Hepatocellular carcinoma patients serum modulates the regenerative capacities of adipose mesenchymal stromal cells.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers causing the highest mortality rate worldwide. Treatment options of surgery, radiation, cytotoxic drugs and liver transplantation suffer significant side effects and a high frequency of relapse. Stem cell therapy has been proposed as a new effective therapy, however, controversial reports are emerging on the role of mesenchymal stem cells in cancer. In this work, we aimed to assess the regenerative capacities of adipose mesenchymal stem cells when exposed to serum from HCC patients, by assessing the effect of the sera on modulating the regenerative capacities of h-AMSCs and the cancer properties in HCC cells. This will pave the way for maximizing the efficacy of MSCs in cancer therapy. Our data show that HCC serum-treated hA-MSCs suffered oncogene-induced senescence as shown by their altered morphology and ameliorated proliferation and differentiation. The cells were enlarged with small irregular nuclei, swollen rough endoplasmic reticulum cisternae, and aging lysosomes typified by dark residual bodies. HCC serum-treated Huh-7 cancer cells on the other hand displayed higher tumor aggressiveness as depicted by altered morphology, increased cellular proliferation and migration, and decreased percentage of early and late apoptotic cells. Our findings provide evidence that exposure of hA-MSCs to the serum of HCC patients decreases their regenerative capacities and should be considered when employed as a potential therapy in HCC patients.

The cross talk between type II diabetic microenvironment and the regenerative capacities of human adipose tissue-derived pericytes: a promising cell therapy.

BACKGROUND: Pericytes (PCs) are multipotent contractile cells that wrap around the endothelial cells (ECs) to maintain the blood vessel's functionality and integrity. The hyperglycemia associated with Type 2 diabetes mellitus (T2DM) was shown to impair the function of PCs and increase the risk of diabetes complications. In this study, we aimed to investigate the deleterious effect of the diabetic microenvironment on the regenerative capacities of human PCs. METHODS: PCs isolated from human adipose tissue were cultured in the presence or absence of serum collected from diabetic patients. The functionality of PCs was analyzed after 6, 14, and 30 days. RESULTS: Microscopic examination of PCs cultured in DS (DS-PCs) showed increased aggregate formation and altered surface topography with hyperbolic invaginations. Compared to PCs cultured in normal serum (NS-PCs), DS-PCs showed more fragmented mitochondria and thicker nuclear membrane. DS caused impaired angiogenic differentiation of PCs as confirmed by tube formation, decreased VEGF-A and IGF-1 gene expression, upregulated TSP1, PF4, actin-related protein 2/3 complex, and downregulated COL21A1 protein expression. These cells suffered more pronounced apoptosis and showed higher expression of Clic4, apoptosis facilitator BCl-2-like protein, serine/threonine protein phosphatase, and caspase-7 proteins. DS-PCs showed dysregulated DNA repair genes CDKN1A, SIRT1, XRCC5 TERF2, and upregulation of the pro-inflammatory genes ICAM1, IL-6, and TNF-α. Further, DS-treated cells also showed disruption in the expression of the focal adhesion and binding proteins TSP1, TGF-ß, fibronectin, and PCDH7. Interestingly, DS-PCs showed resistance mechanisms upon exposure to diabetic microenvironment by maintaining the intracellular reactive oxygen species (ROS) level and upregulation of extracellular matrix (ECM) organizing proteins as vinculin, IQGAP1, and tubulin beta chain. CONCLUSION: These data showed that the diabetic microenvironment exert a deleterious effect on the regenerative capacities of human adipose tissue-derived PCs, and may thus have possible implications on the vascular complications of T2DM. Nevertheless, PCs have shown remarkable protective mechanisms when initially exposed to DS and thus they could provide a promising cellular therapy for T2DM.

Effectiveness of three different local routes of dexamethasone administration on postoperative sequelae following mandibular third molar surgery. A prospective randomised single-blind clinical study / Eficácia de três diferentes vias locais de administração de dexametasona em sequelas pós-operatórias após cirurgia de terceiros molares inferiores. Um estudo clínico prospectivo randomizado simples-cego

Objective: The aim of this study was to provide evidence for comparing the effectiveness of three different routes of local administration of Dexamethasone on the postoperative pain, edema and trismus following surgical removal of impacted mandibular third molar. Material and Methods: Forty-five patients underwent surgical removal of impacted lower third molars and were randomly allocated postoperatively into 3 groups: 8 mg of dexamethasone injected into the submucosa of the vestibule near the surgical site (group I), 8 mg of dexamethasone injected into the pterygomandibular space (group II) and 10 mg of dexamethasone powder applied to the extraction site, after bleeding control (group III). Facial swelling and maximal interincisal opening were measured at preoperatively. Pain was measured by the patient response to a visual analogue scale. Pain perception, Facial edema and trismus were evaluated for one week postoperatively. Results: There was no significant difference between the three groups concerning pain after 1, 2, 5, 7 days of follow up. However, group II showed less pain at 3 and 4 days. The difference between edema measurements was not significant in the three groups at 1, 5, 7 days, though in group I and II edema subsided from day 2. As for trismus, group I and III showed statistically significant lower maximum interincisal opening measurement than group II after two days. Conclusion: Local administration of Dexamethasone through three different routes is beneficial in decreasing postoperative sequelae following third molar surgery. Pterygomandibular space injection of Dexamethasone resulted in earlier resolution of pain, and less facial edema and trismus at the second postoperative day compared to the submucosal injection and transalveolar application. However, at one week the difference in measurements of the three variables between the groups was not significant. (AU)

Objetivo: O objetivo deste estudo foi fornecer evidências para comparar a eficácia de três diferentes vias de administração local de dexametasona na dor pós-operatória, edema e trismo após a remoção cirúrgica do terceiro molar inferior impactado. Material e Métodos: Quarenta e cinco pacientes foram submetidos à remoção cirúrgica de terceiros molares inferiores impactados e distribuídos aleatoriamente no pós-operatório em 3 grupos: 8 mg de dexametasona injetados na submucosa vestíbular próximo ao local da cirurgia (grupo I), 8 mg de dexametasona injetados no espaço pterigomandibular (grupo II) e 10 mg de pó de dexametasona aplicados no local da extração, após o controle do sangramento (grupo III). Edema facial e abertura interincisal máxima foram medidos no pré-operatório. A dor foi medida pela resposta do paciente a uma escala visual analógica. Percepção de dor, edema facial e trismo foram avaliados por uma semana de pós-operatório. Resultados: Não houve diferença significativa entre os três grupos em relação à dor após 1, 2, 5, 7 dias de acompanhamento. No entanto, o grupoII mostrou menos dor em 3 e 4 dias. A diferença entre as medidas de edema não foi significativa nos três grupos em 1, 5, 7 dias, embora nos grupos I e II o edema cedeu a partir do dia 2. Quanto ao trismo, os grupos I e III apresentaram medida de abertura interincisal máxima inferior estatisticamente significativa do que o grupo II depois de dois dias. Conclusão: A administração local de dexametasona por três vias diferentes é benéfica na redução das sequelas pós-operatórias após a cirurgia do terceiro molar. A injeção de dexametasona no espaço pterigomandibular resultou na resolução mais precoce da dor e menos edema facial e trismo no segundo dia de pós-operatório em comparação com a injeção submucosa e a aplicação transalveolar. No entanto, em uma semana, a diferença nas medidas das três variáveis entre os grupos não foi significativa.(AU)

Human Adipose-Derived Pericytes: Biological Characterization and Reprogramming into Induced Pluripotent Stem Cells.

BACKGROUND/AIMS: Pericytes (PCs) are multipotent vascular precursors that play a critical physiological role in the development and maintenance of blood vessel integrity. In this study, we aim to characterize PCs isolated from human abdominal adipose tissue and develop an integration-free induced pluripotent stem cells (iPSCs) using episomal vectors. METHODS: The ultrastructure of adipose tissue-derived PCs was determined using scanning and transmission electron microscopy. The expression of mesenchymal stem cells (MSCs) and pericyte markers were examined using flow cytometry and PCR analysis. PCs were induced to adipogenic, osteogenic and myogenic lineages, and their angiogenic potential was determined using tube formation assay. We further established pericyte reprogramming protocol using episomal vectors. RESULTS: Our data showed that human adipose tissue-derived PCs uniformly expressed MSCs, CD105 and CD73, and PCs markers, desmin, and alpha smooth muscle actin (α-SMA), while lacked the expression of HLA-DR and the hematopoietic markers CD34, CD11b and CD45. Ultrastructure analysis showed typical internal structure for the PCs with a characteristic prominent eccentric nuclei and cytoplasmic invaginations forming a caveolar system. Functional analysis showed efficient differentiation into adipocytes, osteocytes, and myocyte-like cells. Adipose tissue-derived PCs showed angiogenic potential using tube-forming assay. To determine further application of these cells for personalized therapy, we reprogrammed PCs into induced pluripotent stem cells (iPSCs) using episomal vectors. Reprogrammed cells gradually lost their fusiform shape, acquired the epithelial cell morphology and formed colonies. Furthermore, reprogrammed cells successfully expressed the pluripotency markers OCT4, Nanog, SSEA-4, and ß-catenin, an early reprogramming marker. CONCLUSION: The accessibility and abundance of human fat supports the application of adipose derived PCs as a novel and promising source of cell therapy and regenerative medicine.

Eye movements and imaging in vestibular migraine / Movimientos oculares y pruebas de imagen en migraña vestibular

BACKGROUND AND OBJECTIVE: Migrainous dizziness is one of the most frequent complaints. Dizziness associated with migraine may be the result of abnormal eye movements. Brain imaging and changes in eye movements may explain the dizziness and highlight possible pathophysiological substrates in migraine dizziness. Our aim is to evaluate eye movement using videonystagmography (VNG) and video head impulse test (vHIT) and to study the occipital lobe metabolic profile in vestibular migraine patients (VM). MATERIALS AND METHODS: There were 2 groups enrolled in the study; the first group consisted of 25 vestibular migraine patients (VM) according to the recent criteria of Barany society. The second group consisted of 20 age matched healthy subjects. Both groups underwent the following: (1) A detailed history, VNG test protocol, vHIT in three planes. (2) Magnetic resonance imaging (MRI) for the brain and inner ear using 1.5 T magnet and proton magnetic resonance spectroscopy (H1-MRS). RESULTS: Sixty eight percent of the patients complained of spontaneous vertigo and 28% complained of positional vertigo. Non-paroxysmal positional nystagmus was recorded in 92% during their dizzy spell. The brain MRI was unremarkable in 72% of the cases. Chemical shift in the occipital lobe was found in 92% of VM. Lactate peaks were statistically significant related with the presence of non-paroxysmal positional nystagmus. CONCLUSIONS: A statistically significant relationship exists between non-paroxysmal positional nystagmus and presence of lactate peaks in the occipital lobe in VM patients

ANTECEDENTES Y OBJETIVO: El mareo migrañoso es una de las quejas más frecuentes. Las pruebas de imagen del cerebro y los cambios en los movimientos oculares pueden explicar los mareos y destacar los posibles sustratos fisiopatológicos en la migraña vestibular. Nuestro objetivo fue evaluar el movimiento ocular utilizando videonistagmografía (VNG) y la prueba de impulso cefálico por vídeo (vHIT), y estudiar el perfil metabólico del lóbulo occipital en pacientes con migraña vestibular (VM). MATERIALES Y MÉTODOS: Se incluyeron dos grupos en el estudio; el primer grupo consistió en 25 pacientes con VM según los criterios recientes de la sociedad Bárány. El segundo grupo consistió en 20 sujetos sanos emparejados por edad. Ambos grupos se sometieron a lo siguiente: 1) Una historia detallada, protocolo de prueba de VNG y vHIT en 3 planos, y 2) Imágenes de resonancia magnética (IRM) para el cerebro y el oído interno con el imán de 1,5 tesla y la espectroscopía de resonancia magnética de protones (H1-MRS). RESULTADOS: El 68% de los pacientes se quejó de vértigo espontáneo, y el 28% de vértigo posicional. El nistagmo posicional no paroxístico se registró en el 60% de los pacientes durante su mareo. La resonancia magnética cerebral no mostró alteraciones en el 72% de los casos. El cambio químico en el lóbulo occipital se encontró en el 92% de los casos de VM. Los picos de lactato fueron estadísticamente significativos con relación a la presencia de nistagmo posicional no paroxístico. CONCLUSIONES: Existe una relación estadísticamente significativa entre el nistagmo posicional no paroxístico y la presencia de picos de lactato en el lóbulo occipital en pacientes con VM

Eye movements and imaging in vestibular migraine.

BACKGROUND AND OBJECTIVE: Migrainous dizziness is one of the most frequent complaints. Dizziness associated with migraine may be the result of abnormal eye movements. Brain imaging and changes in eye movements may explain the dizziness and highlight possible pathophysiological substrates in migraine dizziness. Our aim is to evaluate eye movement using videonystagmography (VNG) and video head impulse test (vHIT) and to study the occipital lobe metabolic profile in vestibular migraine patients (VM). MATERIALS AND METHODS: There were 2 groups enrolled in the study; the first group consisted of 25 vestibular migraine patients (VM) according to the recent criteria of Barany society. The second group consisted of 20 age matched healthy subjects. Both groups underwent the following: (1) A detailed history, VNG test protocol, vHIT in three planes. (2) Magnetic resonance imaging (MRI) for the brain and inner ear using 1.5T magnet and proton magnetic resonance spectroscopy (H1-MRS). RESULTS: Sixty eight percent of the patients complained of spontaneous vertigo and 28% complained of positional vertigo. Non-paroxysmal positional nystagmus was recorded in 92% during their dizzy spell. The brain MRI was unremarkable in 72% of the cases. Chemical shift in the occipital lobe was found in 92% of VM. Lactate peaks were statistically significant related with the presence of non-paroxysmal positional nystagmus. CONCLUSIONS: A statistically significant relationship exists between non-paroxysmal positional nystagmus and presence of lactate peaks in the occipital lobe in VM patients.

Comparison of different uncoated and starch-coated superparamagnetic iron oxide nanoparticles: Implications for stem cell tracking.

However, labelling of stem cells using nanoparticles (NPs) for tracking purpose has been intensively investigated, the biosafety of these materials needs more clarification. Herein, different forms of iron oxide Fe2O3, Fe3O4, and CoxNi1-x Fe2O4 NPs either uncoated or starch-coated (ST-coated) were prepared. We successfully labelled adipose-derived stem cells (ASCs) using these NPs with the aid of lipofectamine as a transfection agent (TA). We then evaluated the effect of these NPs on stem cell proliferation, viability, migration and angiogenesis. Results showed that ASCs labelled with Fe2O3, Fe3O4, ST-Fe2O3 and ST-Fe3O4 did not show any significant difference in proliferation compared to that of TA-treated cells. Moreover, they have shown a protective effect against apoptosis. Conversely, CoxNi1-x Fe2O4 NPs caused a significant decrease in cell proliferation. Compared to that of the TA-treated cells, the migration capacity of cells labelled with Fe2O3, Fe3O4 and CoxNi1-xFe2O4 was significantly compromised. Interestingly, the ST-coated composites reversed this effect. Among the groups treated with different NPs, the angiogenic potential of the ASCs was most robust in the ST-Fe2O3-treated group. In conclusion, labelling ASCs with ST-Fe2O3 NPs enhanced cell migration and angiogenic potential and conferred higher resistance to apoptosis than labelling the cells with the other tested NPs.

A 2D laser rangefinder scans dataset of standard EUR pallets.

In the past few years, the technology of automated guided vehicles (AGVs) has notably advanced. In particular, in the context of factory and warehouse automation, different approaches have been presented for detecting and localizing pallets inside warehouses and shop-floor environments. In a related research paper Mohamed et al., 2018, we show that an AGVs can detect, localize, and track pallets using machine learning techniques based only on the data of an on-board 2D laser rangefinder. Such sensor is very common in industrial scenarios due to its simplicity and robustness, but it can only provide a limited amount of data. Therefore, it has been neglected in the past in favor of more complex solutions. In this paper, we release to the community the data we collected in Ref. Mohamed et al., 2018 for further research activities in the field of pallet localization and tracking. The dataset comprises a collection of 565 2D scans from real-world environments, which are divided into 340 samples where pallets are present, and 225 samples where they are not. The data have been manually labelled and are provided in different formats.

Video head impulse test (vHIT) in migraine dizziness.

BACKGROUND AND OBJECTIVE: Migraine is an extremely prevalent primary headache disorder that frequently associates parallel symptoms such as dizziness, tinnitus and hearing loss. Our aim is to investigate differences in video head impulse (vHIT) results with patients suffering from vestibular migraine (VM) and healthy people, taking into consideration mean values of vestibule ocular reflex (VOR) gain, occurrence of the compensatory saccades latency and amplitude. According to the results, determine the usefulness of vHIT in vestibular migraine diagnostics. METHODS: A total number of 120 subjects were enrolled in the study, 80 of them were vestibular migraine patients (VM), while the other 40 were a control group of age matched healthy subjects. History was taking during the evaluation; videonystagmography and the video head impulse test were done. RESULTS: The rate of saccades is much more higher in the VM group compared to the healthy subjects group, only 7.5% of the VM group showed a low VOR gain with compensatory saccades denoting a vestibular deficit. CONCLUSION: The refixation saccades are an important sign that could underlie different vestibular problems. vHIT result can contribute to the completion of full mosaic of vestibular migraine diagnostics.

Development of decellularized amniotic membrane as a bioscaffold for bone marrow-derived mesenchymal stem cells: ultrastructural study.

Developing effective stem cell-based therapies requires the design of complex in vitro culture systems for accurate representation of the physiological stem cell niche. Human amniotic membrane (hAM) has been successfully used in clinical grafting applications due to its unique biological and regenerative properties. Decellularized hAM (d-hAM) has been previously applied to the culture of human bone marrow mesenchymal stem cells (hMSCs), promoting their expansion and differentiation into adipogenic and osteogenic lineages. In the present study, hAM was decellularized by NaOH-treatment, to provide the three-dimensional (3D) bioscaffold for culturing hMSCs. The ultrastructural differences between intact hAM and decellularized hAM were characterized using the transmission electron microscope (TEM), as well as the 3D interaction between d-hAM and hMSCs cultured on the membrane. TEM examination of the intact hAM showed many microvilli on the epithelial layer cells, active Golgi apparatus, smooth endolplasmic reticulum and the characteristic pinocytic vesicles. The epithelial layer with its structures was absent in the d-hAM. However, no observable difference was detected in the ultrastructural characteristics of the compact stromal layer of d-hAM compared to intact hAM. Both contained bundles of extra cellular matrix (ECM) proteins, and scattered elastic fibres. Cultured human mesenchymal stem cells (hMSCs) examined by TEM appeared oval to spherical in shape and had a rough and non-uniform surface with distinct protrusions or irregular fillopodia. Their diameter ranged from 20.49 to 21.6 µm. Most of the cellular organelles were also noticed. SEM examination of the prepared samples revealed unique 3D interaction between the hMSC and d-hAM, where the latter seems to envelop the segments of the hMSCs lying on the surrounding membrane. This study shows that the decellularization process affected the epithelial layer only of hAM and had no effect on altering the presence of ECM components present in the stromal layer of the d-hAM. The interaction between hMSCs and d-hAM maybe mediated by hAM components other than human amniotic epithelial cells, such as ECM components or MSCs present in the deeper spongy layer of the membrane or/and the adhesive components of the basement membrane of the removed epithelial layer.

Cancer cell-soluble factors reprogram mesenchymal stromal cells to slow cycling, chemoresistant cells with a more stem-like state.

BACKGROUND: Mesenchymal stem cells (MSCs) play different roles in modulating tumor progression, growth, and metastasis. MSCs are recruited to the tumor site in large numbers and subsequently have an important microenvironmental role in modulating tumor progression and drug sensitivity. However, the effect of the tumor microenvironment on MSC plasticity remains poorly understood. Herein, we report a paracrine effect of cancer cells, in which they secrete soluble factors that promote a more stem-like state in bone marrow mesenchymal stem cells (BM-MSCs). METHODS: The effect of soluble factors secreted from MCF7, Hela, and HepG2 cancer cell lines on BM-MSCs was assessed using a Transwell indirect coculture system. After 5 days of coculture, BM-MSCs were characterized by flow cytometry for surface marker expression, by qPCR for gene expression profile, and by confocal immunofluorescence for marker expression. We then measured the sensitivity of cocultured BM-MSCs to chemotherapeutic agents, their cell cycle profile, and their response to DNA damage. The sphere formation, invasive properties, and in-vivo performance of BM-MSCs after coculture with cancer cells were also measured. RESULTS: Indirect coculture of cancer cells and BM-MSCs, without direct cell contact, generated slow cycling, chemoresistant spheroid stem cells that highly expressed markers of pluripotency, cancer cells, and cancer stem cells (CSCs). They also displayed properties of a side population and enhanced sphere formation in culture. Accordingly, these cells were termed cancer-induced stem cells (CiSCs). CiSCs showed a more mesenchymal phenotype that was further augmented upon TGF-ß stimulation and demonstrated a high expression of the ß-catenin pathway and ALDH1A1. CONCLUSIONS: These findings demonstrate that MSCs, recruited to the tumor microenvironment in large numbers, may display cellular plasticity, acquire a more stem-like state, and acquire some properties of CSCs upon exposure to cancer cell-secreted factors. These acquired characteristics may contribute to tumor progression, survival, and metastasis. Our findings provide new insights into the interactions between MSCs and cancer cells, with the potential to identify novel molecular targets for cancer therapy.

Frequency of Helicobacter pylori infection among Egyptian children presenting with gastrointestinal manifestations.

BACKGROUND AND AIMS: Recurrent abdominal pain (RAP) and other gastrointestinal (GI) symptoms are common complaints among children. The role of Helicobacter pylori in the causation of these complaints remains controversial. The aim of this study was to determine the frequency of H. pylori infection among children presenting with GI manifestations and to determine the most common clinical presentation of the infection in Egyptian children. PATIENTS AND METHODS: This prospective cross-sectional study included 150 consecutive patients aged 5-15 years who presented to the outpatient clinic of Cairo University's Specialized Pediatric Hospital with GI complaints. Screening for H. pylori infection was performed using a 13C-urea breath test (13C-UBT), and in patients whose 13C-UBT was positive, diagnosis was confirmed by visualizing the bacterium in biopsy specimens obtained by GI endoscopy. RESULTS: RAP was the most frequent GI complaint of the study population (82%), followed by anorexia (6.7%), vomiting (6.7%), and chronic diarrhea (4.7%). Seventy percent of these patients were positive for the 13C-UBT. Ninety-one of the patients who complained of RAP had a positive 13C-UBT, whereas the remaining 32 patients who had RAP had a negative 13C-UBT, a difference that was statistically significant (P=0.023). However, no statistically significant differences were found between the 13C-UBT result among patients with anorexia, vomiting, or diarrhea (P=0.153, 1.00, and 0.447, respectively). CONCLUSION AND RECOMMENDATIONS: Screening for H. pylori infection should be performed for school-aged children who have GI complaints, especially for those who complain of RAP.

Histochemical analysis of muscle fiber types of rat superior rectus extraocular muscle.

Extraocular muscles (EOMs) represent a distinctive class among mammalian skeletal muscles in exhibiting unique anatomical and physiological properties. To gain insight into the basis for the unique structural/functional diversity of EOM fiber types and to explain their high fatigue resistance, rat superior rectus muscle (SRM) was studied using histochemical techniques. Muscle fibers were typed with regard to their oxidative and glycolytic profiles generated from succinic dehydrogenase (SDH) and phosphorylase activities in combination with their morphologic characteristics. Superior rectus muscle is organized into two layers, a central global layer (GL) of mainly large diameter fibers and an outer C-shaped orbital layer (OL) of principally small diameter fibers. Five muscle fiber types were recognized within the SRM: I, II, III, IV and V. In the global layer, four muscle fiber types were identified: type I (18.25±0.96µm; 32%) showed intermediate SDH (coarse type) and high phosphorylase activity. Type II fibers (14.45±0.82µm; 22%) exhibited high SDH (fine type) and intermediate phosphorylase activity. Low SDH (granular type) and high phosphorylase activity were demonstrated by type III fibers (22.65±1.73µm; 36%). Type IV fibers (26.24±1.32µm; 10%) were recognized by their low oxidative and glycolytic reactions. In the orbital region, only three muscle fiber types were recognized; fiber types I and II were found to compose approximately two-thirds of the layer. The third orbital fiber type (type V, 10.05±0.99µm) exhibited low SDH and low phosphorylase profiles. In this paper, the functional significance of the histochemical characteristics of the EOM fiber types is discussed.

Stability of insulin during the erosion of poly(lactic acid) and poly(lactic-co-glycolic acid) microspheres.

In recent years, the acylation of peptides during the erosion of poly(lactic acid) and poly(lactic-co-glycolic acid) microspheres has been described in the literature. To investigate whether insulin is prone to the covalent attachment of lactic or glycolic acid, insulin-loaded PLA and PLGA microspheres containing 5% bovine insulin were manufactured using a w/o/w multiple emulsion-solvent evaporation technique. Microspheres were characterized for their insulin encapsulation efficiency and release characteristics in phosphate-buffered saline (PBS) at pH 7.4 and 37 degrees C. Moreover, the stability of the peptide during 18 days of release was evaluated using HPLC and HPLC-MS techniques. The results showed that the insulin loading efficiencies of PLA and PLGA microspheres were 75.18% and 79.63%, respectively. The microspheres were spherical with relatively porous surfaces with an average diameter of 40 and 53 mum, respectively. Insulin release from the microspheres was characterized by an initial burst, which was attributed to the amount of protein located on or close to the microsphere surface. The total ion chromatogram (TIC) of insulin samples extracted after 18 days of erosion in phosphate buffer pH 7.4 at 37 degrees C revealed that deamidation was the major mechanism of instability. Surprisingly, no acylation products were found. Control experiments in concentrated lactic acid solutions confirmed a minimal reactivity of the peptide under these conditions.