RESUMO
As an attractive biomaterial for bone reconstruction, injectable biomaterials have many prominent characteristics such as good biocompatibility and bone-filling ability. However, there are weak as load-bearing scaffolds. In this study, polyvinyl alcohol (PVA) and bioactive glass (BAG) were interpenetrated into sodium alginate (SA) network to obtain self-enhanced injectable hydrogel. The optimum ratio of PVA/SA/BAG hydrogel was determined based on injectability, gelation time and chemical characterization. Results showed that the selected ratio had the shortest gelation time of 3.5min, and the hydrogel had a rough surface and good coagulation property. The hydrogel was capable of carrying 1kg of weight by mineralization for 14 d The compressive strength, compressive modulus, and fracture energy of the hydrogel reached 0.12MPa, 0.376MPa and 17.750kJ m-2, respectively. Meanwhile, the hydrogel had high moisture content and dissolution rate, and it was sensitive to temperature and ionic strength. Hydroxyapatite was generated on the hydrogel surface, and the hydrogel pores increased, and the pore size enlarged. The biocompatibility of PVA/SA/BAG hydrogel was analyzed using hemolysis and cytotoxicity assays. Results revealed its good biocompatibility with low hemolysis rate and no cytotoxicity to MC3T3-E1 cells. The hydrogel was also found to promote the differentiation of MC3T3-E1 cells with significantly increased in ALP activity and expression of relevant differentiation factors. In vitro mineralization assay showed an increase in calcium nodules and calcification area, indicating the ability of hydrogel to promote mineralization MC3T3-E1 cells. These findings indicated that PVA/SA/BAG hydrogel had potential uses in the field of irregular bone-defect repair due to its injectability, cytocompatibility, and tailorable functionality.
Assuntos
Hemólise , Hidrogéis , Humanos , Hidrogéis/química , Materiais Biocompatíveis , Durapatita/química , Diferenciação CelularRESUMO
Antler ossified tissue has been widely used for the extraction of bioactive peptides. In this study, collagen was prepared from antler ossified tissue via acetic acid and pepsin. Five different proteases were used to hydrolyze the collagen and the hydrolysate treated by neutrase (collagen peptide named ACP) showed the highest DPPH radical clearance rate. The extraction process of ACP was optimized by response surface methodology, and the optimal conditions were as follows: a temperature of 52 °C, a pH of 6.1, and an enzyme concentration of 3200 U/g, which resulted in the maximum DPPH clearance rate of 74.41 ± 0.48%. The peptides (ACP-3) with the strongest antioxidant activity were obtained after isolation and purification, and its DPPH free radical clearance rate was 90.58 ± 1.27%; at the same time, it exhibited good scavenging activity for ABTS, hydroxyl radical, and superoxide anion radical. The study investigated the protective effect of ACP-3 on oxidative damage in HaCaT cells. The findings revealed that all groups that received ACP-3 pretreatment exhibited increased activities of SOD, GSH-Px, and CAT compared to the model group. Furthermore, ACP-3 pretreatment reduced the levels of ROS and MDA in HaCaT cells subjected to H2O2-induced oxidative damage. These results suggest that collagen peptides derived from deer antler ossified tissue can effectively mitigate the oxidative damage caused by H2O2 in HaCaT cells, thereby providing a foundation for the utilization of collagen peptides in pharmaceuticals and cosmetics.
Assuntos
Chifres de Veado , Cervos , Animais , Humanos , Antioxidantes/farmacologia , Peróxido de Hidrogênio/farmacologia , Células HaCaT , Estresse Oxidativo , Peptídeos/farmacologia , Colágeno/farmacologiaRESUMO
Insulin-like growth factor (IGF)-1 is a polypeptide hormone with vital biological functions in bone cells. The abnormal expression of IGF-1 has a serious effect on bone growth, particularly bone remodeling. Evidence from animal models and human disease suggested that both IGF-1 deficiency and excess cause changes in bone remodeling equilibrium, resulting in profound alterations in bone mass and development. Here, we first introduced the functions and mechanisms of the members of IGFs in bone. Subsequently, the critical role of IGF-1 in the process of bone remodeling were emphasized from the aspects of bone resorption and bone formation respectively. This review explains the mechanism of IGF-1 in maintaining bone mass and bone homeostasis to a certain extent and provides a theoretical basis for further research.
Assuntos
Reabsorção Óssea , Fator de Crescimento Insulin-Like I , Animais , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Osso e Ossos/metabolismo , Densidade ÓsseaRESUMO
Both vascular endothelial growth factor A (VEGF) and osteopontin (OPN) can directly induce tumor angiogenesis, which is essential for the growth and metastasis of solid tumors. Here we engineered a bispecific antibody (VEGF/OPN-BsAb) using the anti-VEGF-A antibody bevacizumab and the anti-OPN antibody hu1A12. Compared with hu1A12 alone and bevacizumab alone, VEGF/OPN-BsAb was significantly more effective in inhibiting tumor angiogenesis in a highly metastatic human hepatocellular carcinoma nude mouse model. Further study demonstrated that VEGF/OPN-BsAb could effectively suppress primary tumor growth and metastasis to lungs, suggesting that it might be a promising therapeutic agent for treatment of metastatic cancer.
Assuntos
Anticorpos Biespecíficos/farmacologia , Neoplasias/prevenção & controle , Osteopontina/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Bevacizumab , Células CHO , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cricetinae , Cricetulus , Feminino , Humanos , Cinética , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Hepáticas Experimentais/prevenção & controle , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/patologia , Neovascularização Patológica/prevenção & controle , Osteopontina/genética , Osteopontina/imunologia , Ligação Proteica/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carga Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To develop more effective anti-CD20 reagents for B-cell lymphoma, we designed and constructed a bispecific tetravalent anti-CD20 antibody, 11B8/2F2(ScFvHL)(4)-Fc, derived from two fully human monoclonal antibodies (mAb), 2F2 and 11B8. 2F2 is a type I CD20 mAb, which is potent in complement-dependent cytotoxicity (CDC) assays but poor at inducing apoptosis, whereas 11B8 is a type II CD20 mAb, which is effective in induction of apoptosis but ineffective in CDC. Our results showed that 11B8/2F2(ScFvHL)(4)-Fc possessed apoptosis-inducing activity markedly superior to that of 2F2, and even 11B8, 11B8 plus 2F2, and 2F2(ScFvHL)(4)-Fc, a 2F2-derived monospecific tetravalent antibody developed previously. Interestingly, 11B8/2F2(ScFvHL)(4)-Fc displayed a similar ability to mediate CDC as 2F2(ScFvHL)(4)-Fc, although two of its four antigen-binding arms originated from 11B8. To explore why 11B8/2F2(ScFvHL)(4)-Fc was so potent in both CDC and apoptotic activity, a bispecific divalent antibody composed of 2F2 and 11B8, denoted as 11B8/2F2-ScFvFc, was constructed and characterized. Our results partially explained the reason for the potent CDC and apoptosis-inducing activity of 11B8/2F2(ScFvHL)(4)-Fc. Further in vivo therapy studies showed that 11B8/2F2(ScFvHL)(4)-Fc had a significantly more potent antitumor activity compared with 2F2, 11B8, 2F2 plus 11B8, and 2F2(ScFvHL)(4)-Fc. These data suggest that 11B8/2F2(ScFvHL)(4)-Fc may serve as a potential therapeutic agent for B-cell lymphoma.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Antígenos CD20/imunologia , Linfoma de Células B/terapia , Animais , Anticorpos Biespecíficos/biossíntese , Anticorpos Biespecíficos/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Apoptose/imunologia , Células CHO , Processos de Crescimento Celular/imunologia , Cricetinae , Cricetulus , Feminino , Humanos , Fragmentos de Imunoglobulinas/biossíntese , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Linfoma de Células B/imunologia , Camundongos , Camundongos Nus , Camundongos SCID , TransfecçãoRESUMO
The major cause of cancer mortality is the metastatic spread of tumor cells that can occur via multiple routes, including the vascular system and the lymphatic system. In this study, we developed an IgG-like fusion protein molecule [vascular endothelial growth factor (VEGF) receptor 31-immunoglobulin (VEGFR31-Ig)] which could simultaneously bind the angiogenic growth factor VEGF-A and the lymphangiogenic growth factor VEGF-C. Importantly, VEGFR31-Ig exhibited VEGF-A-binding affinity similar to that of VEGFTrap, the most potent VEGF-A binder, and VEGF-C-binding affinity comparable with that of the soluble fusion protein VEGFR3-Ig (sVEGFR3). Pharmacokinetic analysis in mice showed that VEGFR31-Ig had improved pharmacokinetic properties compared with either VEGFTrap or sVEGFR3. In a highly metastatic human hepatocellular carcinoma (HCCLM3) model in severe combined immunodeficient mice, VEGFR31-Ig potently blocked both tumor angiogenesis and lymphangiogenesis, effectively inhibiting primary tumor growth and metastasis to lungs and lymph nodes. In contrast, VEGFTrap only suppressed primary tumor growth and metastasis to lungs by inhibiting tumor angiogenesis, whereas VEGFR3 was only effective in suppressing tumor metastasis to lymph nodes by blocking tumor lymphangiogenesis. Although a combination of VEGFTrap (25 mg/kg twice weekly) and sVEGFR3 (25 mg/kg twice weekly) can achieve the same therapeutic effect as VEGFR31-Ig (25 mg/kg twice weekly) in the HCCLM3 xenograft mouse model, developing two separate receptor-Ig fusion proteins for clinical use as combination therapy is impractical, mainly owing to regulatory hurdles and cost. Taken together, the VEGFR31-Ig fusion protein presented here has been suggested to have great potential for the treatment of metastatic cancer.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Imunoglobulina G/administração & dosagem , Neoplasias Hepáticas/tratamento farmacológico , Receptores de Fatores de Crescimento do Endotélio Vascular/administração & dosagem , Proteínas Recombinantes de Fusão/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator C de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Células CHO , Carcinoma Hepatocelular/irrigação sanguínea , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Feminino , Humanos , Imunoglobulina G/genética , Neoplasias Hepáticas/irrigação sanguínea , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Linfangiogênese/efeitos dos fármacos , Metástase Linfática , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCID , Neovascularização Patológica/tratamento farmacológico , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Proteínas Recombinantes de Fusão/genéticaRESUMO
Despite the effectiveness of the anti-CD20 chimeric antibody (mAb), rituximab, in treating B-cell lymphomas, its efficacy remains variable and often modest. In this study, a humanized anti-CD20 antibody, hu8E4, was generated by complementarity-determining region grafting method. Hu8E4 was as effective as rituximab in mediating antibody-dependent cellular cytotoxicity and inducing apoptosis in B-lymphoma cells, but it exhibited much more potent complement-dependent cytotoxicity than rituximab. Immunotherapeutic studies showed that hu8E4 was significantly more effective than rituximab in prolonging the survival of severe combined immunodeficient mice bearing human B-cell lymphomas, suggesting that it might be a promising therapeutic agent for B-cell lymphomas.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD20/imunologia , Linfoma de Células B/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Citotoxicidade Celular Dependente de Anticorpos , Sequência de Bases , Células CHO , Cricetinae , Cricetulus , Primers do DNA , Humanos , Imunoterapia , Linfoma de Células B/terapia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência de AminoácidosRESUMO
Understanding the evolutionary mechanism that acts at the interfaces of protein-protein complexes is a fundamental issue with high interest for delineating the macromolecular complexes and networks responsible for regulation and complexity in biological systems. To investigate whether the evolution of protein-protein interface acts in a similar way as antibody affinity maturation, we incorporated evolutionary information derived from antibody affinity maturation with common simulation techniques to evaluate prediction success rates of the computational method in affinity improvement in four different systems: antibody-receptor, antibody-peptide, receptor-membrane ligand, and receptor-soluble ligand. It was interesting to find that the same evolutionary information could improve the prediction success rates in all the four protein-protein complexes with an exceptional high accuracy (>57%). One of the most striking findings in our present study is that not only in the antibody-combining site but in other protein-protein interfaces almost all of the affinity-enhancing mutations are located at the germline hotspot sequences (RGYW or WA), indicating that DNA hot spot mechanisms may be widely used in the evolution of protein-protein interfaces. Our data suggest that the evolution of distinct protein-protein interfaces may use the same basic strategy under selection pressure to maintain interactions. Additionally, our data indicate that classical simulation techniques incorporating the evolutionary information derived from in vivo antibody affinity maturation can be utilized as a powerful tool to improve the binding affinity of protein-protein complex with a high accuracy.
Assuntos
Evolução Molecular , Mapeamento de Interação de Proteínas/métodos , Proteínas/genética , Proteínas/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Murinos , Afinidade de Anticorpos/genética , Afinidade de Anticorpos/imunologia , Antígenos CD/química , Antígenos CD/genética , Antígenos CD/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Antígeno CTLA-4 , Simulação por Computador , Cristalografia por Raios X , Subunidade alfa de Receptor de Interleucina-2/química , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/química , Rituximab , Homologia de Sequência de AminoácidosRESUMO
Despite widespread use of the anti-CD20 monoclonal antibody (mAb), rituximab, in treating B-cell lymphomas, its efficacy remains variable and often modest. A better understanding of rituximab-mediated killing mechanisms is essential to develop more effective therapeutic agents. In this study, we modulated the binding property of rituximab by introducing several point mutations in its complementarity-determining regions. The data showed that changing the binding avidity of rituximab in the range from 10(-8) to 10(-10) M could regulate its antibody-dependent cellular cytotoxicity but not affect its complement-dependent cytotoxicity and apoptosis-inducing activity in B-lymphoma cells. Contradictory to previous findings, we found that the complement-dependent cytotoxicity potency of CD20 mAb was independent of the off-rate. Despite still being a type I CD20 mAb, a rituximab triple mutant (H57DE/H102YK/L93NR), which had a similar binding avidity to a double mutant (H57DE/H102YK), was unexpectedly found to have extremely potent apoptosis-inducing activity. Moreover, this triple mutant, which was demonstrated to efficiently initiate both caspase-dependent and -independent apoptosis, exhibited potent in vivo therapeutic efficacy, even in the rituximab-resistant lymphoma model, suggesting that it might be a promising therapeutic agent for B-cell lymphomas.