RESUMEN
OBJECTIVES: The aim of the present study was to characterize the cellular reaction to a xenogeneic resorbable collagen membrane of porcine origin using a subcutaneous implantation model in Wistar rats over 30 days. MATERIALS AND METHODS: Ex vivo, liquid platelet-rich fibrin (PRF), a leukocyte and platelet-rich cell suspension, was used to evaluate the blood cell membrane interaction. The material was implanted subcutaneously in rats. Sham-operated rats without biomaterial displayed physiological wound healing (control group). Histological, immunohistological, and histomorphometric analyses were focused on the inflammatory pattern, vascularization rate, and degradation pattern. RESULTS: The membrane induced a large number of mononuclear cells over the observation period, including lymphocytes, macrophages, and fibroblasts. After 15 days, multinucleated giant cells (MNGCs) were observed on the biomaterial surface. Their number increased significantly, and they proceeded to the center of the biomaterial on day 30. These cells highly expressed CD-68, calcitonin receptor, and MMP-9, but not TRAP or integrin-ß3. Thus, the membrane lost its integrity and underwent disintegration as a consequence of the induction of MNGCs. The significant increase in MNGC number correlated with a high rate of vascularization, which was significantly higher than the control group. Physiological wound healing in the control group did not induce any MNGCs at any time point. Ex vivo blood cells from liquid-PRF did not penetrate the membrane. CONCLUSION: The present study suggests a potential role for MNGCs in biomaterial degradation and questions whether it is beneficial to accept them in clinically approved biomaterials or focus on biomaterials that induce only mononuclear cells. Thus, further studies are necessary to identify the function of biomaterial-induced MNGCs. CLINICAL RELEVANCE: Understanding the cellular reaction to biomaterials is essential to assess their suitability for specific clinical indications and outline the potential benefit of specific group of biomaterials in the respective clinical indications.
Asunto(s)
Materiales Biocompatibles , Fibrina Rica en Plaquetas , Animales , Colágeno , Células Gigantes , Ratas , Ratas Wistar , PorcinosRESUMEN
BACKGROUND: The present study evaluated the cellular tissue reaction of two equine-derived collagen hemostatic sponges (E-CHS), which differed in thickness after pressing, over 30 days in vivo. The inflammatory response during physiological wound healing in sham-operated animals was used as control group. MATERIAL AND METHODS: First, the E-CHS was pressed by applying constant pressure (6.47 ± 0.85 N) for 2 min using a sterile stainless-steel cylinder until the material was uniformly flattened. Consequently, the original (E-CHS), the pressed (P-E-CHS), as well as the control group (CG; sham operation) were studied independently. The 3 groups were evaluated in vivo after subcutaneous implantation in Wistar rats during 3, 15, and 30 days. Histochemical and immunohistochemical methods provided observations of biomaterial degradation rate, cellular inflammatory response, and vascularization pattern. A derivative of human blood known as platelet-rich fibrin (PRF) was used as an ex vivo model to simulate the initial biomaterial-cell interaction. Segments of E-CHS and P-E-CHS were cultivated for 3 and 6 days with PRF, and the release of pro-inflammatory proteins was measured using ELISA. PRF cultivated alone was used as a control group. RESULTS: At day 3, the CG induced a statistically significant higher presence of monocytes/macrophages (CD68+), pro-inflammatory macrophages (M1; CCR7+), and pro-wound healing macrophages (M2; CD206+) compared to E-CHS and P-E-CHS. At the same time point, P-E-CHS induced a statistically significant higher presence of CD68+ cells compared to E-CHS. After 15 days, E-CHS was invaded by cells and vessels and showed a faster disintegration rate compared to P-E-CHS. On the contrary, cells and vessels were located only in the outer region of P-E-CHS and the biomaterial did not lose its structure and accordingly did not undergo disintegration. The experimental groups induced similar inflammatory reaction primarily with positive pro-inflammatory CD68+/CCR7+ macrophages and a low presence of multinucleated giant cells (MNGCs). At this time point, significantly lower CD68+/CCR7+ macrophages and no MNGCs were detected within the CG when compared to the experimental groups (P < 0.05). After 30 days, E-CHS and P-E-CHS were fully degraded. All groups showed similar inflammatory reaction shifted to a higher presence CD206+ macrophages. A low number of CCR7+ MNGCs were still observable in the implantation bed of both experimental groups. In the ex vivo model, the cells and fibrin from PRF penetrated E-CHS. However, in the case of P-E-CHS, the cells and fibrin stayed on the surface and did not penetrate towards materials central regions. The cultivation of P-E-CHS with PRF induced a statically significant higher release of pro-inflammatory proteins compared to the CG and E-CHS after 3 days. CONCLUSION: Altering the original presentation of a hemostatic sponge biomaterial by pressing modified the initial biomaterial-cell interaction, delayed the early biomaterial's degradation rate, and altered the vascularization pattern. A pressed biomaterial seems to induce a higher inflammatory reaction at early time points. However, altering the biomaterial did not modify the polarization pattern of macrophages compared to physiologic wound healing. The ex vivo model using PRF was shown to be an effective model to simulate the initial biomaterial-cell interaction in vivo. CLINICAL RELEVANCE: A pressed hemostatic sponge could be applied for guided tissue regeneration and guided bone regeneration. In that sense, within the limitations of this study, the results show that the same biomaterial may have two specific clinical indications.
Asunto(s)
Macrófagos , Animales , Materiales Biocompatibles , Colágeno , Caballos , Humanos , Fibrina Rica en Plaquetas , Ratas , Ratas WistarRESUMEN
Different tissue engineering techniques are used to support rapid vascularisation. A novel technique is the use of platelet-rich fibrin (PRF), an autologous source of growth factors. This study was the first to investigate the influence of PRF matrices, isolated following different centrifugation protocols, on human dermal vascular endothelial cells (ECs) in mono-culture and co-culture with human primary fibroblasts (HFs) as an in vitro model for tissue regeneration. Focus was placed on vascular structure formation and growth factor release. HFs and ECs were cultivated with PRF prepared using a high (710 ×g) or low (44 ×g) relative centrifugation force (RCF) over 14 d. Immunofluorescence staining and immunohistochemistry were used to evaluate the microvascular formation. Cell culture supernatants were collected for evaluation of growth factor release. The results showed a PRF-mediated effect on the induction of angiogenesis in ECs. Microvessel-like structure formation was promoted when ECs were combined with low-RCF PRF as compared to high-RCF PRF or control group. The percentage of vascular lumen area was significantly higher in low-RCF PRF, especially at day 7, which coincided with statistically significantly higher growth factor [vascular endothelial factor (VEGF), transforming growth factor ß1 (TGF-ß1) and platelet derived growth factor (PDGF)] concentration measured in low-RCF PRF as compared to high-RCF PRF or control group. In conclusion, reducing the RCF according to the low-speed centrifugation concept (LSCC) resulted in increased growth factor release and angiogenic structure formation with EC mono-culture, suggesting that PRF may be a highly beneficial therapeutic tool for tissue engineering applications.
Asunto(s)
Células Endoteliales/metabolismo , Fibroblastos/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Neovascularización Fisiológica/efectos de los fármacos , Fibrina Rica en Plaquetas , Técnicas de Cultivo de Célula , Células Endoteliales/citología , Fibroblastos/citología , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacologíaRESUMEN
Purpose The present study evaluated the platelet distribution pattern and growth factor release (VEGF, TGF-ß1 and EGF) within three PRF (platelet-rich-fibrin) matrices (PRF, A-PRF and A-PRF+) that were prepared using different relative centrifugation forces (RCF) and centrifugation times. Materials and methods immunohistochemistry was conducted to assess the platelet distribution pattern within three PRF matrices. The growth factor release was measured over 10 days using ELISA. Results The VEGF protein content showed the highest release on day 7; A-PRF+ showed a significantly higher rate than A-PRF and PRF. The accumulated release on day 10 was significantly higher in A-PRF+ compared with A-PRF and PRF. TGF-ß1 release in A-PRF and A-PRF+ showed significantly higher values on days 7 and 10 compared with PRF. EGF release revealed a maximum at 24 h in all groups. Toward the end of the study, A-PRF+ demonstrated significantly higher EGF release than PRF. The accumulated growth factor releases of TGF-ß1 and EGF on day 10 were significantly higher in A-PRF+ and A-PRF than in PRF. Moreover, platelets were located homogenously throughout the matrix in the A-PRF and A-PRF+ groups, whereas platelets in PRF were primarily observed within the lower portion. âDiscussion the present results show an increase growthfactor release by decreased RCF. However, further studies must be conducted to examine the extent to which enhancing the amount and the rate of released growth factors influence wound healing and biomaterial-based tissue regeneration. âConclusion These outcomes accentuate the fact that with a reduction of RCF according to the previously LSCC (described low speed centrifugation concept), growth factor release can be increased in leukocytes and platelets within the solid PRF matrices.
Asunto(s)
Plaquetas/metabolismo , Centrifugación/métodos , Factor de Crecimiento Epidérmico/metabolismo , Fibrina Rica en Plaquetas/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adulto , Femenino , Voluntarios Sanos , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Persona de Mediana Edad , Prueba de Estudio Conceptual , Cicatrización de HeridasRESUMEN
PEGylated gold nanoparticles (AuNPs) have an extended circulation time after intravenous injection in vivo and exhibit favorable properties for biosensing, diagnostic imaging, and cancer treatment. No impact of PEGylated AuNPs on the barrier forming properties of endothelial cells (ECs) has been reported, but recent studies demonstrated that unexpected effects on erythrocytes are observed. Almost all studies to date have been with static-cultured ECs. Herein, ECs maintained under physiological cyclic stretch and flow conditions and used to generate a blood-brain barrier model were exposed to 20 nm PEGylated AuNPs. An evaluation of toxic effects, cell stress, the release profile of pro-inflammatory cytokines, and blood-brain barrier properties showed that even under physiological conditions no obvious effects of PEGylated AuNPs on ECs were observed. These findings suggest that 20 nm-sized, PEGylated AuNPs may be a useful tool for biomedical applications, as they do not affect the normal function of healthy ECs after entering the blood stream.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Oro/metabolismo , Nanopartículas/metabolismo , Polietilenglicoles/metabolismo , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/patología , Oro/química , Oro/toxicidad , Células Endoteliales de la Vena Umbilical Humana , Humanos , Nanopartículas/química , Nanopartículas/toxicidad , Tamaño de la Partícula , Polietilenglicoles/química , Polietilenglicoles/toxicidad , PorcinosRESUMEN
The present study analyzes the influence of the addition of monocytes to a biphasic bone substitute with two granule sizes (400-700 µm and 500-1000 µm). The majority of the added monocytes was detectable as mononuclear cells, while also low amounts of (chimeric) multinucleated giant cells (MNGCs) were found. No increase in the total number of MNGCs was established, but a significantly increased percent vascularization. Altogether, the results show that the added monocytes become involved in the tissue response to a biomaterial without marked changes in the overall reaction. Monocyte addition enables an increased implant bed vascularization especially via induction of vessel maturation and, thus intervenes positively in the healing reaction to a biomaterial. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2928-2935, 2016.
Asunto(s)
Sustitutos de Huesos/metabolismo , Hidroxiapatitas/metabolismo , Monocitos/citología , Neovascularización Fisiológica , Animales , Células Cultivadas , Femenino , Células Gigantes/citología , Humanos , Ensayo de Materiales , Ratones SCID , Prótesis e ImplantesRESUMEN
The present study investigated the influence of granule size of 2 biphasic bone substitutes (BoneCeramic® 400-700 µm and 500-1000 µm) on the induction of multinucleated giant cells (MNGCs) and implant bed vascularization in a subcutaneous implantation model in rats. Furthermore, degradation mechanisms and particle phagocytosis of both materials were examined by transmission electron microscopy (TEM). Both granule types induced tissue reactions involving primarily mononuclear cells and only small numbers of MNGCs. Higher numbers of MNGCs were detected in the group with small granules starting on day 30, while higher vascularization was observed only at day 10 in this group. TEM analysis revealed that both mono- and multinucleated cells were involved in the phagocytosis of the materials. Additionally, the results allowed recognition of the MNGCs as the foreign body giant cell phenotype. Histomorphometrical analysis of the size of phagocytosed particles showed no differences between the 2 granule types. The results indicate that granule size seems to have impact on early implant bed vascularization and also on the induction of MNGCs in the late phase of the tissue reaction. Furthermore, the results revealed that a synthetic bone substitute material can induce tissue reactions similar to those of some xenogeneic materials, thus pointing to a need to elucidate their "ideal" physical characteristics. The results also show that granule size in the range studied did not alter phagocytosis by mononuclear cells. Finally, the investigation substantiates the differentiation of material-induced MNGCs, which are of the foreign body giant cell type.
Asunto(s)
Sustitutos de Huesos/farmacología , Huesos/irrigación sanguínea , Huesos/inmunología , Células Gigantes/metabolismo , Hidroxiapatitas/farmacología , Leucocitos Mononucleares/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Ensayo de Materiales , Ratones , Microscopía Electrónica de Rastreo , Neovascularización Fisiológica , Tamaño de la Partícula , Fagocitosis , RatasRESUMEN
BACKGROUND: Wound healing depends on a well-balanced regulation of inflammation and angiogenesis. In chronic wounds the healing process is disturbed and inflammation persists. Regulation of wound closure is controlled by transmembrane and extracellular proteins, the folding and maturation of which occur in the endoplasmic reticulum (ER) by ER-resident chaperone machinery. OBJECTIVES: To study the role of the ER-resident chaperones BiP/Grp78, its cochaperone Mdg1/ERdJ4, and Grp94 in chronic, nonhealing wounds. METHODS: Immunohistochemical staining of these chaperones in individual human biopsies and investigation of the possible role of BiP and Mdg1 in endothelial cells, focusing on their inflammatory response and angiogenic potential. RESULTS: In all chronic wounds investigated, the levels of these ER-resident chaperones were elevated in endothelial cells and leucocytes. The proangiogenic role of BiP has been shown in tumour growth studies before and was confirmed in this study. Proangiogenic activity of the cochaperone Mdg1 has been postulated before but could not be confirmed in this study. The chemokine tumour necrosis factor (TNF)-α was shown to trigger the presentation of proinflammatory adhesion molecules and the release of proinflammatory cytokines. Here we show that TNF-α does not affect endogenous chaperone levels, but that the ER-resident chaperones BiP and Mdg1 modulate the cellular TNF-α-induced proinflammatory response. CONCLUSIONS: According to the presented data we assume that in chronic wounds upregulated levels of ER-resident chaperones might contribute to persistent inflammation in chronic wounds. Therapies to downregulate chaperone levels might provide a tool that switches the imbalanced chronic wound microenvironment from inflammation to healing.
Asunto(s)
Chaperonas Moleculares/fisiología , Cicatrización de Heridas/fisiología , Células Cultivadas , Enfermedad Crónica , Regulación hacia Abajo/fisiología , Retículo Endoplásmico/fisiología , Chaperón BiP del Retículo Endoplásmico , Células Endoteliales/fisiología , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas del Choque Térmico HSP40/fisiología , Proteínas de Choque Térmico/metabolismo , Humanos , Inflamación/fisiopatología , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Chaperonas Moleculares/metabolismo , Neovascularización Fisiológica/fisiología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
INTRODUCTION: Idiopathic immaturity is one of the main reasons for latent placental insufficiency and antenatal hypoxia. Postnatal identification of the immature placental phenotype may help early stratification of a heterogeneous population of newborns and individually identify risk of disease in the immediate postnatal life. The aim of the study was to determine the relevant diagnostic markers associated with pathological placental immaturity. METHODS: 111 tissue samples from normal and pathological term placentas with persisting villous immaturity comprised the comparative immunohistochemical study (CD15, CD34). Positive immunohistochemical reactions were quantitatively assessed in the chorionic plate and vessels of the villi of different histological type. RESULTS: We have shown that pathological villous immaturity is attended by significantly increased CD15-expression in the macro- and microvascular endothelium compared with the normal placenta. CD34-expression was not different from that in normal placentas. DISCUSSION: This paper documents the correlation of CD15+ endothelium in the macrovascular fetoplacental vessels with a severe form of villous immaturity associated with fetal hypoxia/asphyxia and erythroblastosis. Increased CD15-expression only in the microvascular segment of the fetoplacental vessels correlated with moderate villous immaturity and was associated with GDM, idiopathic fetal macrosomia and nonspecific chronic villitis. CONCLUSION: We propose that "immature" CD15+ endothelium is an important diagnostic marker of persisting villous immaturity and chronic placental dysfunction. The level of CD15 expression in the macro- and microvasculature reflects the degree of pathological placental villous immaturity.
Asunto(s)
Antígenos CD34/metabolismo , Fucosiltransferasas/metabolismo , Antígeno Lewis X/metabolismo , Insuficiencia Placentaria/metabolismo , Biomarcadores/metabolismo , Estudios de Casos y Controles , Femenino , Humanos , EmbarazoRESUMEN
OBJECTIVE: Placental growth and villous maturation are critical parameters of placental function at the end of pregnancy. A failure in these processes leads to the development of placental dysfunction, as well as fetal and neonatal mortality and morbidity. The aim of the study was to determine the relevant diagnostic markers associated with pathological placental development. STUDY DESIGN: Forty tissue samples from normal placentas of different gestational age and 68 pathological term placentas with defective villous maturation (GDM, idiopathic IUFD, preeclamsia, HELLP syndrome) comprised the comparative immunohistochemical study (CD15, CD45 and CD34). Positive immunohistochemical reactions were quantitatively assessed in the chorionic plate and vessels of the villi of different histological type. RESULTS: Physiologically immature placentas of the first and second trimester and pathologically immature term placentas were characterized by marked endothelial CD15-immunostaining. A significant loss of CD15-positive endothelium of the placentas was associated with a physiological and accelerated villous maturity. A spatio-temporal correlation was shown for CD15+ endothelial cells (ECs) and the number of CD45+ stromal cells (SCs). A negative temporal correlation was shown for CD15+ ECs and CD15+ myelomonocytes in the fetal blood. CD34 expression in the ECs was stable during the pregnancy. CONCLUSION: A correlation between a transient CD15-positive endothelial phenotype and a physiological and pathological fetoplacental immaturity was demonstrated. Physiological and accelerated placental maturation was accompanied by a significant disappearance of CD15-positive endothelium. We propose that "immature" CD15+ endothelium is an important diagnostic marker of the physiological and pathological fetoplacental immaturity.
Asunto(s)
Antígenos CD34/metabolismo , Células Endoteliales/metabolismo , Fucosiltransferasas/metabolismo , Edad Gestacional , Antígenos Comunes de Leucocito/metabolismo , Antígeno Lewis X/metabolismo , Placentación , Adulto , Estudios de Casos y Controles , Diabetes Gestacional/metabolismo , Células Endoteliales/citología , Femenino , Retardo del Crecimiento Fetal/metabolismo , Síndrome HELLP/metabolismo , Humanos , Inmunohistoquímica , Placenta/citología , Placenta/metabolismo , Preeclampsia/metabolismo , EmbarazoRESUMEN
In the past numerous analyses have studied several aspects of autopsies in particular with regard to the decline of frequency; however, long-term studies spanning more than one decade have rarely been published, especially in recent years. On the occasion of the 100 year jubilee the archive data of the Institute of Pathology of the University of Mainz were analyzed for autopsies performed between 1971 and 2010. In this cohort, we focused on patients over 14 years old (n = 14,724) who died in the University hospital. We compared the number of autopsies with the total number of deceased patients and, in addition, studied several epidemiological aspects with special relevance for the cause of death (COD). In 1973 the peak autopsy frequency was reached with a value of 73.4 % followed by a decrease to 49.1 % in 1980. In the following decade a relatively steady state was achieved (frequency 53.3 % in 1985, and 43.2 % in 1990), followed by a remarkable decline after the 1990s (1997: 26.4 %, 1998: 15.9 % and 2010: 5.6 %). The mean overall age increased during the observation period (59.1 years in 1971 and 67.5 years in 2008). Among the COD groups cardiovascular diseases were predominantly recorded (between 35 % in the 1970s and 39 % in 1995-2010), followed by infectious diseases (between 20 and 25 %). Malignancies represented the third most common COD group with an increase in frequency from approximately 10.5 % in the 1970s to 17 % observed in the last decade. Among the single specific CODs, pulmonary embolism was most often encountered in the 1970s (about 11.5 %), while in the following decades myocardial infarction predominated (up to 15.8 % between 1995 and 2010). In the overall period, lung cancer was the single most common malignancy of the CODs (between 2.5 and 3.9 %). These data confirmed previous studies showing that in Germany the autopsy frequency began to decline remarkably in the 1990s. Besides general aspects, the specific local causes for these phenomena are discussed.
Asunto(s)
Academias e Institutos/historia , Academias e Institutos/estadística & datos numéricos , Autopsia/historia , Autopsia/tendencias , Revisión de Utilización de Recursos/historia , Revisión de Utilización de Recursos/tendencias , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Autopsia/estadística & datos numéricos , Causas de Muerte/tendencias , Alemania , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Persona de Mediana Edad , Patología/tendencias , Adulto JovenRESUMEN
Ameloblastoma is a locally invasive odontogenic tumor with a high recurrence rate. Its local invasiveness is aided by angiogenesis, which can be correctly estimated by CD34. On the other hand, maspin decreases the local invasive and metastatic capability of cancer cells and functions as an angiogenesis inhibitor. We aim to assess the association between maspin expression and microvessel density in ameloblastoma. Twenty-five formalin-fixed paraffin-embedded (FFPE) blocks of ameloblastoma cases were prepared for antibody processing to CD34 and maspin. Positive immunohistochemical staining was marked by brown cytoplasmic/membrane coloration for CD34 and by nuclear/cytoplasmic coloration for maspin. At the ×40 magnification, we counted blood vessels in two areas of dimension; 300 × 400 µm (area A) and 150 × 200 µm (area B) adjacent to the tumor region to assess relative dispersion of the vessels bordering the tumor. The overall approximate microvessel density (MVD) for area A = 11 (minimum 2, maximum 21) and that for area B = 5 (minimum 1, maximum 10). The MVD in the area A of plexiform ameloblastoma was similar to that of the unicystic, while the hemangiomatous variant had the highest MVD for area A. Maspin positivity was present only in the cytoplasm of ameloblast, stellate reticulum, and the fibrous connective tissue in varying proportions. There was no evidence of the anti-angiogenesis effect of maspin in ameloblastoma from this study. The significance of cytoplasmic localization of maspin in the ameloblasts and stellate reticulum cells needs further investigation.
Asunto(s)
Ameloblastoma/irrigación sanguínea , Antígenos CD34/análisis , Neoplasias Maxilomandibulares/irrigación sanguínea , Serpinas/análisis , Adolescente , Adulto , África Occidental , Anciano , Ameloblastoma/química , Niño , Femenino , Humanos , Inmunohistoquímica , Neoplasias Maxilomandibulares/química , Masculino , Persona de Mediana EdadRESUMEN
In the present study, the structure of two allogeneic and three xenogeneic bone blocks, which are used in dental and orthopedic surgery, were histologically analyzed. The ultimate goal was to assess whether the components postulated by the manufacturer can be identified after applying conventional histological and histochemical staining techniques. Three samples of each material, i.e. allogeneic material-1 and -2 as well as xenogeneic material-1, -2 and -3, were obtained commercially. After decalcification and standardized embedding processes, conventional histological staining was performed in order to detect inorganic matrix, cellular or organic matrix components. Allogeneic material-1 showed trabecular bone-like structures, which were free of cellular components as well as of organic matrix. The allogeneic material-2 showed trabecular bone structures, in which connective tissue and cellular remnants were embedded. Additionally, some connective tissue, which resembled fat-like tissue, was found within this material. The xenogeneic material-1 showed trabecular bone-like structures and contained organic components comparable to that demonstrated for the allogeneic material-2. The xenogeneic material-2 showed trabecular bone structures with single cells located in lacunae. The xenogeneic material-3 also showed trabecular structures. Neither cellular nor organic matrix components were found within this material. According to the data of the present study, the allogeneic material-1 and the xenogeneic material-3 were the only investigated materials for which the obtained histological data were in accordance with the manufacturers advertised information. The remaining three materials showed discrepancies-although the manufacturers of all five bone substitute materials stated that their blocks were free of organic/cellular remnants. These data are of great clinical and material science interest. It seems that even patented processing techniques are not always able to deliver reproducible materials. Although the manufacturers of all five bone blocks stated that their blocks were free of organic/cellular remnants, our histological analysis revealed that three out of five bone blocks did contain such remnants. Such specimens might be able to induce an immune response within the recipient.
Asunto(s)
Aloinjertos/química , Sustitutos de Huesos/síntesis química , Sustitutos de Huesos/normas , Xenoinjertos/química , Ensayo de Materiales/normas , Guías de Práctica Clínica como Asunto , Aloinjertos/normas , Trasplante Óseo/normas , Xenoinjertos/normas , InternacionalidadRESUMEN
The successful vascularisation of complex tissue engineered constructs for bone regeneration is still a major challenge in the field of tissue engineering. In this context, co-culture systems of endothelial cells and osteoblasts represent a promising approach to advance the formation of a stable vasculature as well as an excellent in vitro model to identify factors that positively influence bone healing processes, including angiogenesis. Under physiological conditions, the activation phase of angiogenesis is mainly induced by hypoxia or inflammation. Inflammatory cells such as macrophages secrete proinflammatory cytokines and proangiogenic growth factors, finally leading to the formation of new blood vessels. The aim of this study was to investigate if macrophages might positively influence the formation of microvessel-like structures via inflammatory mechanisms in a co-culture system consisting of human outgrowth endothelial cells (OECs) and primary osteoblasts. Treatment of co-cultures with macrophages (induced from THP-1) resulted in a higher number of microvessel-like structures formed by OECs compared to the co-culture. This change correlated with a significantly higher concentration of the proangiogenic VEGF in cell culture supernatants of triple-cultures and was accompanied by an increase in the expression of different proinflammatory cytokines, such as IL-6, IL-8 and TNFα. In addition, the expression of E-selectin and ICAM-1, adhesion molecules which are strongly involved in the interaction between leukocytes and endothelial cells during the process of inflammation was also found to be higher in triple-cultures compared to the double co-cultures, documenting an ongoing proinflammatory stimulus. These results raise the possibility of actively using pro-inflammatory stimuli in a tissue engineering context to accelerate healing mechanisms.
Asunto(s)
Diferenciación Celular , Citocinas/farmacología , Células Endoteliales/efectos de los fármacos , Macrófagos/metabolismo , Neovascularización Fisiológica , Osteoblastos/efectos de los fármacos , Regeneración Ósea , Huesos/irrigación sanguínea , Huesos/fisiología , Línea Celular Tumoral , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Citocinas/metabolismo , Células Endoteliales/citología , Humanos , Microvasos/citología , Microvasos/fisiología , Osteoblastos/citología , Ingeniería de TejidosRESUMEN
According to present knowledge, blood derived endothelial progenitor cells (EPC) might act as proangiogenic myeloid cells, which play a fundamental role in the regulation of angiogenesis and blood vessel reorganisation. In this context, we have evaluated the contribution of endogenous myeloid cells in co-cultures of blood derived outgrowth endothelial cells (OEC) and osteogenic cells. In addition, we investigated the role of EPC as a potential source of myeloid cells in the formation of vascular structures in an in vitro model consisting of mesenchymal stem cells (MSC) and OEC. For this purpose, we added EPCs to co-cultures of MSC and OECs. Vascular structures and the co-localisation of myeloid cells were analysed by confocal laser microscopy (CLSM) for endothelial and myeloid markers and quantitative image analysis. The molecular effects of myeloid cells were evaluated by quantitative real time PCR, ELISA and protein arrays from cell culture supernatants and lysates. Endogenous myeloid cells were significantly co-localised with angiogenic structures in co-cultures of OEC and osteogenic cells. The active addition of EPC to co-cultures of OEC and MSC resulted in a statistically approved increase in the formation of prevascular structures at early stages of the co-culture process. In addition, we observed an increase of endothelial markers, indicating beneficial effects of EPC or myeloid cells on endothelial cell growth. Furthermore, real time PCR indicated high expression levels of CD68, CD11b and CD163 in co-cultures of EPC and MSC indicating that EPC act at least partly as macrophage like-cells.
Asunto(s)
Regeneración Ósea , Huesos/irrigación sanguínea , Diferenciación Celular , Células Progenitoras Mieloides/citología , Neovascularización Fisiológica , Huesos/fisiología , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Células Progenitoras Mieloides/metabolismo , Osteocitos/citología , Osteocitos/metabolismo , Proteoma/genética , Proteoma/metabolismoRESUMEN
Angiotensin I-converting enzyme (kininase II, ACE, and CD143) availability is a determinant of local angiotensin and kinin concentrations and their physiological actions. Until now, it is unclear whether the decrease of pulmonary ACE activity in sepsis-described in clinical studies-is due to an enzyme compensatory downregulation (reduced ACE-mRNA expression) to shedding of ACE or endothelial damage. To address these questions, ACE distribution under septic conditions was studied in vitro by treating pulmonary microvascular endothelial cells (HPMEC) and human umbilical vein endothelial cells (HUVEC) with lipopolysaccharide from Escherichia coli (LPS). Primary isolated HUVEC and HPMEC were compared by detecting ACE activity, membrane-bound ACE, as well as shedding and mRNA production of ACE with and without LPS (1 ng/ml-1 µg/ml). ACE mRNA expression was detected by real-time PCR, and shedded ACE was measured in cell culture supernatant by ELISA. Additionally, membrane-bound protein expression was investigated by immunohistochemistry in situ. In septic ARDS, the distribution of ACE protein was significantly reduced in all lung endothelial cells (p<0.001). After stimulation with LPS, cultivated HPMEC showed more markedly than HUVEC, a concentration-dependent reduction of ACE protein expression compared to the respective untreated controls. Real-time PCR demonstrated a reduced ACE mRNA expression after LPS stimulation, predominantly in HPMEC. Specifically, in HPMEC, a concentration-dependent increase of shedded ACE was shown 24 h after LPS treatment. HPMEC cultures are an apt model for the investigation of pulmonary ACE expression in sepsis. This study suggests that reduced pulmonary microvascular endothelial ACE expression in septic ARDS is caused by two processes: (initial) increased shedding of ACE accompanied by a compensatory downregulation of ACE-mRNA and membrane-bound protein expression.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Peptidil-Dipeptidasa A/biosíntesis , Escherichia coli , Células Endoteliales de la Vena Umbilical Humana , Humanos , Lipopolisacáridos/farmacología , Pulmón/citología , Proteínas de la Membrana/biosíntesis , Peptidil-Dipeptidasa A/genéticaRESUMEN
PURPOSE: The host tissue reaction to the xenogeneic bone substitute Bio-Oss™ (Geistlich Biomaterials, Wolhousen, Switzerland) was investigated focusing on the participating inflammatory cells and implantation bed vascularization. MATERIALS AND METHODS: Bio-Oss™ was implanted subcutaneously into CD1 mice for up to 60 days and analyzed by means of specialized histological and histomorphometrical techniques after explantation. RESULTS: Bio-Oss™ induced within the first 15 days an early high vascularization combined with a marked presence of multinucleated giant cells. The latter cells were associated mainly with the smaller sized granules within the implantation bed. Toward the end of the study the number of multinucleated giant cells decreased while the tissue reaction to the larger granules was mainly mononuclear. CONCLUSION: The results of the present study showed that smaller xenogeneic bone substitute granules induce multinucleated giant cells, whereas the larger-sized ones became integrated within the implantation bed by means of a mononuclear cell-triggered granulation tissue. Obviously, the presence of multinucleated giant cells within biomaterial implantation beds is not only related to the type of synthetic bone substitute material, but also to the granule size of the natural-based xenogeneic bone substitute material.
RESUMEN
OBJECTIVE: Defective placental maturation is associated with restricted functional capacity and adverse perinatal fetal outcomes. The aim of the study was a comparative analysis of the role of mRNA expression of various angiogenic factors in placental maturation defects. STUDY DESIGN: We examined the mRNA expression patterns of prokineticin 1 (PK1), its receptors (PKRs), basic-fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) in tissue from third-trimester placentae that exhibited delayed or accelerated villous maturation. RESULTS: The expression of PK1 and PKR2 was elevated in placental tissue exhibiting accelerated maturation and a predominant differentiation of terminal villi. The opposite was found in tissue exhibiting delayed maturation and deficiency of the terminal villi. In addition, low expression of bFGF correlated with the predominant differentiation of terminal villi, whereas the opposite was observed when terminal villi were deficient. The expression of VEGF, PIGF, and PKR1 showed no significant differences between the groups. CONCLUSION: Defective placental maturation is associated with an imbalance of expression of bFGF and PK1. Our results demonstrate an involvement of the PK1/PKR2-signalling pathway in the regulation of the functional adequate capillarization in late pregnancy. We propose the bFGF/PK1-ratio as a monitor of placental function and a possible indicator of latent clinical problems, such as placental dysfunction leading to fetal hypoxia.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/metabolismo , Hormonas Gastrointestinales/metabolismo , Enfermedades Placentarias/metabolismo , Proteínas Gestacionales/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/metabolismo , Femenino , Humanos , Placenta/patología , Enfermedades Placentarias/patología , Factor de Crecimiento Placentario , Embarazo , Tercer Trimestre del Embarazo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismoRESUMEN
Cobalt-based materials are widely used for coronary stents, as well as bone and joint implants. However, their use is associated with high corrosion incidence. Titanium alloys, by contrast, are more biocompatible owing to the formation of a relatively inactive titanium oxide (TiO2) layer on their surface. This study was aimed at improving Co28Cr6Mo alloy cytocompatibility via sol-gel TiO2 coating to reduce metal corrosion and metal ion release. Owing to their role in inflammation and tissue remodelling around an implant, endothelial cells present a suitable in vitro model for testing the biological response to metallic materials. Primary human endothelial cells seeded on Co28Cr6Mo showed a stress phenotype with numerous F-actin fibres absent on TiO2-coated material. To investigate this effect at the gene expression level, cDNA microarray analysis of in total 1301 genes was performed. Compared with control cells, 247 genes were expressed differentially in the cells grown on Co28Cr6Mo, among them genes involved in proliferation, oxidative stress response and inflammation. TiO2 coating reduced the effects of Co28Cr6Mo on gene expression in endothelial cells, with only 34 genes being differentially expressed. Quantitative real-time polymerase chain reaction and protein analysis confirmed microarray data for selected genes. The effect of TiO2 coating can be, in part, attributed to the reduced release of Co(2+), because addition of CoCl2 resulted in similar cellular responses. TiO2 coating of cobalt-based materials, therefore, could be used in the production of cobalt-based devices for cardiovascular and skeletal applications to reduce the adverse effects of metal corrosion products and to improve the response of endothelial and other cell types.