Corneal transplantation is the most common surgical procedure amongst solid organ transplants with a high survival rate of 86% at 1-year post-grafting. This high success rate has been attributed to the immune privileg...Corneal transplantation is the most common surgical procedure amongst solid organ transplants with a high survival rate of 86% at 1-year post-grafting. This high success rate has been attributed to the immune privilege of the eye. However, mechanisms originally thought to promote immune privilege, such as the lack of antigen presenting cells and vessels in the cornea, are challenged by recent studies. Nevertheless, the immunological and physiological features of the cornea promoting a relatively weak alloimmune response is likely responsible for the high survival rate in "low-risk" settings. Furthermore, although corneal graft survival in "lowrisk" recipients is favourable, the prognosis in "high-risk" recipients for corneal graft is poor. In "high-risk" grafts, the process of indirect allorecognition is accelerated by the enhanced innate and adaptive immune responses due to pre-existing inflammation and neovascularization of the host bed. This leads to the irreversible rejection of the allograft and ultimately graft failure. Many therapeutic measures are being tested in pre-clinical and clinical studies to counter the immunological challenge of "high-risk" recipients. Despite the prevailing dogma, recent data suggest that tissue matching together with use of systemic immunosuppression may increase the likelihood of graft acceptance in "high-risk" recipients. However, immunosuppressive drugs are accompanied with intolerance/side effects and toxicity, and therefore, novel cell-based therapies are in development which target host immune cells and restore immune homeostasis without significant side effect of treatment. In addition, developments in regenerative medicinemay be able to solve both important short comings of allotransplantation:(1) graft rejection and ultimate graft failure; and(2) the lack of suitable donor corneas. The advances in technology and research indicate that wider therapeutic choices for patients may be available to address the worldwide problem of corneal blindness in both "low-risk" and "high-risk" hosts.展开更多
The importance of the extracellular matrix （ECM） in contributing to structural, mechanical, functional and tissue-specific features in the body is well appreciated. While the ECM was previously considered to be a pa...The importance of the extracellular matrix （ECM） in contributing to structural, mechanical, functional and tissue-specific features in the body is well appreciated. While the ECM was previously considered to be a passive bystander, it is now evident that it plays active, dynamic and fexible roles in shaping cell survival, differentiation, migration and death to varying extents depending on the specific site in the body. Dendritic cells （DCs） are recognized as potent antigen presenting cells present in many tissues and in blood, continuously scrutinizing the microenvironment for antigens and mounting local and systemic host responses against harmful agents. DCs also play pivotal roles in maintaining homeostasis to harmless self-antigens, critical for preventing autoimmunity. What is less understood are the complex interactions between DCs and the ECM in maintaining this balance between steady-state tissue residence and DC activation during inflammation. DCs are finely tuned to inflammation-induced variations in fragment length, accessible epitopes and post-translational modifications of individual ECM components and correspondingly interpret these changes appropriately by adjusting their profiles of cognate binding receptors and downstream immune activation. The successful design and composition of novel ECM-based mimetics in regenerative medicine and other applications rely on our improved understanding of DC-ECM interplay in homeostasis and the challenges involved in maintaining it.展开更多
Background:Patients with inflammation or severe corneal pathology are often at high risk of rejecting the human donor corneas that they receive during transplantation.Our goal was to determine whether cell-free implan...Background:Patients with inflammation or severe corneal pathology are often at high risk of rejecting the human donor corneas that they receive during transplantation.Our goal was to determine whether cell-free implants incorporating phosphorylcholine-based polymer,2-methacryloyloxyethyl phosphorylcholine(MPC),which has inflammation suppressing properties,can support repair and regeneration of ulcerated,high-risk corneas.Methods:Interpenetrating networks of recombinant human collagen and MPC(RHC-MPC)were fabricated into corneal implants in a certified and monitored cleanroom.An open-label,first-in-human observational study was conducted following ISO 14971 and 14155:2011,the Declaration of Helsinki and relevant laws of Ukraine and India,after respective ethical approval and trial registration.Seven unilaterally blind patients,aged 36 to 76 years old,diagnosed with conditions putting them at high risk of rejecting conventional corneal transplantation,and capable of providing informed written consent were grafted and followed up for an average of two years.However,RHC like native collagen is a large macromolecule and difficult to process and is non-customisable.Hence,small,customisable analogs to collagen comprising collagen-like peptides(CLP)conjugated to polyethylene glycol(PEG)were developed.These CLP-PEG analogs like RHC were combined with MPC into implants and tested in mini-pig models with alkali burned corneas,a high-risk condition.Results:One patient had an unrelated infection that necessitated re-grafting and was excluded from the study.The RHC-MPC implants in the remaining six patients were stably integrated throughout the entire observational period.There was regeneration of the cornea epithelium and stroma.Significant vision improvement was observed in in patients with damaged corneas due to infection.By two weeks post-operation,RHC-MPC implanted corneas of patients with active ulcers were free from the symptoms of pain,irritation and photophobia.Over the two-year follow-up period,sensitivity to touch improved,suggesting that the implants were able to promote nerve regeneration.The results seen in the clinical trial were reproduced in corneal grafts comprising MPC and the CLP-PEG collagen analog.Both CLP-PEG-MPC and control CLP-PEG only implants promoted regeneration of corneal epithelium,stroma and nerves.However,the alkali-burned corneas grafted with CLP-PEG-MPC implants retained the thickness of their healthy contralateral controls.Control corneas with CLP-PEG implants without the MPC however,were significantly thicker.Conclusions:These results demonstrate that collagen or its synthetic analog comprising CLP-PEG,can promote regeneration.The incorporation of MPC appears to suppress inflammation in pathologies that constitute conditions with a high-risk for graft rejection.Regeneration was able to occur in inflamed corneas as evidenced by CLP-PEG only grafts,but long-term follow-up is needed to determine if the chronic inflammation may influence graft failure over time.展开更多
Background:The sterilization of corneal implants composed of carbodiimide crosslinked recombinant human collagen type III(RHCIII)and phosphorylcholine polymers(RHCIII-MPC)is constrained by the biochemical properties o...Background:The sterilization of corneal implants composed of carbodiimide crosslinked recombinant human collagen type III(RHCIII)and phosphorylcholine polymers(RHCIII-MPC)is constrained by the biochemical properties of RHCIII.Early human trials used 1%chloroform in 0.1 M phosphate buffered saline(C-PBS),but require a stringent wash procedure with antibiotics to remove the chloroform.Irradiation with gamma or electron-beam(e-beam)allows a chemical-free sterilization method,but may result in crosslinking or denaturation.Here,electron-beam irradiation is evaluated as a sterilization method for RHCIII-MPC implants.Methods:Dose-finding study:RHCIII-MPC were cast in round,350µm thick,12 mm diameter molds for corneal implants and 0.5 mm thick dumbbell-shaped molds for mechanical testing.The hydrogels received an irradiation dose of 17,19,or 21 kGy and unirradiated controls were stored in C-PBS,n=3 per group.The hydrogels were tested for sterility and endotoxin,optical and mechanical properties,biodegradation,free radicals,and cell compatibility.Clinical evaluation in rabbits:RHCIII-MPC implants were e-beamed at 17 kGy or kept in C-PBS.One implant from each group was implanted into the right cornea of each rabbit by deep anterior lamellar keratoplasty,n=4 animals per group.Animals underwent preoperative and 6-month post-operative in vivo confocal microscopy(IVCM)to check nerve count and ingrowth of keratocytes.Corneal grafts and controls were assessed via histology and immunohistochemistry.Results:Dose finding study:hydrogels were sterile at all irradiation doses with no evidence of free radicals.There were no significant differences in optical or mechanical properties between the treatment groups and controls.All hydrogels supported cell growth.The 19 and 21 kGy implants had high collagenase degradation for 21 hours until they stabilized,whereas the 17 kGy and C-PBS implants had gradual degradation until 48 hours.Clinical results:the rabbits did not experience post-surgical inflammatory reactions and full epithelial coverage of the implants occurred within the first week of surgery for all animals.Mild neovascularization occurred in all animals,but resolved by 6-month follow-up.A mild 0.5-1.0 grade subepithelial haze was observed in all rabbits,but the implanted grafts remained transparent.Re-innervation occurred in all grafts with no significant differences between sterilization methods.All regenerated corneas had mucin production and were positive for cytokeratin 3 and 12.Grafted and control corneas were negative for macrophages and blood vessels.Conclusions:E-beam sterilization is a safe and effective form of sterilization for RHCIII-MPC implants.展开更多
Background:Regeneration of nerves or nerve bundles is problematic mainly due to issues of nerves finding their target tissues.A very clear example of this is the lack of treatments for traumatic injury to the optic ne...Background:Regeneration of nerves or nerve bundles is problematic mainly due to issues of nerves finding their target tissues.A very clear example of this is the lack of treatments for traumatic injury to the optic nerve,something that is associated with surgery or trauma to the skull.Nerve guides have been used to support this for the better part of the last century,unfortunately the clinical improvements in patients receiving this sort of treatment is poor.Large improvements to the type of nerve guides used are needed to make this a viable solution for repair.It has been shown that electrostimulation of cells on conductive polymers can have positive effects on nerve regeneration.There are several material innovations that improve on speed of nerve regeneration;conductive polymer coatings being one example.There are constant improvements on solutions for nerve regeneration in many fields,unfortunately combining these different solutions is often slow.We combine electrospinning,3D printing and surface modification.Electrospinning allows control over fibrous structures.We tune the surface properties using conductive polymer coatings.The conductive fibrous structure can be integrated in a larger 3D printed scaffold that takes the role of guiding the nerve bundle.Methods:For manufacture of aligned fibers,PCL in chloroform was electrospun on a rotating mandrel.Random fibers were collected on a flat stationary collector.Dip coating was performed by submerging the fibrous scaffold in a solution of PEDOT:PSS in water and isopropanol.An outer layer of PEDOT:tosylate was added using vapor phase polymerization(VPP).3D printing was performed using an ink consisting of 0.25%alginate and 8.75%gelatin.The ink was cross-linked after printing using 0.4%CaCl2.Cell cultures were performed using chick dorsal root ganglia and a mouse neuroblastoma cell line.Ganglia and cells were seeded on the fibrous scaffolds.Electrostimulation was performed using a custom set up at constant DC current and slow pulsed DC current(1 min on off cycle)Materials were imaged using scanning electron microscopy.Cell cultures were stained using ICC and imaged with fluorescence microscopy.Results:All of the materials supported cell growth and neurite extension to some degree.The materials that were coated with PEDOT:tosylate and a combination of PEDOT:tosylate+PEDOT:PSS outperformed the PSS only group.Stimulation with a slow pulsed or constant DC current increased neurite extension on the negative pole,while there was inhibition of neurite growth on the opposite pole.The 3D printed outer layer serves as a biocompatible,bioactive support and guide for the bundle of neurites.Conclusions:The nerve guides can guide nerve growth.The 3D printed scaffold is cell friendly.The construct allows electrostimulation to increase speed of regeneration.展开更多
基金Supported by Saving Sight in Grampian,Development Trust of University of Aberdeen,United KingdomAction Medical Research United Kingdom(grant SP4328)Link?ping University,Sweden
文摘Corneal transplantation is the most common surgical procedure amongst solid organ transplants with a high survival rate of 86% at 1-year post-grafting. This high success rate has been attributed to the immune privilege of the eye. However, mechanisms originally thought to promote immune privilege, such as the lack of antigen presenting cells and vessels in the cornea, are challenged by recent studies. Nevertheless, the immunological and physiological features of the cornea promoting a relatively weak alloimmune response is likely responsible for the high survival rate in "low-risk" settings. Furthermore, although corneal graft survival in "lowrisk" recipients is favourable, the prognosis in "high-risk" recipients for corneal graft is poor. In "high-risk" grafts, the process of indirect allorecognition is accelerated by the enhanced innate and adaptive immune responses due to pre-existing inflammation and neovascularization of the host bed. This leads to the irreversible rejection of the allograft and ultimately graft failure. Many therapeutic measures are being tested in pre-clinical and clinical studies to counter the immunological challenge of "high-risk" recipients. Despite the prevailing dogma, recent data suggest that tissue matching together with use of systemic immunosuppression may increase the likelihood of graft acceptance in "high-risk" recipients. However, immunosuppressive drugs are accompanied with intolerance/side effects and toxicity, and therefore, novel cell-based therapies are in development which target host immune cells and restore immune homeostasis without significant side effect of treatment. In addition, developments in regenerative medicinemay be able to solve both important short comings of allotransplantation:(1) graft rejection and ultimate graft failure; and(2) the lack of suitable donor corneas. The advances in technology and research indicate that wider therapeutic choices for patients may be available to address the worldwide problem of corneal blindness in both "low-risk" and "high-risk" hosts.
基金Supported by The Royal College of Surgeons of Edinburgh
文摘The importance of the extracellular matrix （ECM） in contributing to structural, mechanical, functional and tissue-specific features in the body is well appreciated. While the ECM was previously considered to be a passive bystander, it is now evident that it plays active, dynamic and fexible roles in shaping cell survival, differentiation, migration and death to varying extents depending on the specific site in the body. Dendritic cells （DCs） are recognized as potent antigen presenting cells present in many tissues and in blood, continuously scrutinizing the microenvironment for antigens and mounting local and systemic host responses against harmful agents. DCs also play pivotal roles in maintaining homeostasis to harmless self-antigens, critical for preventing autoimmunity. What is less understood are the complex interactions between DCs and the ECM in maintaining this balance between steady-state tissue residence and DC activation during inflammation. DCs are finely tuned to inflammation-induced variations in fragment length, accessible epitopes and post-translational modifications of individual ECM components and correspondingly interpret these changes appropriately by adjusting their profiles of cognate binding receptors and downstream immune activation. The successful design and composition of novel ECM-based mimetics in regenerative medicine and other applications rely on our improved understanding of DC-ECM interplay in homeostasis and the challenges involved in maintaining it.
文摘Background:Patients with inflammation or severe corneal pathology are often at high risk of rejecting the human donor corneas that they receive during transplantation.Our goal was to determine whether cell-free implants incorporating phosphorylcholine-based polymer,2-methacryloyloxyethyl phosphorylcholine(MPC),which has inflammation suppressing properties,can support repair and regeneration of ulcerated,high-risk corneas.Methods:Interpenetrating networks of recombinant human collagen and MPC(RHC-MPC)were fabricated into corneal implants in a certified and monitored cleanroom.An open-label,first-in-human observational study was conducted following ISO 14971 and 14155:2011,the Declaration of Helsinki and relevant laws of Ukraine and India,after respective ethical approval and trial registration.Seven unilaterally blind patients,aged 36 to 76 years old,diagnosed with conditions putting them at high risk of rejecting conventional corneal transplantation,and capable of providing informed written consent were grafted and followed up for an average of two years.However,RHC like native collagen is a large macromolecule and difficult to process and is non-customisable.Hence,small,customisable analogs to collagen comprising collagen-like peptides(CLP)conjugated to polyethylene glycol(PEG)were developed.These CLP-PEG analogs like RHC were combined with MPC into implants and tested in mini-pig models with alkali burned corneas,a high-risk condition.Results:One patient had an unrelated infection that necessitated re-grafting and was excluded from the study.The RHC-MPC implants in the remaining six patients were stably integrated throughout the entire observational period.There was regeneration of the cornea epithelium and stroma.Significant vision improvement was observed in in patients with damaged corneas due to infection.By two weeks post-operation,RHC-MPC implanted corneas of patients with active ulcers were free from the symptoms of pain,irritation and photophobia.Over the two-year follow-up period,sensitivity to touch improved,suggesting that the implants were able to promote nerve regeneration.The results seen in the clinical trial were reproduced in corneal grafts comprising MPC and the CLP-PEG collagen analog.Both CLP-PEG-MPC and control CLP-PEG only implants promoted regeneration of corneal epithelium,stroma and nerves.However,the alkali-burned corneas grafted with CLP-PEG-MPC implants retained the thickness of their healthy contralateral controls.Control corneas with CLP-PEG implants without the MPC however,were significantly thicker.Conclusions:These results demonstrate that collagen or its synthetic analog comprising CLP-PEG,can promote regeneration.The incorporation of MPC appears to suppress inflammation in pathologies that constitute conditions with a high-risk for graft rejection.Regeneration was able to occur in inflamed corneas as evidenced by CLP-PEG only grafts,but long-term follow-up is needed to determine if the chronic inflammation may influence graft failure over time.
文摘Background:The sterilization of corneal implants composed of carbodiimide crosslinked recombinant human collagen type III(RHCIII)and phosphorylcholine polymers(RHCIII-MPC)is constrained by the biochemical properties of RHCIII.Early human trials used 1%chloroform in 0.1 M phosphate buffered saline(C-PBS),but require a stringent wash procedure with antibiotics to remove the chloroform.Irradiation with gamma or electron-beam(e-beam)allows a chemical-free sterilization method,but may result in crosslinking or denaturation.Here,electron-beam irradiation is evaluated as a sterilization method for RHCIII-MPC implants.Methods:Dose-finding study:RHCIII-MPC were cast in round,350µm thick,12 mm diameter molds for corneal implants and 0.5 mm thick dumbbell-shaped molds for mechanical testing.The hydrogels received an irradiation dose of 17,19,or 21 kGy and unirradiated controls were stored in C-PBS,n=3 per group.The hydrogels were tested for sterility and endotoxin,optical and mechanical properties,biodegradation,free radicals,and cell compatibility.Clinical evaluation in rabbits:RHCIII-MPC implants were e-beamed at 17 kGy or kept in C-PBS.One implant from each group was implanted into the right cornea of each rabbit by deep anterior lamellar keratoplasty,n=4 animals per group.Animals underwent preoperative and 6-month post-operative in vivo confocal microscopy(IVCM)to check nerve count and ingrowth of keratocytes.Corneal grafts and controls were assessed via histology and immunohistochemistry.Results:Dose finding study:hydrogels were sterile at all irradiation doses with no evidence of free radicals.There were no significant differences in optical or mechanical properties between the treatment groups and controls.All hydrogels supported cell growth.The 19 and 21 kGy implants had high collagenase degradation for 21 hours until they stabilized,whereas the 17 kGy and C-PBS implants had gradual degradation until 48 hours.Clinical results:the rabbits did not experience post-surgical inflammatory reactions and full epithelial coverage of the implants occurred within the first week of surgery for all animals.Mild neovascularization occurred in all animals,but resolved by 6-month follow-up.A mild 0.5-1.0 grade subepithelial haze was observed in all rabbits,but the implanted grafts remained transparent.Re-innervation occurred in all grafts with no significant differences between sterilization methods.All regenerated corneas had mucin production and were positive for cytokeratin 3 and 12.Grafted and control corneas were negative for macrophages and blood vessels.Conclusions:E-beam sterilization is a safe and effective form of sterilization for RHCIII-MPC implants.
文摘Background:Regeneration of nerves or nerve bundles is problematic mainly due to issues of nerves finding their target tissues.A very clear example of this is the lack of treatments for traumatic injury to the optic nerve,something that is associated with surgery or trauma to the skull.Nerve guides have been used to support this for the better part of the last century,unfortunately the clinical improvements in patients receiving this sort of treatment is poor.Large improvements to the type of nerve guides used are needed to make this a viable solution for repair.It has been shown that electrostimulation of cells on conductive polymers can have positive effects on nerve regeneration.There are several material innovations that improve on speed of nerve regeneration;conductive polymer coatings being one example.There are constant improvements on solutions for nerve regeneration in many fields,unfortunately combining these different solutions is often slow.We combine electrospinning,3D printing and surface modification.Electrospinning allows control over fibrous structures.We tune the surface properties using conductive polymer coatings.The conductive fibrous structure can be integrated in a larger 3D printed scaffold that takes the role of guiding the nerve bundle.Methods:For manufacture of aligned fibers,PCL in chloroform was electrospun on a rotating mandrel.Random fibers were collected on a flat stationary collector.Dip coating was performed by submerging the fibrous scaffold in a solution of PEDOT:PSS in water and isopropanol.An outer layer of PEDOT:tosylate was added using vapor phase polymerization(VPP).3D printing was performed using an ink consisting of 0.25%alginate and 8.75%gelatin.The ink was cross-linked after printing using 0.4%CaCl2.Cell cultures were performed using chick dorsal root ganglia and a mouse neuroblastoma cell line.Ganglia and cells were seeded on the fibrous scaffolds.Electrostimulation was performed using a custom set up at constant DC current and slow pulsed DC current(1 min on off cycle)Materials were imaged using scanning electron microscopy.Cell cultures were stained using ICC and imaged with fluorescence microscopy.Results:All of the materials supported cell growth and neurite extension to some degree.The materials that were coated with PEDOT:tosylate and a combination of PEDOT:tosylate+PEDOT:PSS outperformed the PSS only group.Stimulation with a slow pulsed or constant DC current increased neurite extension on the negative pole,while there was inhibition of neurite growth on the opposite pole.The 3D printed outer layer serves as a biocompatible,bioactive support and guide for the bundle of neurites.Conclusions:The nerve guides can guide nerve growth.The 3D printed scaffold is cell friendly.The construct allows electrostimulation to increase speed of regeneration.
