímero natural es una estrategia muy interesante para obtener un biomaterial con posibles aplicaciones en medicina regenerativa y que podría extenderse a otros sistemas estructuralmente relacionados. (AU)

In the present work, the preparation of a biomaterial from a synthetic terpolymer (TerP) and a natural polymer, physically crosslinked, is shown. In order to evaluate the new material for bone tissue regeneration, physicochemical and biological characterizations were performed. The membranes were obtained by solvent casting and characterized using FTIR spectroscopy, swelling tests, contact angle measurements, and scanning electron microscopy (SEM). It was found that the compatibility between the polymers is stable at physiological pH and the incorporation of a higher amount of TerP into the matrix increases hydrophobicity and porosity.Furthermore, considering the intended application of these materials, studies of biocompatibility and cytotoxicity were conducted with Bone Marrow Progenitor Cells (BMPCs) and RAW264.7 cells, respectively. Cell proliferation, NO production and release into the culture medium for 24 and 48 hours, and proinflammatory cytokine expression of IL-1ß and TNF-α from cells grown on the biomaterials while varying the amount of the synthetic polymer were evaluated. Greater cell proliferation and lower NO production were found on matrices containing a lower proportion of TerP, in addition to better biocompatibility. The results of this study demonstrate that the obtained terpolymer and its combination with a natural polymer is a highly interesting strategy for biomaterial preparation with potential applications in regenerative medicine. This approach could be extended to other structurally related systems. (AU)

Feasibility of corneal epithelial transplantation with polyethylene glycol hydrogel membrane as a carrier for limbal stem cell deficiency / 中华实验眼科杂志

Objective:To investigate whether polyethylene glycol hydrogel films (PHFs) can be used as a carrier for the expansion of corneal epithelial cells (CECs) in vitro and whether PHFs can be used in the treatment of limbal stem cell deficiency (LSCD). Methods:Sebacoyl chloride, dihydroxyl PCL and glycerol ethoxylate were used to synthesize PHFs.The thickness, transmittance and mechanical tensile properties of PHFs were measured.Four clean-grade New Zealand white rabbits were selected to culture primary limbal epithelial cells.The expression of keratin marker AE1/AE3 and stem cell marker p63 in the cultured cells were observed under a fluorescence microscope.The cells were divided into negative control group cultured with common cell culture solution, positive control group cultured with cell culture solution containing 100 μmol/L H 2O 2, and PHFs+ CECs group lined with PHFs cultured with common cell culture solution for 24 hours.The proliferation and apoptosis of cells in the three groups were observed by MTT and TUNEL staining, respectively.Fifteen clean-grade New Zealand white rabbits were divided into control group, PHFs group and PHFs+ CECs group by random number table method, with 5 rabbits in each group.LSCD model was constructed in the three groups.The control group was not given any treatment after modeling.In PHFs group, empty PHFs were placed on the corneal surface of rabbits.In PHFs+ CECs group, tissue-engineered grafts constructed with CECs after passage implanted on PHFs were placed on the corneal surface of rabbits.The corneal defect area of rabbits was detected and scored by fluorescein sodium staining.The histological characteristics of rabbits corneal epithelium was observed by hematoxylin-eosin staining.The use and care of animals complied with Guide for the Care and Use of Laboratory Animals by the U. S.National Research Council.The experimental protocol was approved by the Research and Clinical Trial Ethics Committee of The First Affiliated Hospital of Harbin Medical University (No.2021006). Results:The synthetic PHFs were with a thickness ≤150 μm, a tensile strength about 6 MPa, and a transmittance over than 99% in the range of 400-700 nm.Most of the cells from primary culture of limbal tissue were positive for AE1/AE3 and p63.MTT test results showed that the A490 value of PHFs+ CECs group, negative control group and positive control group was 0.59±0.01, 0.65±0.07 and 0.06±0.04, respectively, showing a statistically significant overall difference ( F=12.25, P<0.05). The A490 values of PHFs+ CECs group and negative control group were significantly higher than that of positive control group, and the differences were statistically significant (both at P<0.05). TUNEL test results showed that there was a significant difference in the TUNEL-positive cell rate among the three groups ( F=13.45, P<0.05), and the rates of TUNEL-positive cells in PHFs+ CECs group and negative control group were significantly lower than that in positive control group (both at P<0.05). Fluorescein sodium staining results showed that with the extension of postoperative period, the corneal fluorescein sodium staining score of the three groups decreased, which decreased successively in control group, PHFs group and PHFs+ CECs group.Hematoxylin-eosin staining showed fewer irregularly shaped corneal epithelial cells in the control group, and sparse single layer of corneal epithelial cells in some areas of the PHFs group.In PHFs+ CECs group, the corneal epithelium coverage was the largest, and the cell layers increased to 3-5, and the cells were with regular morphology and in close arrangement. Conclusions:PHFs have enough toughness, high transmittance and can expand corneal epithelium in vitro.PHFs are suitable for corneal epithelial transplantation and can promote the repair of corneal epithelium in rabbit model of LSCD.

A comparative study of antihyperglycemic effect of Gymnema sylvestre and metformin in streptozotocin induced diabetic rats

Background: Diabetes mellitus (DM) is a metabolic disorder that has the phenotype of hyperglycemia. According to World Health Organization (WHO) there were 65.1 million diabetics in India in 2013, International Diabetes Federation estimates this to increase to 190 million by 2035. Although a number of drugs are available for treatment of DM, their cost and safety profile are major concern. Medicinal plants are used by clinicians for treatment of diabetes. Gymnema sylvestre (GS) extract has been reported to increase insulin levels in diabetic rats. This study was designed to compare the antihyperglycemic effect of Gymnema sylvestre with metformin.Methods: Diabetes was induced in Sprague-Dawley rats using streptozotocin 45mg/kg. Methanolic extract of Gymnema sylvestre 120mg/kg p.o. prepared using Soxhlet apparatus.Results: GS extract reduced blood glucose levels but not statistically significant. GS extract increased HDL and triglycerides, reduced both serum ALT and AST but no statistical significance seen. Metformin significantly increased serum urea, which was not seen in GS extract group. GS extract showed regenerative changes in pancreas, liver and kidney.Conclusions: The study investigation demonstrates that methanolic extract of GS possesses antihyperglycemic and hypolipidaemic activity and so it can be considered as a promising natural remedy in a prediabetic state and in mild hyperlipidaemia to prevent its progression. Increase in ? cell regeneration activity could be a probable mechanism of action. However, further long term clinical studies are recommended to define its possible role in diabetes mellitus and hyperlipidaemia. Role of GS as a potential hepatoprotective agent also needs further evaluation.

Combination of stromal vascular fraction and Ad-COMP-Ang1 gene therapy improves long-term therapeutic efficacy for diabetes-induced erectile dysfunction / 亚洲男科学杂志(英文版)

Men with diabetic erectile dysfunction (ED) respond poorly to the currently available oral phosphodiesterase-5 inhibitors. Therefore, functional therapies for diabetic ED are needed. Stromal vascular fraction (SVF) and the adenovirus-mediated cartilage oligomeric matrix angiopoietin-1 (Ad-COMP-Ang1) gene are known to play critical roles in penile erection. We previously reported that SVF and Ad-COMP-Ang1 have only a short-term effect in restoring erectile function. Further improvements to ED therapy are needed for long-lasting effects. In the present study, we aimed to test if the combination of SVF and Ad-COMP-Ang1 could extend the erection effect in diabetic ED. We found that the combination therapy showed a long-term effect in restoring erectile function through enhanced penile endothelial and neural cell regeneration. Combination therapy with SVF and Ad-COMP-Ang1 notably restored cavernous endothelial cell numbers, pericyte numbers, endothelial cell-cell junctions, decreased cavernous endothelial cell permeability, and promoted neural regeneration for at least 4 weeks in diabetic mice. In summary, this is an initial description of the long-term effect of combination therapy with SVF and Ad-COMP-Ang1 in restoring erectile function through a dual effect on endothelial and neural cell regeneration. Such combination therapy may have therapeutic potential for the treatment of diabetic ED.

Células madre: buscando marcadores de superficie celular que predispongan compromiso de diferenciación cardiaca / Stem cells: searching predisposition to cardiac commitment by surface markers expression

Resumen Actualmente las enfermedades cardiovasculares se han convertido en un serio problema para los sistemas de salud de todo el mundo, ya que son la principal causa de muerte y representan una enorme carga económica. Este problema ha sido abordado con diferentes estrategias, entre ellas con la ayuda de terapia celular, aunque sin resultados contundentes. Durante más de 20 años, se ha utilizado una gran variedad de células madre en diferentes modelos de infarto del miocardio. El uso de células madre cardiacas (CSC) parece ser la mejor opción, pero la inaccesibilidad y la escasez de estas células hacen que su uso sea muy limitado. Además, existe un riesgo elevado pues tienen que obtenerse directamente del corazón del paciente. A diferencia de las CSC, las células madre adultas derivadas de médula ósea o tejido adiposo, entre otras, representan una opción atractiva debido a su fácil accesibilidad y abundancia, pero sobre todo a la probable existencia de progenitores cardiacos entre sus diferentes subpoblaciones. En esta revisión hacemos un análisis de los marcadores de superficie presentes en CSC en comparación con otras células madre adultas, y sugerimos la preexistencia de células que comparten marcadores de superficie específicos con CSC, la presencia de un inmunofenotipo predecible, aunque en proporciones bajas, pero con un potencial de diferenciación cardiaca similar a las CSC, lo cual podría aumentar su valor terapéutico. Este estudio revela las nuevas perspectivas con respecto a la presencia de dichos marcadores, los cuales comprometerían algunas de estas subpoblaciones a diferenciarse a tejido cardiaco.

Abstract It is well-known that cardiovascular diseases are the leading cause of death world- wide, and represent an important economic burden to health systems. In an attempt to solve this problem, stem cell therapy has emerged as a therapeutic option. Within the last 20 years, a great variety of stem cells have been used in different myocardial infarction models. Up until now, the use of cardiac stem cells (CSCs) has seemed to be the best option, but the inaccessibility and scarcity of these cells make their use unreliable. Additionally, there is a high risk as they have to be obtained directly from the heart of the patient. Unlike CSCs, adult stem cells originating from bone marrow or adipose tissue, among others, appear to be an attractive option due to their easier accessibility and abundance, but particularly due to the probable existence of cardiac progenitors among their different sub-populations. In this review an analysis is made of the surface markers present in CSCs compared with other adult stem cells. This suggested the pre-existence of cells sharing specific surface markers with CSCs, a predictable immunophenotype present in some cells, although in low proportions, and with a potential of cardiac differentiation that could be similar to CSCs, thus increasing their therapeutic value. This study highlights new perspectives regarding MSCs that would enable some of these sub-populations to be differentiated at cardiac tissue level.

Advances in the biological restoration of the hearing / 复旦学报（医学版）

Sensorineural deafness is the most common disorders of senses,which severely impact the life quality and bring heavy burden to the family as well as the society.Current state-of-art treatments focus on sound amplification and implanted electrodes that stimulate the auditory nerve.These strategies offer partial recovery of function for a limited patient population but do not come close to restoring natural hearing.Clearly,there is strong need for development of biological treatments for the hearing restoration through correcting the genetic defects or regenerating the new hair cells on the damaged cochlear sensory epithelium.We reviewed the advances in the biological restoration of the hearing,including the gene therapy and the hair cell regeneration aspects.

Advances in pancreatic β cell regeneration and transformation / 国际儿科学杂志

Type 1 diabetes mellitus (T1DM) is an organ-specific autoimmune disease caused by absolute lack of insulin secretion and progressive pancreatic β cell damage.Patients require lifelong subcutaneous injection of insulin.However,this treatment could induce great pain without improving long-term prognosis.Along with the medicine development,comprehensive methods including genetic technology are adopted to achieve pancreatic β cell regeneration and transformation.Protecting the function of remaining pancreatic β cells for T1DM patients provides a new insight in prevention of acute and chronic T1DM complications.Currents studies of islet β cell reconstruction focus on islet transplantation,stem cell transplantation and immune regulator applications.Unfortunately,owing to their limitations,these methods cannot satisfy clinical application.While transdifferentiation of pancreatic islet δ cells has become a hope recently because of its unique advantages.In this paper,we will summarize the technical methods,influence factors and possible mechanisms of the transdifferentiation of pancreatic islet δ cells.

Development of the endocrine pancreas and novel strategies for beta-cell mass restoration and diabetes therapy

Diabetes mellitus represents a serious public health problem owing to its global prevalence in the last decade. The causes of this metabolic disease include dysfunction and/or insufficient number of β cells. Existing diabetes mellitus treatments do not reverse or control the disease. Therefore, β-cell mass restoration might be a promising treatment. Several restoration approaches have been developed: inducing the proliferation of remaining insulin-producing cells, de novo islet formation from pancreatic progenitor cells (neogenesis), and converting non-β cells within the pancreas to β cells (transdifferentiation) are the most direct, simple, and least invasive ways to increase β-cell mass. However, their clinical significance is yet to be determined. Hypothetically, β cells or islet transplantation methods might be curative strategies for diabetes mellitus; however, the scarcity of donors limits the clinical application of these approaches. Thus, alternative cell sources for β-cell replacement could include embryonic stem cells, induced pluripotent stem cells, and mesenchymal stem cells. However, most differentiated cells obtained using these techniques are functionally immature and show poor glucose-stimulated insulin secretion compared with native β cells. Currently, their clinical use is still hampered by ethical issues and the risk of tumor development post transplantation. In this review, we briefly summarize the current knowledge of mouse pancreas organogenesis, morphogenesis, and maturation, including the molecular mechanisms involved. We then discuss two possible approaches of β-cell mass restoration for diabetes mellitus therapy: β-cell regeneration and β-cell replacement. We critically analyze each strategy with respect to the accessibility of the cells, potential risk to patients, and possible clinical outcomes.

Experimental research on the characteristics of pancreatic cells regeneration in an animal model for isolated pancreatic trauma / 重庆医学

Objective To study the relationships between tissue damage and the ability of the pancreatic cells to regenerate ,and analyze the alteration of the pancreatic cells regeneration .Methods Sixty rats were divided into two groups :impact group(the pan‐creas was injured by a BIM‐Ⅲ biotical impact machine ,40 rats) and control group(sham operated ,20 rats) .All rats were sacrificed at 6 h ,24 h ,72 h ,7 d after operation .The level of AMS ,LPS in the serum were detected by spectrophotometry ,pancreatic cells re‐generation were examined and analyzed by TUNEL staining and flow cytomertry ,and the Bcl‐2 and Bax expression were measured by Western blot .Results In the impact groups ,LPS was activated later than AMS ,and lasted persistently .The results from TUNEL stain ,flow cytometry and Western blot indicated that pancreatic trauma induces cell death and the compensatory prolifera‐tion of pancreatic cells .The characteristics of pancreatic cells regeneration in the animal model of isolated pancreatic trauma indicate that the proper remedial time is in the first 24h after the pancreatic trauma .Conclusion Detecting AMS and LPS at the same time can help us to determine the exocrine function of pancrease .

Human Bone Marrow Subpopulations Sustain Human Islet Function and Viability In vitro.

Aims: Allogeneic bone marrow (BM) has been shown to support human islet survival and function in long-term culture by initiating human islet vascularization and β-cell regeneration. Various BM subpopulations may play different roles in human islet functions and survival. In this paper we investigated the effects of BM and its subpopulations, endothelial progenitor cells (E) and mesenchymal (M) cells on human islet’s β-cell function and regeneration. Study Design: Isolation and identification of subpopulations from human bone marrow and culture with allogeneic human islet to investigate effects of different cell population on human islet function and regeneration. Place and Duration of Study: Department of Medicine, Center for Stem Cell & Diabetes Research, RWMC, Providence, RI, USA, between 2010 - 2014. Methodology: Human islets were distributed from Integrated Islet Distribution Program (IIDP) and human bone marrow (BM) was harvested by Bone marrow transplantation center at Roger Williams Hospital. BM subpopulation was identified cell surface markers through Fluorescenceactivated cell sorting, applied in flow cytometry (FACS), islet function was evaluated by human ELISA kit and β cell regeneration was evaluated by three methods of Cre-Loxp cell tracing, β cell sorting and RT-PCR for gene expression. Results: Four different BM and seven different islet donates contributed human tissues. We observed islet β-cell having self regeneration capability in short term culture (3~5 days) using a Cre-Loxp cell tracing. BM and its subtype E, M have similar benefits on β cell function during coculture with human islet comparison to islet only. However, only whole BM enables to sustain the capability of islet β-cell self regeneration resulting in increasing β cell population while single E and M individual do not significantly affect on that. Mechanism approach to explore β-cell self regeneration by evaluating transcription factor expressions, we found that BM significantly increases the activations of β-cell regeneration relative transcription factors, the LIM homeodomain protein (Isl1), homologue to zebrafish somite MAF1 (MAFa), the NK-homeodomain factor 6.1 (NKX6.1), the paired box family factors 6 (PAX6), insulin promoter factor 1 (IPF1) and kinesin family member 4A (KIF4a). Conclusion: These results suggest that BM and its derived M and E cells enable to support human islet β-cell function. However, only BM can sustain the capability of β-cell self regeneration through initiating β-cell transcriptional factors but not individual E and M cells suggesting pure E and M cells less supportive for islet long-term survival in vitro.

In vitro Enhancement of Pancreatic  Cells MIN6 Proliferation by Insulinotropic Gymnema sylvestre Aqueous Extracts: Evidence-based Regenerative Therapeutic Capacity of a Medicinal Herb.

Aim: To show that Gymnema sylvestre (Roxb.) Asclepiadaceae not only has antidiabetic propensities, but it most likely works by regeneration of pancreatic  cells which is imperative in anti-obesity-diabetes therapeutic applications of medicinal plants. Study Design: The present study design investigated the effects of G. sylvestre leaves crude aqueous extracts (AEs), traditionally utilized in diabetes treatment, on the pancreatic β-cell MIN6 proliferation and insulin secretion and extrapancreatic dietary carbohydrate and lipid digestion Place and Duration of Study: Faculty of Pharmacy, The University of Jordan, 2008-2012. Results: Comparable to GLP-1 (500 nM) pancreatic proliferative capacity; G. sylvestre AE concentrations (0.01 and 0.1 mg/mL) induced MIN6 monolayers expansion by respective 130.3% and 127.4% (P<0.001 vs. spontaneous control). Like L-alanine (10 mM) insulinotropic efficacy and without exerting cytotoxicity, glucose-stimulated insulin secretion was potentiated by G. sylvestre AEs (5, 10 and 25 mg/mL) (711.0%, 843.0% and 906.5%, respectively, P<0.001 vs. basal control). The potent plants’ insulin secretory bioactivities were abolished in the depleted Ca2+ conditions (P<0.001). Similar to orlistat antilipolytic efficacy, pancreatic lipase IC50 value for G. sylvestre AEs was 106.3±7.2 μg/mL. Unlike acarbose (100 μg/mL) dual inhibition of α-amylase/α-glucosidase, G. sylvestre AE was inactive at used doses. Dissimilar to guar gum (50 mg/mL) diffusional hindrance in a simple dialysis model, G. sylvestre AEs (10, 25 and 50 mg/mL) proved inactive. This in vitro ineffectiveness was mirrored in respective in vivo oral carbohydrate tolerance tests in overnight fasting normoglycemic rats. Conclusion: This evaluation has revealed that G. sylvestre leaves AEs augmented β-cell expansion and potentiated glucose-evoked Ca2+-regulated insulin secretion; combined with impressive antilipolytic activity. These actions depend on the bioactive water soluble phytoprinciples intact absorption in vivo. Future directives may assess the potential of G. sylvestre as a new alternative for anti-obesity-diabetes pharmacotherapy and prevention.

Evaluation of antidiabetic antihyperlipidemic and pancreatic regeneration, potential of aerial parts of Clitoria ternatea

The aim of the present study was to investigate the pancreatic regeneration potential of of diferent fractions of the ethanol extract Clitoria ternatea L., Fabaceae. The antidiabetic and antihyperlipidemic potential was evaluated in streptozotocin-induced diabetic rats and correlated with its in vivo and in vitro antioxidant activity. The extract and its fractions were initially screened for acute and sub-chronic antidiabetic activity in the dose range of 100200 mg/kg. The most potent extract and fractions were further evaluated for pancreatic β-cells regeneration activity along with antioxidant and antihyperlipidemic activity. The polyphenolic, flavonoid and flavanone contents were assessed and correlated with its antidiabetic activity. The most significant pancreatic regeneration activity, antidiabetic and antihyperlipidemic activity and was shown by ethanol extract and butanol soluble fraction at a dose level of 200 mg/kg, while rutin was found to be least potent. In conclusion, pancreatic regeneration studies of ethanol extract treated rats show nesidioblastosis. It is also suggested that the factors causing regeneration are present within the pancreas. The newly generated islets may have formed from the ductal precursor cells and reduced oxidative stress helps in restoration of β-cell function.

The Expression of Ephrin A2 during the Reinnervation and the Regeneration of the Hair Cells in the Chick Cochlea following Kanamycin Ototoxicity / 听力学及言语疾病杂志

Objective To investigate the role of Ephrin A2 in the regeneration and reinnervation of hair cells in the chick cochlea following kanamycin ototoxicity.Methods 66 newly hatched Roman chickens (3 days old) were randomly divided into experimental group and control groups. Experimental chickens (n=48) received intramuscular kanamycin (200 mg/kg:Sigma,St Louis,MO) for 10 consecutive days and were subsequently sacrificed 2 days before the last injection,and 1,3,7,10,15,21,30,and 60 days after the last injection (n=6 per subgroup). Control chickens (n=18) were untreated and sacrificed 3,13 and 43 days after hatching (n=6 per subgroup). Ephrin A2 protein expression in acoustic ganglia was determined by western blot analysis in all chickens after sacrifice. Results Ephrin A2 protein expression was found and the protein level was almost same in acoustical ganglia of all normal chickens. After kanamycin exposure,the Ephrin A2 protein expression level in the cochlea of the experiment chickens from 2 to 7 days after last kanamycin injection was lower than that in control chickens,respectively. Ephrin A2 expression increased obviously at 15 days after kanamycin last injection. By 30 days after the cessation of kanamycin treatment,the level of Ephrin A2 protein approximated to that in normal control group.Conclusion The expression of Ephrin A2 protein in the acoustical ganglia basically synchronizes with the regeneration and the reinnervation of the hair cells in the chicken cochlea following kanamycin damage,indicating that Ephrin A2 may play an important role in this process.